Chapter 3
Architecture and Assembly of Virus Particles SUMMARY Knowledge of the detailed structure of virus particles is an essential prerequisite to our understanding of many aspects of virology. This outer shell has to be robust enough to protect the viral genome while the particle is outside the cell yet to be labile enough to enable the genome to be released into a newly infected cell; furthermore, its construction should have minimal energy requirements. In recent years the development of the use of virus particles in nanotechnology (Chapter 15) has been based on an understanding of virus particle structure. Knowledge of virus architecture has increased greatly in recent years, due to a wide range of techniques which have given more detailed chemical information, and to the application of more refined electron microscopic, optical diffraction, and X-ray crystallographic procedures. This chapter describes the architecture of plant viruses, in many cases in molecular detail, and how the virus particles are assembled.
BOX 3.1 Terms Used in Virus Structure Capsid: The closed shell or tube of a virus Capsomere: The clusters of subunits on the capsid as seen in electron micrographs; also termed Morphological subunit. Encapsidation (or encapsulation): The process of enclosing the viral genomic nucleic acid in virus-encoded protein usually to form a virus particle. Morphological subunit: groups of protein subunits revealed by electron microscopy and X-ray crystallography. Multicomponent virus: Infectious virus genome divided between several nucleic acid segments which are separately encapsidated. Negative stain: The virus particle is embedded in an electron dense material, such as phosphotungic acid or uranyl acetate which forms a dense background against which the virus particle appears translucent in the electron microscope. This method is capable of providing information about structural details often finer than those visible in thin sections, replicas, or shadowed specimens and has the advantage of speed and simplicity. Nucleocapsid: The inner nucleoprotein core of membrane-bound viruses. Nucleoprotein: A complex between the viral genomic nucleic acid and virus-encoded protein which may or may not have a defined structure Particle (virus particle): The virus genome enclosed in a capsid, and for some viruses also a lipid membrane. Protein subunit: Individual virus-encoded protein molecule that makes up the capsomere or nucleoprotein; also termed Structural subunit. Virion: The mature virus which may be membrane bound; term interchangeable with virus particle (Caspar et al., 1962).
Many different terms are used in virus structure, and there has been some confusion in their use over the years. I will use the terms listed in Box 3.1. The implications of virus structure for virus self-assembly are discussed later in this chapter. Virus disassembly is discussed in Chapter 6 as it is usually closely related to initial expression of the encapsulated genome. Figure 1.3 shows the range of sizes and shapes found among plant viruses.
I. METHODS A large number of methods are used not only to determine the shape and size of virus particles but also to show the distribution of the component proteins and nucleic acids (and lipid membranes in some cases). Information on the molecular structure of the component proteins and on the stabilizing bonds that hold the capsid together is important in understanding how particles assemble, their stability, and how they can be dissociated to release the encapsulated genome.
A. Chemical and Biochemical Methods Knowledge of the size and nature of the viral nucleic acid and of the proteins and other components occurring in a Plant Virology, Fifth Edition. © 2014 2012 Elsevier Inc. All rights reserved.
virus particle is essential to an understanding of its architecture. Chemical and enzymatic studies may give various kinds of information about virus structure. For example, the fact that carboxypeptidase A removed the terminal threonine from intact TMV1 indicated that the C-terminus of the polypeptide was exposed at the surface of the virus (Harris and Knight, 1955). Using methyl picolinimidate, which reacts 1
Acronyms of virus names are shown in Appendix D.
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with the exposed amino group of lysine residues, with several TMV mutants Perham (1973) determined which residues were at the surface of the virus and which were buried. In the technique of tritium planigraphy the macromolecular target is exposed to a beam of tritium atoms and the distribution of tritium labeling is analyzed along the length of the macromolecule. Applying this technique Dobrov et al. (2004) proposed structural models for PVX and PVA and showed fine structural differences between wild-type TMV and a temperature-sensitive mutant. Tritium planigraphy is especially useful with targets that for some reason cannot be assayed by other methods, such as X-ray analysis, nuclear magnetic resonance, or cryoelectron microscopy. With improvements in analytic methods (e.g., mass spectroscopy), tritium planigraphy may also increase in resolution to allow the details of nucleic acid–protein interactions in various nucleoproteins to be assessed directly (Dobrov et al., 2007). For viruses with more complex structures, partial degradation by chemical or physical means—for example, removal of the outer envelope—can be used to establish where particular proteins are located within the particle (Jackson, 1978; Lu et al., 1998). Studies on the stability of a virus under various pH and ionic or other conditions may give clues as to the structure and the nature of the bonds holding the structure together. Many studies of this sort have been carried out, but results are often difficult to interpret in a definitive way (Kaper, 1975). Some examples of this approach are given later in this chapter.
B. Methods for Studying Size of Viruses 1. Hydrodynamic Measurements Various properties (e.g., diameter) of the virus particle can be measured in solution in contrast to many other methods which give nonhydrated properties. The classic procedure for hydrodynamic measurements has been to use the Svedberg equation (Schachman, 1959). M
RTs D(1 υ * ρ )
where R is the gas constant per mole (8.134 J mole−1 K−1), T is the absolute temperature in degrees Kelvin, s is the sedimentation coefficient, D, the diffusion coefficient, and v*, the partial specific volume for the virus being studied. D and v* are rather troublesome to determine by classic procedures. s can be determined readily in the analytical ultracentrifuge. For this reason many workers go no further than determining s for a new virus. The term molecular weight is widely used with reference to viruses. I will follow this usage, but particle weight would be strictly a more appropriate term. s may be conveniently determined in a preparative ultracentrifuge by sedimenting the virus in a linear sucrose
density gradient and comparing the distance it sediments to internal markers of known s. The markers and unknown should have similar sedimentation rates. If not, other methods of calculation can be used (Clark, 1976). Laser light scattering has been used to determine the radii of several approximately spherical viruses with a high degree of precision (Harvey, 1973; Camerini-Otero et al., 1974). This method is of particular interest since it gives an estimate of the hydrated diameter whereas the Svedberg equation and electron microscopy give a diameter for the dehydrated virus. Comparison of the two approaches can give a measure of the hydration of the virus particle. The dynamic properties of crystalline arrays of STNV particles were measured under hydrated and air-dried conditions by Brillouin light scattering (Stephanidis et al., 2007). On air-drying the crystals stiffened up, illustrating differences between hydrated and nonhydrated conditions.
2. Transmission Electron Microscopy Measurements made on electron micrographs of isolated virus particles, or thin sections of infected cells, offer very convenient estimates of the size and shapes of viruses. For some of the large viruses and for the rod-shaped viruses such measurements may be the best available, but they are subject to significant errors. There are various magnification errors inherent in measurements on electron micrographs which may be overcome in part, but not entirely, by using a well-characterized, stable, and distinctive virus, such as TMV as an internal standard (Bos, 1975). However, Markham et al. (1964) pointed out that TMV rods may not be absolutely uniform in length. The width of the rods may offer a better standard. Flattening of particles on the supporting film, which can be detected with a tilting stage (Serwer, 1977), may cause significant errors. If films not coated with carbon are used, there may be significant distortion of individual virus particles probably due to stretching of the film (Ronald et al., 1977). When length distributions of rods are being prepared, it is necessary to combine individual measurements into size classes. If the size classes chosen are too large then the presence of more than one length of particle may be obscured. There are additional difficulties in measuring the contour lengths of flexuous rods (de Leeuw, 1975). Hatta (1976) pointed out that measurements of interparticle distances in arrays of particles seen in thin sections of crystals of purified small isometric viruses will always be less than the true diameter of the particles because particles overlap in the arrays.
3. X-Ray Crystallography For viruses that can be obtained as stable crystalline preparations, X-ray crystallography can give accurate and
Chapter | 3 Architecture and Assembly of Virus Particles
unambiguous estimates of the radius of the particles in the crystalline state (usually unhydrated). The technique is limited to viruses that are stable or can be made stable in the salt solutions necessary to produce crystals.
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with lipoprotein bilayer membranes. Although Steere (1969) illustrated arrays of freeze-fractured PYDV particles within an infected cell, no novel structural information has yet been obtained for plant viruses using this procedure.
4. Neutron Small-Angle Scattering Neutron small-angle scattering (see Section I, F) can give accurate estimates of the radius of icosahedral viruses in solutions containing relatively low salt concentrations. The method has confirmed that particles of some small icosahedral viruses, such as BMV, in solution under conditions where they are stable, may vary in size (Chauvin et al., 1978) and has shown the swelling of TBSV particles under certain conditions (Aramayo et al., 2005) (see Section V, B, 4, d).
5. Atomic Force Microscopy TMV particles, when dried on glass substrates, assemble into characteristic patterns that can be studied using atomic force microscopy (AFM) (Maeda, 1997). In highly orientated regions, the particle length was measured as 301 nm and the width as 14.7 nm; the latter measurement shows intercalation of packed particles. The particles are not flattened, and their depth was 16.8–18.6 nm. AFM can also be used to study the nucleation, growth, and potential disorders in crystal formation (Kuznetsov et al., 2000; Malkin et al., 2002; Kuznetsov and McPherson, 2011).
C. Fine Structure Determination: Electron Microscopy (see Sections II and IV for details of fine structures). Horne (1985) and De Carlo and Harris (2011) give accounts of the application of electron microscopy to the study of plant virus structure.
1. Metal Shadowed Preparations In early electron microscope studies of virus particles, shadowing with heavy metals was used to enhance contrast. Such shadowing tended to obscure surface details but gave information on overall size and shape of the dry particle. Much more information can be obtained if specimens are freeze-dried and then shadowed in a very high vacuum (Hatta and Francki, 1977). For example, Roberts (1988), using this kind of procedure, was able to show that TRSV had a structure resembling that of models made up of 60 subunits in clusters of five arranged in a T = 1 structure.
2. Freeze Etching This technique can give useful information about the surfaces and substructure of larger viruses—particularly those
3. Negative Staining The use of electron-dense stains has proved of much greater value than metal shadowing in revealing the detailed morphology of virus particles. Such stains may be positive or negative. Positive stains [e.g., various osmium, lead, and uranyl compounds and phosphotungstic acid (under certain conditions)] react chemically with and are bound to the virus. Chemical combination may lead to alteration in, or disintegration of, the virus structure. In negative staining, on the other hand, the electron-dense material does not react with the virus but penetrates available spaces on the surface or within the virus particle. This is now the preferred technique for examining virus structure by electron microscopy. Common negative stains are potassium phosphotungstate (KPT) at pH 7.0, uranyl acetate or uranyl formate used near pH 5.0 and ammonium molybdate at the desired pH. The virus structure stands out against the surrounding electrondense material. The carbon substrates used to mount stained specimens have a granular background. Structural detail can often be seen most clearly in images of particles in a film of stain suspended over holes in the carbon grid. The stain may or may not penetrate any hollow space within the virus particle. For a review on technical improvements of the basic method of negative staining, see Laue (2010). With helical rod-shaped particles, the central hollow is frequently revealed by the stain. With the small spherical viruses, which often have associated with them empty protein shells, it was assumed by some workers that stained particles showing a dense inner region represented empty shells in the preparation. However, staining conditions may lead to loss of RNA from a proportion of the intact virus particles, allowing stain to penetrate, while stain may not enter some empty shells. Stains differ in the extent to which they destroy or alter a virus structure, and the extent of such changes depends closely on the conditions used. The particles of many viruses that are stabilized by protein:RNA bonds are disrupted by KPT at pHs above 7.0 (Section I, J). Even in the best electron micrographs, the finer details of virus structure tend to be obscured, first, by noise due to minor irregularities in the actual virus structure and other factors, such as irregularities in the stain, and second, by the fact that contrast due to the stain is often developed on both sides of the particle to varying degrees. To overcome these difficulties and to extract more reliable and detailed information from images of negatively stained individual virus particles, several methods have been used.
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FIGURE 3.1 (A) Part of a 2D hexagonal array of TroV showing regular packing of the particles within the lattice. The central light region of individual capsids can be clearly seen together with a few particles where the interior has been completely penetrated by stain. The shape and size of the capsids in this orientation within the hexagonal array are more regular than those observed in a square or skewed array. (B) Optical diffraction pattern obtained from material in (A) showing typical spectra present in TRoV hexagonal arrays. The first order spots a-a correspond to the center-to-center particle spacing of 27.5 nm. From Horne et al. (1977) with permission of the publishers.
The main procedures have been photographic superposition (Markham et al., 1963, 1964; Finch and Holmes, 1967), shadowgraphs (Finch and Klug, 1966, 1967), and optical transforms (Klug and Berger, 1964; Crowther and Klug, 1975; Steven et al., 1981). Vogel and Provencher (1988) developed a computational procedure for threedimensional (3D) reconstruction from projections of a disordered collection of single particles and have applied it to TBSV. Optical diffraction has been applied to in vitro crystalline arrays of isometric viruses viewed under the electron microscope (Horne and Pasquali-Ronchetti, 1974; Horne et al., 1977) (Figure 3.1). With developments facilitating the application of cryoelectron microscopy and
X-ray crystallographic analysis to virus structure these procedures will probably not be widely used, except for viruses to which X-ray analysis cannot yet be applied.
4. 3D Images from Electron Microscopy (reviewed by Jonic et al., 2008) Several techniques have been developed for determining the 3D structure of biological specimens using transmission electron microscopy (TEM). Each 3D TEM technique requires a particular image acquisition protocol and a computational method for reconstruction of the 3D structure from the acquired images. The structure of highly
Chapter | 3 Architecture and Assembly of Virus Particles
symmetrical specimens, such as helical structures (e.g., filamentous viruses) or icosahedral structures (e.g., protein capsid of some viruses) can be solved at high resolution by 3D TEM methods that do not require two-dimensional (2D) crystals but rely on symmetry properties of the particle. Two 3D TEM techniques, single-particle analysis (SPA) and electron tomography (ET) (reviewed by Fu and Johnson, 2011) are becoming more widely used. Both techniques have been applied to viruses but not so much plant viruses. SPA is used for studies of macromolecules and macromolecular assemblies whose structure and dynamic interactions can be analyzed in vitro, in isolation (e.g., viruses). The data collection for this approach consists in taking 2D projections of a sample containing many identical but differently oriented copies of the same object. Thus, when an even distribution of single particle orientations is observed on the specimen grid, this technique allows collecting all necessary data for the computation of a 3D average structure of the studied particles. For ET a beam of electrons is passed through the sample at incremental degrees of rotation around the centre of the target sample. This information is collected and used to assemble a 3D image of the target. Current resolutions of ET systems are in the 5–20 nm range, suitable for examining heterogeneous, complex structured, and pleomorphic viruses (Subramaniam et al., 2007).
5. Thin Sections Some aspects of the structure of the enveloped viruses, particularly bilayer membranes, can be studied using thin sections of infected cells or of a pellet containing the virus (MacLeod et al., 1966).
6. Cryoelectron Microscopy (reviewed by Laue, 2010; Hryc et al., 2011) Cryoelectron microscopy (cryo-EM), which involves the extremely rapid freezing of samples in an aqueous medium, allows the imaging of symmetrical particles in the absence of stain and under conditions that preserve their symmetry. This technique has been widely used for isometric viruses and has been used sometimes for rod-shaped viruses (Jeng et al., 1989). For these particles, the images have to be reconstructed to give a 3D object from a 2D image using a range of approaches, for example, Fuller et al. (1996), Lanczycki et al. (1998), and Rossmann and Tao (1999). This leads to information, which complements that from X-ray crystallography and also allows the detailed analysis of viruses that are not amenable to crystal formation. For instance, the structure of CaMV has been resolved by cryoEM to 30 Å resolution (Cheng et al., 1992). Recent advances in the technique include single-particle cryo-EM, which makes it possible to visualize large
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macromolecular structures to near-atomic resolutions (3.3–4.6 Å) (Sachse et al., 2007; Baker et al., 2010) and the use of a CCD camera which enhances the signal-to-noise ratio (Clare and Orlova, 2010). This technique has been used mainly on animal viruses but is equally applicable to plant viruses.
7. Cryoelectron Microscopy Compared with Negative Staining There has been considerable discussion as to the relative advantages and disadvantages of cryo-EM and negative staining. The basic points are: (i) the contrast of unstained biological materials used for cryo-EM is low leading to problems in focusing and in the signal-to-noise ratio; (ii) fully hydrated specimens are electron-beam sensitive; (iii) cryo-EM is considered to be difficult and costly; and (iv) the resolution of cryo-EM combined with image analyses is often greater than negative staining. Adrian et al. (1998) have developed a technique, termed cryo-negative staining, that combines the advantages of the two techniques. The vitrified samples are prepared with ammonium molybdate and are blotted onto holey carbon supports.
8. Atomic Force Microscopy (reviewed by McPherson and Kuznetsov, 2011) Atomic force microscopy (AFM) or scanning force microscopy (SFM) was developed from scanning tunnel microscopy and has a resolution on the order of fractions of a nanometer. The information is gathered by “feeling” the surface with a mechanical probe. Piezoelectric elements that facilitate tiny but accurate and precise movements on (electronic) command enable the very precise scanning. AFM is increasingly being used to study viruses and virus-like particles (reviewed by Baclayon et al., 2010). The RNA-CP interaction in TMV particles have been studied by using AFM-based single-molecule force microscopy (Liu et al., 2010).
D. X-Ray Crystallographic Analysis Where it can be applied, X-ray crystallography provides a powerful means of obtaining information about structures that are regularly arrayed in three dimensions. It can give detailed information on both the structure of virus particles and that of the protein subunits. Finch and Holmes (1967) give an introductory account of the methods involved. Over about the past 30 years or so there have been significant advances that have allowed the application of X-ray crystallographic analysis to more viruses, and at higher resolutions. With the definition of structures at the atomic level, it has become possible to interpret structure
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in more detail in relation to biological function. The major advances have been: (i) improved methods of crystallization (Lorber, 2008; Lorber and Witz, 2008; Lorber et al., 2008); (ii) an increase in the capacity and speed of computers, together with a reduction in cost; (iii) noncrystallographic symmetry averaging. Icosahedral viruses, such as TBSV and STNV have 60-fold symmetry in the virus particle. The degree to which this coincides with the symmetry of the crystal used for analysis depends on the crystal form that happens to be obtained. For example, in TBSV the smallest crystal repeating unit (the asymmetric unit) consists of five trimers of the CP which are exactly related by crystallographic symmetry. Thus, symmetry averaging for this virus takes place around a chosen fivefold axis. Successive recalculations from an initial electron density map remove noise and enhance detail in the map. An example of the procedure is given by Olson et al. (1983) for TBSV; (iv) the development of computer graphics. This technology has now replaced the laborious manual model building of the past, which was necessary for the refinement of a structure (Olson et al., 1985). Computer graphics are also extremely useful for exploring particular aspects of a 3D structure and for communicating structural ideas in a more readily comprehensible form, by such means as color coding and selective omission of detail (Namba et al., 1984, 1988); (v) the introduction of site-directed mutagenesis to protein crystallography. The possibility of exchanging one amino acid for another in any chosen site in a protein changes crystallography from a passive technique to one in which the relation between structure and function can be studied in a systematic and rational fashion. X-ray crystallography analysis has two main limitations as far as virus structures are concerned: (i) for nearly all icosahedral viruses most of the nucleic acid is not arranged in a regular manner in relation to the symmetries in the protein shell. However, for some isometric viruses it is possible to obtain information on the distribution of nucleic acid within the particle (Figure 3.37); (ii) larger viruses such as members of the Rhabdoviridae cannot be crystallized. In addition, it is probable that many features of their structure are not strictly regular. For such viruses, electron microscopy provides the best information.
E. Fiber Diffraction (reviewed by Stubbs et al., 2008) Filamentous viruses do not form ordered crystals and therefore their structure cannot be studied by X-ray crystallography. However, if the particles can be orientated so that their long axes are parallel to each other, they can be examined by fiber diffraction. In this technique, the fibers are probed by X-rays and the scattering data analyzed in a manner similar to X-ray crystallography.
Plant Virology
F. Neutron Small-Angle Scattering Neutron scattering by virus solutions is a method by which low-resolution information can be obtained about the structure of small isometric viruses and in particular about the radial dimensions of the RNA or DNA and the protein shell. The effects of different conditions in solution on these virus dimensions can be determined readily. The method takes advantage of the fact that H2O-D2O mixtures can be used that match either the RNA or the protein in scattering power. Analysis of the neutron diffraction at small angles gives a set of data from which models can be built. Neutron scattering, in combination with other techniques, has been used for studying swelling of virus particles (Aramayo et al., 2005).
G. Mass Spectrometry As well as measuring the mass of viral proteins and particles, mass spectrometry can be used to identify viral protein posttranslational modifications, such as myristoylation, phosphorylation, and disulfide bridging (reviewed by Siuzdak, 1998) and cleavage sites of a polyprotein (Marmey et al., 1999). When this technique is used in conjunction with other techniques (e.g., X-ray crystallography or hydrogen exchange), the mobility of the capsid can be studied (Wang et al., 2001; Lucas et al., 2002a). Similarly, nuclearmagnetic-resonance spectroscopy can detect mobile elements on the surface of virus particles (Brierley et al., 1993).
H. Raman Optical Activity Raman optical activity is a powerful probe of the aqueous solution structure of proteins. It has been used to give information on the structure of the coat proteins (CPs) of some rod-shaped (PVX, NMV, and TRV) which are not amenable to studying by high-resolution X-ray methods (Zhu et al., 2006).
I. Serological Methods The reaction of specific antibodies with intact viruses or dissociated viral CPs has been used to obtain information that is relevant to virus structure. For instance, the terminal location of the minor CP on the flexuous rod-shaped particles of closteroviruses and criniviruses was recognized using polyclonal antibodies (Figure 3.11). Monoclonal antibodies (MAbs) are proving to be particularly useful for this kind of investigation, although there are significant limitations (reviewed by Kekuda et al., 1995). Some other examples of the use of MAbs are in the determination of the antigenic structure of PMTV (Pereira et al., 1994) and of PVA (Moravec et al., 1998). For a description of the type of epitopes that induce antibodies see Chapter 13, Section III, A.
Chapter | 3 Architecture and Assembly of Virus Particles
Reactivity of determinants with MAb of group
Schematic representation of determinants ( ) on undisturbed (a) and partially denatured (b) subunits
(a) I
(b)
(a) II
(b)
(a) III (b)
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Properties of determinants
These determinants are located outside the protruding N terminus and are destroyed by extensive denaturation; they become masked in partially denatured preparations containing the protruding N terminus
These determinants are located on the protruding N terminus: they are lost when the virus is treated with trypsin or proteases in crude plant sap: they become inaccessible or are destroyed in partially denatured preparations.
These determinants are located outside the protruding N terminus and are exposed in the B, CsAg and M strains only after partial denaturation of the virus particles. They are not destroyed by extensive denaturation, e.g. with SDS.
FIGURE 3.2 Schematic representation of the three kinds of antigenic determinants distinguished in the coat protein of PVX by their differential reactivity to a set of MAbs. From Koenig and Torrance (1986) with permission of the publishers.
Structural interpretation of the results of cross-reactions between closely related proteins can be confused by the fact that the conformation of any antigenic determinant may be changed by an amino acid substitution occurring elsewhere in the protein. Furthermore, the ability of different MAbs to detect residue exchanges may be extremely variable as was found for a set of TMV mutants (Al-Moudallal et al., 1982). Nevertheless, serological techniques have produced some structural information, especially with viruses for which the detailed protein structure has not been determined by X-ray crystallography. For example, at least three different antigenic determinants were distinguished on the CP of PVX with a set of MAbs (Koenig and Torrance, 1986). The results are summarized in Figure 3.2. The surface location of the N terminus was confirmed by Sõber et al. (1988).
The N-terminal and C-terminal regions of the CP of TYMV were interpreted to be at the surface of the virus (Quesniaux et al., 1983a,b). Similarly, immunological evidence confirmed that these N-terminal regions are also at the virus surface of potyviruses (Shukla et al., 1988). A surface location for both N and C termini appears to be a common feature in other rod-shaped viruses. Caution must be used in interpreting the results of ELISA tests in structural terms when the whole virus is the antigen, as the following results demonstrate. Antibodies against a synthetic peptide of TBSV making up part of the flexible N-terminal arm (amino acids 28–40) reacted with whole virus as antigen in ELISA tests (Jaegle et al., 1988). Since the N-terminal arm is in the interior of a very compact shell (Section V, B, 4, d), this result must mean that virus structure was opened up sufficiently on the ELISA
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(Dore et al., 1989). This result confirms in a graphic way a fact already known from X-ray crystallographic analysis, namely, that the CP in the TMV rod presents a chemically different aspect on its upper and lower surfaces. Using a similar technique, Lesemann et al. (1990) distinguished three groups of MAbs reacting with BNYVV. One reacted along the entire length of the particles. The other two groups reacted with antigenic sites on opposite ends of the particles.
J. Methods for Studying Stabilizing Bonds
FIGURE 3.3 Electron micrographs showing the binding of a goldlabeled MAb to one extremity of TMV rods. Bar = 100 nm. From Dore et al. (1988) with permission of the publishers.
plate to allow antibodies to react with the normally buried arm. Similar results have been obtained for SBMV (MacKenzie and Tremaine, 1986). As was indicated by Dore et al. (1987), a definitive delineation of antigenic sites requires knowledge of the 3D structure of the polypeptides. In most cases, if the 3D structure is known, what is the point, in relation to their structure, of determining the antigenic sites for plant viruses? The situation is quite different for viruses infecting vertebrates, where knowledge of such sites may be very important for vaccine development. However, with recombinant DNA technology an understanding of the surface structure of a plant virus can lead to identification of sites at which foreign epitopes can be introduced and the recombinant virus used for vaccination (Chapter 15, Section IV, B). Dore et al. (1988) developed an elegant procedure involving ELISA reactions on electron microscope grids and gold-labeled antibody. Using this procedure, they showed that anti-TMV MAbs that reacted with both virus particles and the CP subunits bound to the virus rods only at one end (Figure 3.3). Further studies have shown that the MAbs bind to the surface of the protein sub unit that contains the right radial and left radial α-helices
The primary structures of viral CPs and nucleic acids depend on covalent bonds. In the final structure of the simple geometric viruses, these two major components are held together in a precise manner by a variety of noncovalent bonds. Three kinds of interactions are involved: protein:protein, protein:RNA, and RNA:RNA. In addition, small molecules, such as divalent metal ions (Ca2+ in particular) may have a marked effect on the stability of some viruses. Knowledge of these interactions is important for understanding the stability of the virus particle in various environments, how it might be assembled during virus synthesis, and how the nucleic acid might be released following infection of a cell. The stabilizing interactions are hydrophobic bonds, hydrogen bonds, salt linkages, and various other long- and short-range interactions. A variety of physical and chemical methods has been used in attempts to refine our understanding of the role of these bonds in virus structure.
1. X-Ray Crystallographic Analysis Models built to a resolution of less than 3 Å, based on X-ray crystallographic analysis and knowledge of the primary structure of the CP provide a detailed understanding of the bonds in the secondary and tertiary structure of the protein subunit. In addition, the bonds between subunits that make up the quaternary structure of the virus, and the role of accessory molecules and ions, mainly water and Ca2+, can be defined. A virus structure examined to atomic resolution provides a vast amount of detail that cannot be encompassed in a book of this size. The highlights of several such structures are discussed in later sections.
2. Stability to Chemical and Physical Agents The effects of pH, ionic strength, kind of ion, temperature, compounds, such as phenol and detergents, and hydrogen bond-breaking agents, such as urea, on the stability of viruses have been studied in many laboratories (Kaper, 1975). Such experiments can give us information only of a general kind about the bonds involved in virus stability.
Chapter | 3 Architecture and Assembly of Virus Particles
Among the small isometric viruses there is a wide range of stabilities. Viruses like TYMV with strong protein:protein interactions are the most stable. The other end of the spectrum is illustrated by CMV and AMV, where protein:RNA interactions predominate (Kaper, 1975). Protein:protein interactions are obviously very important for the stability of TYMV since empty protein shells are quite stable. Hydrophobic bonding between subunits is a significant factor in this stability because (i) TYMV is very stable at high ionic strengths; (ii) it is readily degraded by phenol and ethanol; and (iii) degradation of the virus and empty protein shell by urea, organic mercurial compounds, and other chemicals can be interpreted on the basis that the predominant protein:protein interaction is via hydrophobic bonding (Kaper, 1975). Extreme conditions, such as high pH (Keeling and Matthews, 1982) and freezing and thawing (Katouzian-Safadi and Haenni, 1986) have been used to study release of RNA from TYMV particles. Alkaline conditions have been used to demonstrate the removal of protein subunits from the TMV rod, beginning at the 5′ end, and to reveal intermediates in the stripping process, these being attributed to regions of unusually strong interaction between the protein and RNA (Perham and Wilson, 1978).
3. Modification of the Coat Protein Particular amino acid residues in the CP can be modified by the chemical addition of a side chain, and the effects of such substitution on stability of the virus can be examined (e.g., Wilson and Perham, 1985, for TMV). The CP can also be modified by mutation of the coding region in infectious cDNA clones of a virus.
4. Removal of Ions For small isometric viruses whose structures are partially stabilized by Ca2+ ions, removal of these ions, usually by EDTA or EGTA, leads to swelling of the virus particle (see Section V, B, 4, d, i). A study of the swelling phenomenon can give information about the kinds of bonds important for stability. A variety of techniques has been used for monitoring swelling. For example, Krüse et al. (1982) used small-angle X-ray and neutron scattering, analytical centrifugation, and fluorescence techniques to study swelling of TBSV. Swelling of STNV in the presence of EDTA was demonstrated by ultracentrifugation and X-ray crystallography (Unge et al., 1986). In swollen virus an amino-terminal peptide that was normally well buried in the structure became susceptible to trypsin. The kinetics of swelling of SBMV was studied using photon correlation spectroscopy (Brisco et al., 1986). Durham et al. (1984) used hydrogen-ion titration curves to follow the reversible swelling and contraction of particles of TBSV, CCMV, and TCV. They drew conclusions regarding the semipermeability of various protein shells.
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Virus particle swelling and of other conformational changes involved in the disassembly of viruses are discussed in Chapter 6, Sections III, D and E.
5. Circular Dichroism Circular dichroism spectra can be used to obtain estimates of the extent of α-helix and β structure in a viral protein subunit (e.g., Denloye et al., 1978 for TRoV; Odumosu et al., 1981, for SBMV). Vibrational circular dichroism of TMV, PapMV, NMV, and PVX has shown helical structures in the CP but not in the RNA (Shanmugam et al., 2005).
6. Methods Applicable to Nucleic Acid Within the Virus Particle Several methods are available to give approximate estimates of the degree of helical base-pairing or other ordered arrangement of the nucleic acid within the virus. These include relative absorbency at 260 nm (e.g., Haselkorn, 1962, for TYMV), laser Raman spectroscopy (e.g., Hartman et al., 1978, for TYMV), circular dichroism spectra (e.g., Odumosu et al., 1981, for SBMV), and magnetic birefringence (e.g., Torbet et al., 1986, for CaMV).
K. Discussion There is a wide range of techniques which can address specific questions of virus shape and structure (Table 3.1) The application of combinations of different methodologies (“hybrid approaches”) (Steven and Baumeister, 2008) coupled with advances in computation methods is giving a greater insight into details of viral structures and a better understanding of how viruses function.
II. ARCHITECTURE OF ROD-SHAPED VIRUSES A. Introduction Crick and Watson (1956) put forward a hypothesis concerning the structure of small viruses, which has since been generally confirmed. Using the knowledge then available for TYMV and TMV, namely, that the viral RNA was enclosed in a coat of protein and (for TMV only, at that stage) that the naked RNA was infectious, they assumed that the basic structural requirement for a small virus was the provision of a shell of protein to protect its RNA. They considered that the relatively large protein coat might be made most efficiently by the virus controlling production in the cell of a large number of identical small protein molecules, rather than in one or a few very large ones. They pointed out that if the same bonding arrangement is to be used repeatedly in the particle the small protein
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TABLE 3.1 Some Methods Used for Studying Virus Structurea Method
What Determined Particle Composition
Particle Size
Particle Structure
Coat Protein Coat Protein Particle Stabilizing Nucleic Acid in Size Structure Bonds Particle
Chemical
+
−
+
−
−
+
+
Enzymatic
+
−
+
+
−
+
+
pH
−
−
−
−
−
+
−
Electrophoresis
+
+
−
+
−
−
−
Sedimentation/ diffusion
−
+
−
−
−
+
+
Laser light scattering
−
+
−
−
−
−
−
Neutron small angle scattering
−
+
+
−
+
+
+
Transmission electron microscopy
−
+
+
−
−
+
+
Cryo-electron microscopy
−
+
+
−
−
+
+
Atomic force microscopy
−
+
+
−
−
+
+
X-ray crystallography
−
+
+
−
+
+
+
Fiber diffraction
−
+
+
−
+
−
−
Mass spectrometry
−
+
+
+
+
+
−
Raman optical activity
−
−
+
−
+
−
+
Circular dichroism
−
−
+
−
+
+
+
Serology
+
−
−
+
−
−
−
a
Indication of use of techniques. Modifications of the technique might increase the rage of use.
molecules would then aggregate around the RNA in a regular manner. There are only a limited number of ways in which the subunits can be arranged. The structures of all the geometric viruses are based on the principles that govern either rod-shaped or spherical particles. In rod-shaped viruses, the protein subunits are arranged in a helical manner. There is no theoretical restriction on the number of protein subunits that can pack into a helical array in rod-shaped viruses. There are basically two types of rod-shaped particles, rigid rods and flexuous rods; some properties of these are given in Table 3.2
B. Rigid Rod-Shaped Particles 1. Tobamovirus Genus a. General Features The particle of TMV is a rigid helical rod, 300 nm long and 18 nm in diameter with a central canal of ~4 nm diameter
that becomes filled with stain in negatively stained preparations (Figure 3.4A). The composition of the particle is approximately 95% protein and approximately 5% RNA. It is an extremely stable structure, having been reported to retain infectivity in nonsterile extracts at room temperature for at least 50 years (Silber and Burk, 1965). The stability of naked TMV RNA is no greater than that of any other ssRNA. Thus, stability of the virus with respect to infectivity is a consequence of the interactions between neighboring protein subunits and between the protein and the RNA. X-ray diffraction analyses have given us a detailed picture of the arrangement of the protein subunits and the RNA in the virus rod. The particle comprises approximately 2130 subunits that are closely packed in a helical array. The pitch of the helix has been precisely determined X-ray fiber diffraction methods to be 22.92 Å (Kendall et al., 2007), and the RNA chain is compactly coiled in a helix following that of the protein subunits (Figure 3.3B, C). There are three
Chapter | 3 Architecture and Assembly of Virus Particles
79
TABLE 3.2 Properties of Characterized Rod-Shaped Viruses Virus Genus
Particle Length (nm)
Particle Diameter (nm)
Rod Typea
Helix Pitch (nm)
Turns/ Repeat
Subunits/ Axial Hole Turn
Tobamovirus
300–310
18
R
2.29
3
16, 33
Yd
This text
c
Reference
Tobravirus
180–215, 46–115(m)b
21.3–23.1
R
2.5
ND
25 or 32
Y
King et al. (2012)
Hordeivirus
110–150 (m)
20
R
2.5
ND
ND
Y
Lawrence et al. (2000)
Furovirus
140–160, 260–300 (m)
20
R
2.4–2.5
ND
ND
Y
Torrance (2000)
Pomovirus
65–80, 150–160, 290–310 (m)
18–20
R
2.4–2.5
ND
ND
Y
Koenig and Lesemann (2000a)
Pecluvirus
190, 245 (m)
21
R
2.6
ND
ND
Y
King et al. (2012)
Benyvirus
85, 100, 265, 390 (m)
20
R
2.6
4
2.25
Y
Koenig and Lesemann (2000b)
Varicosavirus
320–360
18
R
5
ND
ND
Y
King et al. (2012)
Potexvirus
470–580
13
F
3.3–3.7
4–11
8–9
Y
Tollin and Wilson (1988)
Carlavirus
610–700
12–15
F
3.4
ND
ND
ND
Tollin and Wilson (1988)
Potyvirus
680–900
11–13
F
3.4–3.5
ND
ND
ND
Tollin and Wilson (1988)
Tritimovirus
690–700
12–13
F
3.3–3.4
ND
6.9
Y
Parker et al., (2005)
Capillovirus
640–700
10–12
F
3.4–3.8
9–10
ND
ND
Tollin and Wilson (1988)
Trichovirus
640–890
12
F
3.3–3.5
c10
9.3–9.8
ND
Tollin and Wilson (1988)
Vitivirus
725–825
12
F
3.3–3.5
5
c10
ND
Tollin and Wilson (1988), Tollin et al. (1992)
Closterovirus
1250–2000
12
F
3.4–3,8
5
9–10
ND
Tollin and Wilson (1988), Tollin et al. (1992)
R = rigid rod; F = flexuous rod. (m) = multicomponent virus. ND = not determined or, for axial hole, not detected. d Y = yes. a
b c
nucleotides of RNA associated with each protein subunit, and there are 49 nucleotides and 16 and 1/3 protein subunits per turn. The phosphates of the RNA are at about 4 nm from the rod axis. By tilting negatively stained TMV particles in the electron beam and noting changes in the edge appearance of the rods, Finch (1972) established that the basic helix of TMV is right-handed. In a proportion of negatively stained particles, one end of the rod can be seen as concave, and the other end is convex. The 3′ end of the RNA is at the convex end and the 5′ at the concave end (Wilson et al., 1976; Butler et al., 1977). b. Short Rods Most purified preparations of TMV contain a proportion of rods that are of variable length and less than 300 nm. Study of these short rods may be complicated by the problem of
aggregation. Both intact TMV rods and the shorter rods have a tendency to aggregate end to end under appropriate conditions, giving a wide range of lengths and the observed length distribution is very dependent on the precise history of the virus preparation. Some factors affecting the length distribution are pH, ionic strength, nature of ions present, temperature, and length of storage, but not all the factors involved are as yet fully understood. Many of the shorter rods are probably of no special significance. They may be virus particles that were only partially assembled at the time of isolation, or parts of rods fractured during isolation. However, in beans infected with SHMV the RNA containing the CP gene (~1 kb), which is synthesized as a discrete species during virus replication, is assembled into a rod about 40 nm long (Higgins et al., 1976).
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(A)
Outer face of the virus
C N
Beta-sheet
LS RR RS
LR
(C)
Particle axis
Short loop
Maximum radius Mean radius
Long loop
90 Å 75 Å
(B)
23 Å
RNA binding site
Pitch of helix
20 Å
Nucleic acid
FIGURE 3.4 Structure of TMV. (A) Electron micrograph of negatively stained TMV particles; bar = 100 nm. From Hull (2002) with permission. (B) Drawing showing relationship of the RNA and protein subunits; note that the RNA shown free of protein could not maintain that configuration in the absence of protein; (C). Photograph of a model of TMV with the major dimensions indicated. B and C from Klug and Caspar (1961) with permission.
c. Properties of the Coat Protein The CP comprises 158 amino acids giving it a molecular weight of 17- to 18-kDa. Fiber diffraction studies have determined the structure to 2.9 Å resolution (Namba et al., 1989) (Figure 3.5). The protein has a high proportion of secondary structure with 50% of the residues forming four α-helices and 10% of the residues in β-structures, in addition to numerous reverse turns. The four closely parallel or antiparallel α-helices (residues 20–32, 38–48, 74–88, and
Inner face of virus FIGURE 3.5 Ribbon representation of the α-carbon tracing of TMV coat protein. At the core of the protein is a right-handed helical bundle of four α-helices, the left-slewed (LS), the left-radial (LR), the right-slewed (RS), and the right-radial (RRP). The long loop connects the LR and RR helices. From Taraporewala and Culver (1997) with permission of the publishers.
114–134) make up the core of the subunit and the distal ends of the four helices are connected transversely by a narrow and twisted strip of β-sheet. The central part of the subunit distal to the β-sheet is a cluster of aromatic residues (Phe12, Trp17, Phe62, Tyr70, Tyr139, Phe144) forming a hydrophobic patch. The N- and C-termini of the protein are to the outside of the particle. The polypeptide chain is in a flexible or disordered state below a radius in the particle of about 4 nm so that no structure is revealed in this region. d. Structure of the Double Disk One of the reassembly products of TMV protein subunits (Section III, A) is a double disk containing two rings of 17 protein subunits that is of particular interest in that the details of inter-subunit contacts can be determined. Under appropriate conditions, the disks form true 3D crystals. Although the repeating unit is very large, X-ray crystallographic procedures can be applied. This was the approach taken by Klug and colleagues, which after 12 years work led to an elucidation of the structure of the protein subunit and the double disk to 2.8-Å resolution (Bloomer et al., 1978).
Chapter | 3 Architecture and Assembly of Virus Particles
Disk axis
C
59 50
A ring
LS 89
N
54
RS
37
13 152
27
RR
114
LR
6 127
RNA site
74
50
LS 89
114
20
C N
66
19
152
RS
37
B ring
147
134
40
27
RR LR
8 12
60
147
134 74
80 Radius (Å)
FIGURE 3.6 Side view of a sector through the disk of TMV showing the relative disposition of subunits in the two rings and the axial contacts between them. There are three regions of contact indicated by a solid line for the hydrophobic contact of Pro 54 with Ala 74 and Val 75, by dashed lines for the hydrogen bonds between Thr 59 and serines 147 and 148, and further dashed lines for the extended salt-bridge system. The lowradius region of the chain, which has no ordered structure in the absence of RNA, is shown schematically with an indication of an additional 1–2 turns extending the LR helix before it turns upwards into the vertical column. The primary nucleotide-binding site is probably below the RR helix. From Bloomer et al. (1978) with permission of the publishers.
Viewed in section, the subunits of the upper ring in the disk are flat. Those of the lower ring are tilted downward toward the center of the disk with three regions of contact vertically between subunits (Figure 3.6). The outermost contact is a polar interaction between Serines 147 and 148 and Thr59. At lower radius the largest contact region is a salt-bridge system involving a complex hydrogen bonded 3D network in which two water molecules also participate. The innermost contact is a small hydrophobic patch where Ala74 and Val75 both touch the ring of Pro54. Thus, the two disks, in contact at the outer part, open toward the center like a pair of jaws. The flexible inner parts of the folded chains are indicated by dotted lines in Figure 3.6. Other physical studies show that there is thermal motional disorder in this region rather than static disorder (Jardetzky et al., 1978). e. Virus Structure Intact TMV does not form 3D regular crystalline arrays in solution. For this reason structural analysis has not yet proceeded to the detail available for the double disk. However, using fiber diffraction methods, Stubbs et al. (1977) solved the structure to a resolution of 4 Å. Using this, together with the other data already available, they produced a model for the virus (Figure 3.7). Namba and Stubbs (1986) established a structure at 3.6 Å that refined the structure of Stubbs et al. (1977) in certain
81
details that are particularly relevant to our understanding of virus assembly. Namba et al. (1989) produced an even more refined structure at 2.9 Å that has generated a great deal of further information. The following discussion is based mainly on the structure of Namba and Stubbs (1986). The following are important features: i. The outer surface. As noted above, the N- and C-termini are at the virus surface. However, the very C- terminal residues 155–158 were not located on the density map and are therefore assumed to be somewhat disordered (Namba and Stubbs, 1986). ii. The inner surface. The presence of the RNA stabilizes the inner part of the protein subunit in the virus so that its position can be established. The highest peak in the radial density distribution is at about 2.3 nm. This is the region occupied by the vertical chains containing the V helices (Namba and Stubbs, 1986). These chains fill a space 0.9 nm wide by 2.3 nm high, and they are packed closely together to form a dense wall around the axial hole, protecting the RNA from the medium. iii. The RNA binding site. The binding site is in two parts, being formed by the top of one subunit and the bottom of the next. The three bases associated with each protein subunit form a claw that grips the left radial helix of the top subunit. The left radial helix has a large number of aliphatic residues between positions 117 and 128, which form three faces for the bases. The bases lie flat against the hydrophobic side chains and each face can accommodate any base. The other part of the RNA binding site is mostly on the right slewed helix. Two phosphate groups form ion pairs with Arg90 and Arg92. The third phosphate appears to form a hydrogen bond with Thr37 (Namba and Stubbs, 1986). Arg41 extends toward the same phosphate as Arg90 but does not approach as closely. Namba et al. (1989) suggest that the electrostatic interactions between protein and RNA are best considered as complementarity between the electrostatic surfaces of protein and RNA, rather than as simple ion pairs between arginines and phosphates. iv. Electrostatic interactions involved in assembly and disassembly. Caspar (1963) and Butler et al. (1972) obtained results indicating that pairs of carboxyl groups with anomalous pK values (near pH 7.0) (termed “Caspar” carboxylate pairs) were present in TMV. Namba and Stubbs (1986) identified two inter-subunit pairs of carboxyl groups in their model that may be those proposed by Caspar: Glu95-Glu106 at 2.5 nm radius and in a sideto-side interface, and Glu50-Asp77 at 5.8 nm radius in the top-to-bottom interface. These groups may play a critical role in assembly and disassembly of the virus (Section III, A and Chapter 6, Section III, B).
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RS LS RR LR
N C
3 1
2
FIGURE 3.7 Secondary structure in TMV coat protein. Backbone structures of 2 protein subunits and 3 RNA nucleotides (labeled 1, 2 and 3), represented as GAA, are illustrated. The RNA is enlarged at lower left with each nucleotide shaded differently. α-Helices are designated as in Figure 3.5, N (N-terminal, substantially obscured in this view) and C (C-terminal). The viral axis is vertical, to the left of the Figure. From Namba et al. (1989) with permission of the publishers.
Namba et al. (1989) identified three sites where negative charges from different molecules are juxtaposed in subunit interfaces: – a low-radius carboxyl-carboxylate pair appears to bind calcium; – a phosphate-carboxylate pair that also appears to bind calcium; – a high-radius carboxyl-carboxylate pair in the axial interface. This could not bind calcium but could bind a proton and thus titrate with an anomalous pK. These sites create an electrostatic potential that could be used to drive disassembly: However, this model of the “Caspar” carboxylate pairs gives some problems in understanding disassembly. In a fresh look at TMV structure using single particle cryoEM and iterative real-space helical reconstruction, Sachse
et al. (2007) developed a model that differed from that of Namba et al. (1989) in the region of residues 88–109. This is discussed in more detail in Section III, A, 2 and in Figure 3.15. These differences may be due to cryo-EM showing details of the hydrated form of the virus which X-ray crystallography may not (Clare and Orlova, 2010). v. Water structure. Water molecules are distributed throughout the surface of the protein subunit, both on the inner and outer surfaces of the virus and in the subunit interfaces (Namba et al., 1989). vi. Specificity of TMV protein for RNA. Gallie et al. (1987) showed that TMV protein does not assemble with DNA even if the origin of assembly sequence is included. Namba et al. (1989) concluded that this specificity must involve interactions made by the ribose hydroxyl groups, because all three base-binding sites could easily accommodate thymine.
Chapter | 3 Architecture and Assembly of Virus Particles
83
particle diameter (Figure 3.9). Differences in the helical structure could serve to fill the extra volume required by the large diameter of the cylindrical TRV particles relative to those of TMV (Blanch et al., 2001). Like TMV CP, TRV protein can exist in various discrete states of aggregation in solution. In particular, a double disk structure has been observed which have a diameter of 24.8 nm (similar to that of the virus particle) and comprise three turns of a right-hand helix with a pitch of 3.1 nm (different from that of the particle) (Roberts and Mayo, 1980); these may well play a part in virus assembly. FIGURE 3.8 Electron micrograph of TRV showing long and short rods negatively stained with uranyl formats. From Offord (1966) with permission of the publishers.
2. Tobravirus Genus TRV has rigid cylindrical rod-shaped particles built on the same general plan as TMV. TRV genome is divided between two particles, a long rod and a shorter one (Appendix A, Profile 84). Here I shall consider the structure of the long rods. The short rods are thought to be constructed on the same plan using the same protein subunit. Most information about the structure of this virus has been obtained by electron microscopy of virus particles stained in uranyl formate (Offord, 1966) (Figure 3.8). Some information was obtained by optical diffraction. For the strains studied by Offord, the length of the infectious particle was approximately 191 nm and its diameter was 25.6 nm. The pitch of the helix was 2.55 nm with 76 subunits in three turns (Table 3.2). The radius of the central hole was about 2.7 nm. An annular feature was observed at a radius of about 8.2 nm, which might represent the RNA chain. A densely stained ring has been observed at about this radius in cross-sections of rods (Tollin and Wilson, 1971). The strain studied by Offord had about 72 turns of the helix with probably 4 nucleotides per protein subunit, giving 7100 nucleotides. Harrison and Woods (1966) noted that in many of their electron micrographs of different TRV isolates the two ends of the particle had different shapes. One end was slightly convex, while the other end of the axial canal was slightly flared. They suggest that this appearance of the ends of the rods might be due to the protein subunits being banana-shaped with the long axes not inclined exactly 90° to the long axis of the particle. Comparisons of the CP sequences of tobraviruses with those of tobamoviruses (Goulden et al., 1992; Legorburu et al., 1996) reveal many common features suggesting that the predicted structures are similar. Goulden et al. (1992) proposed that the basic structure of tobravirus particles was similar to that of tombusviruses but based on a greater
3. Other Viruses with Rigid Rod-Shaped Particles Structural details of other genera of viruses with rigid rod-shaped particles (Hordeivirus, Furovirus, Pomovirus, Pecluvirus, Benyvirus, and Varicosavirus) are given in Table 3.2 and electron micrographs of some of these viruses are shown in the appropriate profiles in Appendix A. For many of these viruses there is no information on the details of the helical arrangement of the CP subunits, but it is likely that they have basic structures similar to those of TMV and TRV. The antigenic structures of two viruses with rigid rods, the furovirus SBWMV and the pomovirus PMTV have been determined using MAbs (Pereira et al., 1994; Chen et al., 1997). Both these studies showed that, as with TMV, the termini of the CP subunits were at or near the surface of the particle.
C. Flexuous Rod-Shaped Particles Some basic properties of flexuous rod-shaped virus particles are given in Table 3.2 and some details of representative members of the families are given in Appendix A. For many of these viruses there is little or no detailed information on the particle structure as they are difficult viruses for analysis (Kendall et al., 2008). Notable features are that the flexuous rods have a smaller diameter (about 11–13 nm) than the rigid rods (about 20 nm) and that the helix pitches of the flexuous rods (3.3–3.8 nm) are greater than that of rigid rods (2.4–2.6 nm). The flexibility of the flexuous rods is likely to result from their looser structure.
1. Family Alphaflexidae Most of the structural studies of the Flexiviridae have been done on three members of the genus Potexvirus, namely PVX, NMV, and PapMV (reviewed by Atabekov et al., 2007, Kendall et al., 2013). The basic properties of these viruses are listed in Table 3.2. In PVX particles about 1300 identical CP subunits are arranged in a helix of pitch 36 Å with the viral RNA located
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Plant Virology
Å
140 120 100 80 60 40 20
+
0
+
20 40 60 80 100 120 (A) TMV
140
(B) TRV
FIGURE 3.9 Comparison of TMV structure with that suggested for tobraviruses. In (A) the profile of the coat protein from Figure 1d of Namba et al., (1985) is rotated 22° (360°/16⅓) about the cross, which represents the center of the axial canal. In (B) the profile was moved radially out 40 Å, the lowradius insertion represented by the dotted line, and rotated 14° (360°/25⅓) about the analogous point. In both diagrams, the heavy circumferential line represents the position of the RNA backbone. The scale of the diagrams in Å is indicated by the central bar. From Goulden et al. (1992) with permission of the publishers.
between the turns of the helix. Each turn of the helix has 8.9 subunits (Parker et al., 2002; Kendall et al., 2008) with five nucleotides associated with each protein subunit. The RNA backbone is at a radial position of 33–35 Å. The structure determination of PVX CP, which comprises 236 amino acids, suggests that it consists of seven α-helices arranged in two domains, an inner four-helix bundle (similar to TMV) and an outer α/β domain, and six β-strands (Figure 3.10A) (Dobrov et al., 2007; Nemykh et al., 2008). However, there is a problem with this model for PVX CP in that the predicted “length” of the subunit is greater than the radius of the virus particle. It is suggested that the subunits within the virion either contain a kink or are stacked at a fairly large angle to the long axis of the virion; the former suggestion is considered to be the most probable (Dobrov et al., 2007). The N-terminal segment of PVX CP is glycosylated by a single monosaccharide residue (galactose or fructose)
(Baratova et al., 2004) which is proposed to induce the formation of a columnar-type shell of bound water molecules around the virion and which plays a major in maintaining the virion surface structure. In NMV the particle diameter is 110 ± 10 Å, the pitch of the helix 34.45 ± 0.5 Å with 8.8 subunits per turn (Kendall et al., 2008). Vibrational circular dichroism studies show that the NMV CP has a similar structure to that of PVX (Shanmugam et al, 2005).
2. Family Potyviridae Studies have been made on several members of the genus Potyvirus, and on one member each from the genera Rymovirus and Tritimovirus. Basic properties of these virus particles are listed in Table 3.2. Fiber diffraction patterns showed that the structures of five potyviruses, PVY, SMV, TVMV, BCMV, and BCMMV were generally similar (McDonald et al., 2010).
Chapter | 3 Architecture and Assembly of Virus Particles
85
3. Family Closteroviridae The closterovirus BYV encodes a protein (p24) that is structurally related to, but somewhat larger than (with an N-terminal addition), the major CP (p22) (Appendix A, Profile 57). Agranovsky et al. (1995) probed BYV particles with CP (p22) antiserum and an antiserum to the N-terminal part of p24. A 75 nm segment at the 3′ end of the flexuous particle (Zinovkin et al., 1999) was consistently labeled with both types of antibody and the N-terminal antiserum did not label the rest of the particle (Figure 3.11) giving a “rattlesnake” tail structure. The main body of BYV particles is made up of subunits of p22 arranged in a helix. The tail contains four viruscoded proteins (Box 3.2). The rattlesnake tail probably pertains to all closteroviruses; it is also found in the crinivirus, LIYV (Tian et al., 1999). Mention should also be made of tenuiviruses that show folded, branched, or coiled threadlike particles in electron micrographs (Toriyama, 1986). Preparations of these particles are infectious. FIGURE 3.10 Proposed 3D organization of PVX and PVA CP subunits in the virions. The left side of the figure corresponds to the virion surface. (A) The PVX CP subunit: α-helices are shown as cylinders and β-strands as arrows; starts and ends of α-helices and β-strands are numbered; The N- and C- termini of the protein chain are indicated and the 35 to 39 and the 136 to 144 hinges are highlighted. (B) The PVA CP subunit (with modifications from Baratova et al., 2001). From Nemykh et al. (2008) with permission from the publishers.
The CPs of all these viruses (sizes 261–287 amino acids) have a significant α-helical content. Using data from tritium bombardment, Baratova et al. (2001) proposed a model of the potyvirus PVA CP (269 amino acids) structure (Figure 3.10B). The structure resembles that of PVX having an inner four-helix bundle and an outer α/β domain but differs in also having an N-terminal ββα motif. Examination of the particles of PVY and PVA by atomic force microscopy showed an unusual structure at one end of about 10% of the particles (Torrance et al., 2006). The end had a protruding tip which immunogold electron microscopy revealed labeled with antibodies to two potyvirus encoded proteins, VPg which is covalently linked to the 5′ end of the genomic RNA and HC-Pro which has numerous functions (Chapters 6, 9, 12, and Box 16.1). Torrance et al. (2006) suggested that the “tip” structure is at the 5′ end of the virus particle and has several functions, such as initiation of translation of the viral RNA, virus cell-to-cell movement and aphid transmission. The structures of the particles of the rymovirus, AgMV, and the tritimovirus, WSMV, are similar to those of the potyviruses described above (McDonald et al., 2010). However, there are some differences in detail with WSMV showing the largest differences.
III. ASSEMBLY OF ROD-SHAPED VIRUSES Since the early experiments on TMV structure, many workers have studied the mechanism of assembly of rod-shaped virus particles, for there is considerable general interest in the problem especially recently with the uses of virus particles for biomedical and nanotechnological applications (Chapter 15). Disassembly of these viruses is described in Chapter 6 as it is often closely related to genome expression.
A. TMV The pioneering work of Fraenkel-Conrat and Williams (1955) who disassembled TMV into CP subunits and RNA by dialyzing virus preparations against alkaline buffers and separating the protein from the nucleic acid by ammonium sulfate precipitation has led to much study of the factors involved in in vitro virus disassembly and reassembly. Disassembly of TMV in the plant is described in Chapter 6, Section III, B.
1. Assembly of TMV Coat Protein The protein monomer can aggregate in solution in various ways depending on pH, ionic strength, and temperature. The major forms are summarized in Figure 3.12; a form of aggregate known as the stacked disk (not illustrated in Figure 3.12) consists of three or more pairs of rings of subunits (Raghavendra et al., 1986). Experiments with MAbs show that both ends of stacked disks expose the same protein subunit surface. Thus, each
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BOX 3.2 Rattlesnake Tails of Closteroviridae The long flexuous virions of the Closteroviridae have a unique bipolar architecture made up of two coat proteins. With more than 95% of the helical nucleocapsid encapsidated by the major CP and a tail at one end encapsidated four virus-coded proteins (Figure 3.11); for closterovirus genome organization see Appendix A, Profile 57. The tail is about 90 nm long and is made up of approximately 659 nucleotides encapsidated by the minor CP (CPm) and three other proteins (Peremyslov et al., 2004; Satyanarayana et al., 2004). The virus-coded cell-to-cell movement protein (p64 in BYV, p61 in CTV) and the HSP70 homolog (HSP70h) also required for cell-to-cell movement of the virus (Chapter 10, Section IV, B, 1, g) are involved in the assembly of the tails and become an integral part of the virus structure (Alzhanova et al., 2001; Napuli et al., 2000, 2003; Satyanarayana et al., 2004). The BYV virus-coded p20 is required for long-distance virus transport in the plant; p20 binds to HSP70h and is also incorporated into the tail structure (Prokhnevsky et al., 2002). CPm on its own can encapsidate the full-length infectious CTV viral genome. Co-expression of HSP70h and p61 with CPm in protoplasts restricted encapsidation to the 5′ approximately 630 nucleotides which is similar to the boundary of the tail; however, the presence of HSP70h or p61 alone did not limit encapsidation (Satyanarayana et al., 2004). Similarly BYV required CPm, HSP70h and p64 for coordinated of the tails (Alzhanova et al., 2007). Mutations within the 5′ nontranslated region of CTV RNA showed that the origin of assembly overlaps a stem-loop structure that also functions as cis-acting elements required for RNA synthesis (Gowda et al., 2003; Satyanarayana et al., 2004).
FIGURE 3.11 “Rattlesnake” tails on closterovirus particles. (A–D) BYV particles. (A) Particle labeled with mouse polyclonal antiserum to the N-terminal peptide of BYV p24 protein. (B) Labeling with rabbit anti-BYV serum and immunogold labeling with secondary goat ant-rabbit 10-nm gold conjugate. (C) Unlabeled (uranyl acetate stained) particle with distinct terminal structure. (D) The terminal structure shown in C but at a higher magnification. Arrows indicate distinct virus tail. (E–F) CTV particles. (E) Particle gold labeled with p27 antibodies. (F) Particles gold labeled with coat protein antibodies. Magnification markers, A-C 300 nm, D-F 100 nm. From A–D Agranovsky et al. (1995) with permission of the publishers; E and F Febres et al. (1996) with permission of the publishers.
two-layer unit in the stack must be bipolar (i.e., facing in opposite directions) (Dore et al., 1989). A four-layered disk aggregate has been crystallized and its structure solved to atomic resolution (Diaz-Avalos and Caspar, 1998). This showed that it was made up of pairs of disks.
The existence of these various aggregates has been important both for our understanding of how the virus is assembled and also for the X-ray analysis that has led to a detailed understanding of the virus structure. The helical protein rods that are produced at low pH are of two kinds, one with 16 and 1/3 subunits per turn of the helix, as in the virus, and one with 17 and 1/3. In both of these forms the protein subunit structure is very similar to that in the virus. The RNA is replaced by at least one anion binding near a phosphate-binding site (Mandelkow et al., 1981). The main features of the in vitro aggregation behavior of TMV CP were explained by a statistical mechanical model based in the principle of mass action (Kegel and van der Schoot, 2006). The model showed that there are three competing factors, hydrophobic interactions, electrostatic interactions, and the formation of the “Caspar” carboxylate pairs (Section II, B, 1, e), regulating the transitions between the different kinds of aggregation state of the CP.
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Temperature (°C)
30 25
Single helix Disk
20
A protein
15 10 5 0.7
Ionic strength
0.6
“Lockwasher”
0.5 0.4 0.3 0.2 0.1 4.0
5.0
6.0
7.0
8.0
9.0
pH FIGURE 3.12 Some aggregation states of TMV coat protein. Effects of pH, ionic strength, and temperature. Upper figure modified from Richards and Williams (1976) with permission of the publisher; lower figure modified from Durham et al. (1971) with permission of the publisher. The helical rod of protein, the A protein, and the 20S disk have been well characterized (Champness et al., 1976). The lockwasher is the proposed intermediate in the initiation of the assembly of TMV (Durham et al., 1971) (Figure 3.14). Although the lockwasher form has never been isolated, the “nicked” protein helices formed when the pH is lowered rapidly give direct support to the existence of such a form. (Durham and Finch, 1972).
2. Assembly of the TMV Particle a. Assembly In Vitro In their classic experiments, Fraenkel-Conrat and Williams (1955) showed that it was possible to reassemble intact virus particles from TMV CP and RNA. TMV RNA alone had an infectivity about 0.1% that of intact virus. Reconstitution of virus rods gave greatly increased specific infectivity (about 10–80% that of the native virus) and the infectivity was resistant to RNase attack. The 3D structure of the CP is known in atomic detail (Figures 3.5–3.7) and the complete nucleotide sequence of several strains of the virus and related viruses is known. The system therefore provides a useful model for studying interactions during the formation of a macromolecular assembly from protein and RNA. There are four aspects of rod assembly to be considered: the site on the RNA where rod formation begins; the initial nucleating event that begins rod formation; rod
extension in the 5′ direction; and rod extension in the 3′ direction. There is a general consensus concerning most of the details of the initiation site and the initial event, but the nature of the elongation processes remains somewhat controversial. A central problem has been the fact that the CP monomer can exist in a variety of aggregation states, the existence of which is closely dependent on conditions in the medium (Figure 3.12). Equilibria exist between different aggregates so that, while one species may dominate under a given set of experimental conditions, others may be present in smaller amounts. The subject has been reviewed by Wilson and McNicol (1995), Butler (1999), and Klug (1999) and has the following major features: i. The Assembly Origin in the RNA CP does not begin association with the RNA in a random manner. Zimmern and Wilson (1976) located the origin of assembly between 900 and 1300 nucleotides from the 3′ end. Butler et al.
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FIGURE 3.13 Proposed secondary structure for TMV origin of assembly extending from bases 5290 to 5527 of the viral sequence. From Zimmern (1983) with permission of the publisher.
(1977) and Lebeurier et al. (1977) produced evidence showing that the longer RNA tail loops back down the axial hole of the rod. Lebeurier et al (1977). used electron microscopy to show that, in partially assembled rods, a long and a short tail of RNA both protrude from one end of the rod. As the rod lengthens the longer tail disappears. The short tail is then incorporated, completing rod formation. These and other experiments (Otsuki et al., 1977) demonstrated an internal origin of assembly. The nucleotide sequence of the origin of assembly was established by various workers for the common strains of TMV (Jonard et al., 1977; Zimmern, 1977). Nucleotide sequences near the initiating site can form quite extensive regions of internal base-pairing as is illustrated in Figure 3.13. The origin of assembly is located elsewhere in some tobamoviruses and, when in the region that is present in the CP subgenomic mRNA, can give rise to short rods. Loop 1 in Figure 3.13, with the sequence AAGAAGUCG, combines first with a 20S aggregate of CP. Steckert and Schuster (1982) assayed the binding of 25 different
trinucleoside diphosphates to polymerized TMV CP at low temperatures and pH. 5′-AAG-3′ bound most strongly. Under conditions used for virus reconstitution, longer oligomers ((AAG)2 or AAGAAGUUG) were required for strong binding (Turner et al., 1986). The importance of various aspects of loop 1 have been established in more detail by site-directed mutagenesis (Turner and Butler, 1986; Turner et al., 1988). These studies have shown that specific assembly initiation occurs in the absence of loops 2 and 3 of Figure 3.13, but loop 1 is essential. Deletion or alteration of the unpaired sequence in loop 1 abolishes rapid packaging. The binding of loop 1 is mainly due to the regularly spaced G residues. The sequences (UUG)3 and (GUG)3 are as effective as the natural sequence. However, sequences, such as (CCG)3 and (CUG)3 reduced the assembly initiation rate. Thus, there is some, but not complete, latitude in the bases in the first two positions. Base sequence in the stem of loop 1 is not critical, except that shortening the stem reduces the rate of protein binding, as do changes that alter the RNA folding close to the loop (Turner et al., 1988). Overall stability of loop 1 is
Chapter | 3 Architecture and Assembly of Virus Particles
important because base changes that made the stem either more or less strongly base-paired were detrimental to protein binding. The small loop at the base of the stem is also important, but its phasing to the top loop is not critical. The preceding discussion refers to the vulgare strain of TMV. Other tobamoviruses have somewhat different sequences in the stem-loop structure. From comparing these sequences, Okada (1986) suggested a different target sequence in the loop, namely, GAAGUUG. Some other tobamoviruses have the assembly origin in different sites on the genome. For instance, the assembly origin sites are in the CP gene of SHMV (Takamatsu et al., 1983) and the sgRNA for the CP becomes encapsidated. ii. The Initial Nucleating Event Since Butler and Klug (1971) showed that a 20S polymer of CP was responsible for initiating TMV rod assembly, the finding has been confirmed in various ways by many workers. The structure of the 20S double disk is known to atomic resolution (Figure 3.6). For many years it has been assumed that this was the configuration that initiated assembly and that on interaction with the origin of assembly sequence in the RNA, the disk converts to a protohelical form. The structure in Figure 3.6 was derived from 20S aggregates crystallized in solutions of high ionic strength, and it has been assumed that the double disk was also the favored structure under reconstitution conditions. Studies using sedimentation equilibrium (Correia et al., 1985), near-UV circular dichroism (Raghavendra et al., 1985), and electron microscopy (Raghavendra et al., 1986) under salt and temperature conditions used in rod reconstitution experiments suggested that the 20S species observed is a helical aggregate of 39 ± 2 subunits. However, using the rapid-freeze technique, it has been shown directly that under conditions in solution favoring most rapid TMV assembly, about 80% of the CP is in a structure that is compatible only with an aggregate of two rings (Butler, 1999). Thus the predominant structure in solution is likely to be very similar to that found in the crystal (Figure 3.6). Certainly, the model proposed by Champness et al. (1976) appeals on functional grounds. The disks can form an elongated helical rod of indefinite length at lower pH values. The transition between helix and double disk is mainly controlled by a switching mechanism involving abnormally titrating carboxyl groups. At low pH, the protein can form a helix on its own because the carboxyl groups become protonated. When the protein is in the helical state either at low pH or in combination with RNA in the virus, the interlocking vertical helices are present. In this condition, the abnormally titrating carboxyl groups are assumed to be forced together. Champness et al (1976). proposed that the inner part of the two-layered disk acts as a pair of jaws (Figure 3.6) to bind specifically to the origin-of-assembly loop in the RNA (Figure 3.13), in the process converting each disk of the double disk into a “lock
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washer” and forming a protohelix. A model for the early steps in the assembly of TMV is shown in Figure 3.14. Sachse et al. (2007) assumed that assembly is a reversible process that proceeds in the opposite direction of the disassembly process and developed a model from the viewpoint of disassembly (Figure 3.15). As noted above, this model differed from that of Namba et al. (1989) in the region of residues 88–109. They concluded that this region, together with the RNA that binds here, is important for the conversion between helical and disk aggregate forms of the virus, appeared more ordered with welldefined secondary structure. The RNA phosphate backbone is sandwiched between two arginine side chains (residues 90 and 92), stabilizing the interaction between the RNA and CP. A cluster of two or three carboxylates (Glu95 in one subunit and Asp109 and Asp116 from the adjacent subunit; Glu97, Glu106, and Asp109 from the same subunit) is buried in a hydrophobic environment isolating it from neighboring subunits. iii. Rod Extension in the 5′ Direction Following initiation of rod assembly there is rapid growth of the rod in the 5′ direction (Zimmern, 1977) “pulling” the RNA through the central hole until about 300 nucleotides are coated. Zimmern (1983) proposed a model for this initial rapid assembly involving two additional hairpin loops located 5′ to the origin of assembly loop (Figure 3.13). The spacing of these loops is consistent with the idea that they interact successively with three double disks. Various workers have isolated partially assembled rods of definite length classes. At least some of these classes are probably due to regions of RNA secondary structure that delay rod elongation at particular rod lengths (Godchaux and Schuster, 1987). It is generally agreed that rod extension is faster in the 5′ than in the 3′ direction but there has been considerable disagreement as to other aspects. Butler’s group believed that the 20S aggregate is used in 5′ extension and the A protein in the 3′ direction with complete rods being formed in 5–7 min (see Figure 3.12 for aggregation states of the CP monomer). Okada’s group (Fukuda and Okada, 1985, 1987) claimed that 5′ extension uses 4 S protein while 3′ extension uses 20S aggregates, with complete rods taking 40–60 min to form. Some of the earlier relevant experiments are discussed in detail by Butler (1984), Lomonossoff and Wilson (1985), and Okada (1986). The experiments of Fukuda et al. (1978) show that for the strain of virus and assembly conditions they used, fulllength rods were formed only after 40–60 min. Fukuda and Okada (1985) found that rod elongation in the 5′ direction is complete in 5–7 min giving rise to a 260-nm rod, which subsequently elongates in the 3′ direction to give the 300nm rod. They used a Japanese strain of TMV and a higher ionic strength than Butler’s group, which may account for some of the differences.
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FIGURE 3.14 Model for the assembly of TMV; (A–C) initiation; (D–H) elongation. (A) The hairpin loop inserts into the central hole of the 20S disk. This insertion is from the lower side of the disk. It is not yet apparent how the correct side for entry is chosen. (B) The loop opens up as it intercalates between the two layers of subunits. (C) This protein–RNA interaction causes the disk to switch to the helical lockwasher form (a protohelix). Both RNA tails protrude from the same end. The lockwasher–RNA complex is the beginning of the helical rod. (D) A second double disk can add to the first on the side away from the RNA tails. As it does so it switches to helical form and two more turns of the RNA become entrapped. (E–H) Growth of the helical rod continues in the 5′ direction as the loop of RNA receives successive disks, and the 5′ tail of the RNA is drawn through the axial hole. In each drawing, the 3D state of the RNA strand is indicated. (Courtesy of P.J.G. Butler).
However, it is now generally accepted that under optimum assembly conditions, growth of the TMV rod in the 5′ direction is mainly by the addition of double disks, or sometimes single disks of CP (Turner et al., 1989). They prepared in vitro RNA transcripts containing various heterologous nonviral RNAs 5′ to the TMV origin of assembly sequence, instead of the natural TMV sequence. There was no sequence 3′ to the origin of assembly. They then determined the lengths of RNA fragments protected from
nuclease attack after allowing a short period for rod assembly. They found a series of lengths in steps of slightly over 100 nucleotides, or occasionally of just over 50 nucleotides. These are approximately the lengths expected to be protected by two (or one) turns of protein helix. They found such steps in length whatever heterologous RNA was used. This experiment rules out the possibility that the steps are due to some regularity in the RNA sequence that slows assembly at certain distances along the molecule.
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FIGURE 3.15 Summary of the putative disassembly/assembly mechanism of TMV. The structural conversion from the helical into the disk-aggregate form can be induced by a change in pH. The atomic models of two adjacent subunits of the coat protein from the lower and upper layer of the three determined structures are displayed. Negatively and positively (red and blue) charged residues are highlighted because of their importance in the dis assembly/assembly process. The grey color of the disk-structure main chain at the inner wall presents the disordered backbone with a B-factor greater than 100 Å2. (A) The cryo-EM structure (PDB code 2OM3) might represent the stable state of the helical TMV because of its high secondary structure order: the intrasubunit carboxylate cluster (Glu97, Glu106 and possibly Asp109) acts as a metastable switch—here in the spring-loaded off position. Upon a change in the environmental milieu (rise in pH) protons are lost from this cluster of residues and the switch is turned on, initiating the opening of the extended RR helical turn at lower radius. (B) The 2TMV structure can be interpreted as a transitional state between the helical form of TMV and the disk form: the loss of secondary structure in the lower-radius region affects the binding between adjacent subunits because of electrostatic repulsion between Glu95, Asp109 and Asp116 that are located on adjacent subunits. The loss of secondary structure also loosens the RNA binding to the coat protein that is mediated by the positively charged Arg90, Arg92, Arg112 and Arg113. The conformational change in the lower-radius region ultimately leads to RNA dissociation. (C) 1EI7 structure of the disk aggregate: the lower-radius regions of the protein subunits in the disk structure are farther apart from each other compared with their arrangement in the helical forms of the virus. A decrease in pH is thought to promote RNA binding and reversal of the disassembly process. From Sachse et al. (2007) with permission of the publishers. A more detailed version of this figure can be found on http:// booksite.elsevier.com/9780123848710
There is uncertainty as to how the 5′ cap structure of the RNA is encapsidated. Disassembly by ribosomes (Chapter 6, Section III, B) and in vivo would suggest that the structure at the extreme 5′ end might differ from that over most of the virus particle. iv. Rod Extension in the 3′ Direction Fairall et al. (1986) studied reassembly with RNAs that had been blocked at various sites by short lengths of hybridized and cross-linked cDNA probes. They found that even when 5′ extension was incomplete due to the blocked sequence, 3′ extension was completed. However, lengths of rods were determined after 20 min of incubation so the data are not relevant to the question of whether 3′ elongation is completed in 5–7 min. Fukuda and Okada (1987) prepared an ss cDNA probe that extended from the origin of assembly to the 3′ terminus and that was complementary to TMV RNA. They used this to determine the length of rod extension in the 3′ direction. The results showed that significant rod extension in the 3′ direction did not occur at least in the first 4 min. It was first observed at 8 min and was still increasing between 15 and 40 min. At 4 min there was substantial encapsidation of RNA in the 5′ direction. Fukuda and Okada (1987) found a series of discrete sizes of RNA in the RNAprotected material in the 3′ direction and suggest that these differed by about 100 nucleotides in length. However, the results illustrated in their paper indicate a very uneven
increment in length from about 55–135 nucleotides. This scatter in lengths may be due to the effects of the nucleotide sequence on assembly using A protein and on nuclease specificity rather than to the addition of double disks. Turner et al. (1989) carried out assembly experiments on RNA with heterologous sequences inserted 3′ to the TMV origin of assembly sequence. They found no evidence for banding in the protected RNA, giving strong support to earlier work indicating that extension of the rod in the 3′ direction is by the addition of small A protein aggregates. The assembly of other strains of TMV and other tobamoviruses has not been studied as intensively as that of the type strain, but the available evidence indicates that the same basic assembly mechanism operates for all these viruses. However, the structures involved may differ in detail. Studies on reassembly in vitro in which the TMV origin of assembly was embedded in various positions support the ideas that rod extension is much faster in the 5′ than the 3′ direction, that 5′ extension is probably completed before 3′ extension begins, and that the two extension reactions are different from each other (Gaddipati and Siegel, 1990). Artificial RNAs have been constructed that combine the TMV origin of assembly and the mRNA for a foreign gene. This RNA has been assembled into a rod with TMV CP and used to introduce the mRNA into plant cells. Constructs with the origin of assembly are also used in nanotechnology (Chapter 15, Section IV, C).
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b. Assembly In Vivo Very short rods have been seen in electron micrographs of infected leaf extracts, but the 20S aggregate has not been definitively established as occurring in vivo. Nevertheless, the following evidence shows that the process involved in the initiation of assembly outlined earlier is almost certainly used in vivo: (i) the conditions under which assembly occurs most efficiently in vitro (pH 7.0, 0.1 M ionic strength, and 20°C) can be regarded as reasonably physiological; (ii) the correlation between the location of the origin of assembly in different tobamoviruses and the encapsulation, or not, of short rods containing the CP mRNA (Section III, A, 2, a) strongly suggests that the origin of assembly found in vitro is used in vivo; (iii) the mutant Ni2519, which is ts for viral assembly in vivo, has a single base change that is at position 5332 (Zimmern, 1983). This change weakens the secondary structure near the origin-of-assembly loop; (iv) tobacco plants transgenic for the CAT gene with the TMV origin of assembly inserted next to the 3′ terminus give rise to RNA transcripts that can be assembled into virus-like rods with the TMV CP when the plants are systemically infected with TMV (Sleat et al., 1988). It is known from in vitro experiments that TMV CP can form rods with other RNAs. In vivo there appears to be substantial specificity in that most rods formed contain the homologous RNA. In vivo this specificity may be due, first, to specific recognition of the correct RNA by the 20S disk, second, because rods are assembled at an intracellular site where the homologous viral RNA predominates and third, coordinated RNA replication and virion assembly. Nevertheless, fidelity in in vivo assembly is not total. There is no evidence that establishes the method by which the TMV rod elongates in vivo, but there is no reason to suppose that it differs from the mechanism that has been proposed for in vitro assembly.
B. Other Rod-Shaped Viruses There is much less understanding of the processes of virion assembly for other rod-shaped viruses. Much of the information about the assembly of potexvirus particles come from in vitro studies on PVX and PapMV (reviewed by Atabekov et al., 2007). CP subunits isolated either by acetic acid treatment of purified virus or expressed in E. coli formed 450 kDa disks comprising about 20 subunits arranged in two turns of a helix (Erickson et al., 1978; Tremblay et al., 2006). It is considered that these are the building blocks for virion assembly (Tremblay et al., 2006). The origin of assembly is considered to be a stem-loop structure in the 5′ nontranslatable region of the genomic RNA (Kwon et al., 2005). The N-terminus of the PVX CP plays a role in virion assembly and stability (Lukashina et al. (2012) but the N-terminal 14
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amino acid region of PlAMV CP is dispensable for virion assembly (Ozeki et al., 2009). Potyvirus CP subunits also form stacked ring structures under certain conditions (Goodman et al., 1976; McDonald et al., 1976) that, on addition of viral RNA, yield virus-like particles that are shorter than native virus and are noninfectious (McDonald and Bancroft, 1977). The evidence that the origin of assembly is close to the 5′ end of the RNA comes from in vivo studies using RNase protection (Wu and Shaw, 1998) that showed that assembly took place between 30 and 45 min after inoculation of protoplasts with TVMV. Potyvirus CP assembles into virus-like particles when expressed in E. coli, yeast and, mediated by recombinant vaccinia virus, in mammalian cells (Jagadish et al., 1991; Hammond et al., 1998; Hema et al., 2008). This gives a new tool to the studies of the factors involved in virion assembly. For instance, using expression in E. coli, Jagadish et al. (1993) showed that Arg194 and Asp238 of JGMV are required for the assembly process but were not necessarily involved in forming the predicted salt bridge. The N-terminal 26 amino acids of PapMV CP are required for binding RNA and the N-terminus, especially Phe13, is involved in self-assembly of CP subunits into nucleocapsid-like particles (Laliberté et al., 2008). The C-terminal domain of SMV CP is required for CP-CP self-interaction (Kang et al., 2006). Although the surface-exposed N- and C-terminal residues of PVBV CP can be removed from virus particles by trypsinization without affecting their stability such truncated CP subunits are unable to reassemble to for virus-like particles (Anindya and Savithri, 2003). In contrast, mutant TEV CP from which up to 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles, though these structures were more rigid and had smaller diameter than those produced by unmodified CP (Voloudakis et al., 2004). Ideas about the assembly of closteroviruses are discussed above (Section II, C, 3 and Box 3.2).
IV. ARCHITECTURE OF ISOMETRIC VIRUSES A. Introduction From crystallographic considerations, Crick and Watson concluded that the protein shell of a small “spherical” virus could be constructed from identical protein subunits arranged with cubic symmetry, for which case the number of subunits would be a multiple of 12. Crick and Watson (1956) pointed out that cubic symmetry was most likely to lead to an isometric virus particle. There are three types of cubic symmetry: tetrahedral (2:3), octahedral (4:3:2), and icosahedral (5:3:2); thus, an icosahedron has fivefold, threefold, and twofold rotational
Chapter | 3 Architecture and Assembly of Virus Particles
symmetry. For a virus particle, the three types of cubic symmetry would imply 12, 24, or 60 identical subunits arranged identically on the surface of a sphere. These subunits could be of any shape. Klug and Caspar (1961) realized that many viruses probably had shells made up of subunits arranged with icosahedral symmetry which would generate the maximum enclosed space for shells comprising subunits of a given size. Horne and Wildy (1961) discussed possible models for the arrangement of protein subunits in icosahedral shells with 5:3:2 symmetry. They considered possible packing arrangements for clusters of five and six protein subunits. Pawley (1962) enumerated the plane groups that can be fitted on polyhedra and suggested that these may have application in the study of virus structure. Caspar and Klug (1962) further developed the principles of virus construction, particularly with respect to the isometric viruses based on icosahedral structures. A shell made up of many small identical protein molecules makes most efficient use of a virus’ genetic material. Figure 3.16 shows a regular icosahedron (20 faces). With three units in identical positions on each face, this icosahedron gives 60 identical subunits. This is the largest number of subunits that can be located in identical positions in an isometric shell. Some viruses have this structure, but many have much larger numbers of subunits, so that not all subunits can be in identical environments.
B. Possible Icosahedra A problem arises in developing potential structures for isometric viruses with the limitations of 60 protein subunits dictated by the basic icosahedron. A very simple infectious genome would code for a capsid protein (about 1200 nucleotides) and a polymerase (about 2500 nucleotides) which would require a minimum containing space or “hole” of radius about 9 nm. An icosahedron composed of 60 subunit of about 20–30 kDa (the usual size of a viral CP) would have an internal “hole” of about 6 nm and thus would not be sufficiently large to encapsidate most viral genomes. Caspar and Klug (1962) enumerated all the possible icosahedral surface lattices and the number of structural subunits involved. The basic icosahedron (Figure 3.16), with 20 × 3 = 60 structural subunits, can be sub-triangulated (Box 3.3). The process outlined in Box 3.3 leads to expansion of the particle by adding groups of six subunits (hexamers) in predetermined positions between pentamer clusters (Figure 3.17A, panels 1–4). This gives a series of potential spheres of increasing size depending on the T number. The formula in Box 3.3 gives limitations for the values of T, and the most common ones found in viruses are listed in Table 3.3. From this table it can be seen that with increasing particle size the number of pentamers remains constant (12) and the number of hexamers increases.
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FIGURE 3.16 The regular icosahedron. This solid has 12 vertices with fivefold rotational symmetry; the center of each triangular face is on a threefold symmetry axis, and the midpoint of each edge is on a twofold symmetry axis. There are 20 identical triangular faces. Three structural units of any shape can be placed in identical positions on each face, giving 60 structural units. Some of the smallest viruses have 60 subunits arranged in this way.
One other way of viewing the distribution of pentamers and hexamers is that pentamers give the 3D curvature of the structure and hexamers the 2D aspect.
C. Clustering of Subunits The actual detailed structure of the virus surface will depend on how the physical subunits are packed together. For example, three clustering possibilities for the basic icosahedron are shown in Figure 3.18A. In fact, many smaller plant viruses are based on the P = 3, f = 1, T = 3 icosahedron. In this structure, the structural subunits are commonly clustered about the vertices to give pentamers interspersed with hexamers of the subunits. These are the morphological subunits seen in electron micrographs of negatively stained particles (Figures 3.17B and 3.29C). Since there are always 12 vertices with fivefold symmetry in icosahedra, we can calculate the number of morphological subunits (M) (assuming clustering into pentamers and hexamers) as follows: M
[(60T 10(T
60) / 6] hexamers (60 / 5) pentamers 1) hexamers 12 pentamers
In photographs of virus particles where the pentamers and hexamers can be unambiguously recognized (e.g., one-sided images of negatively stained particles or
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BOX 3.3 Basic Icosahedral Symmetry Structure The basic icosahedron of 60 structural subunits can be subtriangulated according to the formula: T PAf 2 where T is called the triangulation number. Parameter f is based on the fact that the basic triangular face can be subdivided by lines joining equally spaced divisions on each side.
(e.g., cross-hatching) and then joining the cut edges to give a vertex with fivefold symmetry. However, if each vertex is joined to another by a line not passing through the nearest vertex, other triangulations of the surface are obtained. In the simplest case the “next but one” vertices are joined. C
t=3
t=1 Division of sides into two gives four triangles
Division of sides into three gives nine triangles
Basic face
Thus, f is the number of subdivisions of each side and is the number of smaller triangles formed. There is another way in which sub-triangulation can be made and this is represented by P. It is easier to consider a plane network of equilateral triangles. A
D
C
B
Such a sheet can be folded down to give the basic icosahedron by cutting out one triangle from a hexagon
freeze-fracture replicas of the outer faces of larger viruses) the parameters h and k might be used to establish the icosahedral class of the particle. This procedure would be particularly useful for shells containing large numbers of hexamers. h and k represent the numbers of hexamers that must be traversed to move by the shortest route from one pentamer to the next. Thus, we must identify two adjacent pentamers. For example, the following was observed on the surface of freeze-etch replicas of phage X (Bayer and Bocharov, 1973) (Figure 3.18B). This indicates a skew icosahedron with “right-handed” skewness.
B
D
t=2
This gives a new array of equilateral triangles and the plane net can be folded to give the solid shown in Figure 3.** A 3 by removing the shaded triangle from each of the original vertices and then folding to give a vertex with fivefold symmetry. It can be shown by simple trigonometry that each of the small triangles has one-third the area of the original faces. This can be seen by inspection by noting that there are six new half triangles within one original face (dashed lines C-B-D.). In this example P = 3. In general, P
h2
hk
k2
where h and k are any integers having no common factor. For h = 1, k = 0, P = 1 For h = 1, k = 1, P = 3 For h = 2, k = 1, P = 7 where p ≥ 7 the icosahedra are skew, and right-handed and left-handed versions are possible. The physical meaning of h and k in a virus structure is illustrated in Fig. 3.18. Since each of the triangles formed with the P parameter can be further subdivided into f2 smaller triangles, T gives the total number of subdivisions of the original faces and 20T the total number of triangles.
D. Quasi-Equivalence Caspar and Klug (1962), with their theory of quasiequivalence, laid the basis for a further understanding of the way the shells of many smaller isometric viruses are constructed of more than 60 identical subunits. In general terms, they assumed that not all the chemical subunits in the shell need be arrayed in a strictly mathematically equivalent way, but only quasi-equivalently. They also assumed that the shell is held together by the same type of bonds throughout, but that the bonds may be deformed
OUTER FACE OF THE VIRUS C N
Beta-sheet FIGURE 3.17 Icosahedral symmetry. (A) Ways of sub-triangulation of the triangular faces of the basic icosahedron shown in Figure 3.16.; 1. the basic icosahedron with T = 1; 2, with T = 4; 3, with T = 3; 4, with T = 12. From Caspar and Klug (1962) with permission of the publishers. (B) Structure of T = 3 particles of TYMV with reconstructed density distribution on particles showing 1, twofold axis; 2, fivefold axis; 3, threefold axis. From Mellema and Amos (1972) with permission of the publishers.
LS
TABLE 3.3 Triangulation Numbers and Sizes of Isometric Virus Particles Triangulation Number T
No of Subunits
No of Pentamers
1
60
12
3
180
12
Number of Hexamers
0 RR
20
Approximate Diameter (nm)
Example
17
Satellite tobacco necrosis virus
30
Cowpea mosaic virusa
28
Poliovirusa
25–30
Tomato bushy stunt virus
30
LR 4
7
240
420
12
12
30
60
780
12
120
Norwalk virus
27
Enterobacteria phage MS2
40
Nudaurelia capensis ω virus
40
Sindbis virus core
50
Cauliflower mosaic virus
75– 75
a
Pseudo T=3 (see text).
Flock House virus
25
45 13
RS
virus 40 ShortSimianloop
Wound tumor virus Bluetongue virus core
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(A)
.
.
.
different polypeptides in different symmetry environments within the shell. Bacilliform virus particles, such as those of AMV and badnaviruses, are also based on icosahedral symmetry (Sections V, B, 2, a and V, B. 5).
E. “True” and “Quasi” Symmetries (B)
h
k
h = 4, k = 1, T = 21 FIGURE 3.18 Clustering of subunits and skewness of icosahedra; see text for details.
in slightly different ways in different nonsymmetry-related environments. They calculated that the degree of deformation necessary would be physically acceptable. Quasiequivalence would occur in all icosahedra except the basic structure (Figure 3.16). As noted above, pentamers give the 3D curvature, 12 giving a closed icosahedron whereas hexamers give a 2D curvature (or tubular structure). Thus, the conceptual basis of quasi-equivalence is the interchangeable formation of hexamers and pentamers by the same protein subunit. However, more recent detailed information on the arrangement of subunits in the shells of small viruses has altered the original view of quasi-equivalence (Johnson and Speir, 1997). A series of molecular switches, often involving a segment of 10–30 residues of the subunit poly peptide, have been identified. The switches enable the subunit polypeptide to show icosahedral symmetry at some interfaces and to be disordered at similar, but not symmetrically equivalent interfaces. The switches include specific segments of the subunit polypeptides, ss or ds RNA, and divalent cations. For instance, viruses such as TBSV have multidomain subunits that, to a large extent, adjust to the different symmetry-related positions in the shell by means other than distortion of intersubunit bonds (Section V, B, 4, d). Some capsids undergo buckling transitions, where the capsid, once assembled, undergoes a change in morphology from being more spherical to a more faceted (or more ‘‘icosahedron-like’’) form (Mannige and Brooks, 2009). Even particles made up of 60 identical subunits may have some variation in the detailed structure if the subunits, possibly associated by their interactions with the genomic nucleic acid (Chapman, 1998). Other viruses have evolved different variations on the icosahedral theme. For example, comoviruses (Section V, B, 6, a) and members of the Reoviridae (Section VI) have
In the basic icosahedron (Figure 3.16) a feature located halfway along any edge of a triangular face is positioned on an axis of rotation for the whole solid. Thus, it is on a “true” or icosahedral symmetry axis; this is a true dyad. In any more complex icosahedron (T>1) there is more than one kind of twofold symmetry. For example, in a P = 3, T = 3 shell (Figure 3.17 panel A subpanel 3), the center of one edge on each of the 60 triangular faces is on a true dyad axis relating to the solid as a whole. The center positions of the other two edges of a face have only local twofold symmetry. These are called “quasi” dyads. On the 3′ axis of the “quasi” symmetry the three chemically identical but structurally independent subunits in an icosahedral asymmetric unit are designated A, B, and C (Harrison et al., 1978) (Figure 3.19).
F. Other Structure Theories The structure of some isometric viruses, for example, polyoma virus which has a capsid composed solely of pentamer subunits rather than the expected pentamer–hexamer clusters, does not fit the Caspar and Klug icosahedral theory; currently, no plant viruses are known to have such a structure. However, in some plant viruses the detailed structure is not based on icosahedral symmetry at all radii of the particles, which has called into question a strict interpretation of the Caspar and Klug theory. This has led to various other theories to explain viral structures and self-assembly processes. Lorman and Rochal (2008) used the Landau theory of crystallization to describe icosahedral viral shells selfassembled from identical asymmetric proteins. They predicted the centers of mass for the proteins thus generating protein positions and described in a uniform way both the structures satisfying the Caspar and Klug geometric model for capsid construction and those violating it. In a novel approach, Twarock (2004) suggested that application of the tiling theory could describe the protein stoichiometry of all icosahedral viruses. In the tiling theory, surfaces are tessellated by a basic set of tiles that predict the locations of the protein subunits. Interactions between two subunits (dimers) and three subunits (trimers) are represented geometrically as shapes called tiles. Details of how this generalization of the Caspar–Klug theory is applied to icosahedral structures are described in Twarock (2004, 2005). The basic tiling approach can be adapted to consider cross-linking in the capsid and the dynamics
Chapter | 3 Architecture and Assembly of Virus Particles
A5
A2 A
B5 C3
C
C3
B2
B C2
FIGURE 3.19 Arrangement of protein subunits as found in several T = 3 plant and animal viruses. The nomenclature for the chemically identical subunits follows Harrison et al. (1978). From Krishna et al. (1999) with permission of the publishers.
of conformational changes within capsids as well as the static properties of the basic capsid structure (Twarock and Hendrix, 2006; Peeters and Taormina, 2009). Keef and Twarock (2009) describe a mathematical framework to allow description of tertiary structure of capsids and the organization of the viral genome within the capsid.
G. Bacilliform Particles Some virus particles, for instance those of AMV and badnaviruses, are bacilliform with rounded ends separated by a tubular section. Hull (1976a) suggested that the structure of these particles is based on icosahedral symmetry. The rounded ends would have constraints of icosahedra with the 3D curvature determined by 12 pentamers, 6 at each end. The 2D structure of the tubular section would be made up of hexamers. Hull derived various hexamer structures from icosahedra cut across the twofold, threefold, fivefold, and interlattice axes. Luque and Reguera (2010) and Luque et al. (2010) suggested that, as with spherical viruses, the structures of bacilliform ones (also called elongated, prolate, or tubular) adopt precise geometries because they are free-energy minima of a very generic interaction between the capsomeres. The energetic optimal structures depend strongly on the morphology of the end caps and the way the cylindrical part fits into them. Thus, for T = 1 particles, those with end caps based on threefold structure have a slightly lower energy than those based on fivefold structure. However, for T = 3 particles, those based on fivefold end caps have a lower energy than those with threefold end caps.
V. SMALL ICOSAHEDRAL VIRUSES A. General Features The coat protein subunits of most small icosahedral viruses are in the range of 20- to 40-kDa; some are larger but fold to
97
give effective “pseudomolecules” within this range (Section V, B, 6). In contrast to rod-shaped viruses, the subunits of most small icosahedral viruses have a relatively high proportion of β-sheet structure and a low proportion of α-helix (Denloye et al., 1978; Odumosu et al., 1981) and have the same basic structure. This comprises an eight-stranded antiparallel β sandwich, often termed a β-barrel or “jelly-roll β-barrel”, which is shown schematically in Figure 3.20. The overall shape is a 3D wedge with the B-C, H-I, D-E, and F-G turns being at the narrow (interior) end. Most variation between the subunit sizes occurs at the Nand C-termini and between the strands of β-sheet at the broad end of the subunit. It is the detailed positioning of the elements of the β barrel and of the N- and C-termini that give the flexibility to overcome the quasi-equivalence problems. This can lead to a different architecture at the virion surface (which is visualized by electron microscopy) and the interior of the virion and is illustrated in the detailed structures described below. The CP subunits form one, two or three structural domains, the S (shell) domain, the R domain (randombinding) and the P (protruding) domain; all viruses have the S domain. The R domain is somewhat of a misnomer but defines an N-terminal highly basic region of the polypeptide chain that associates with the viral RNA. As it is random, no structure can be determined by X-ray crystallography. The P domain gives surface protuberances on some viruses. Belyi and Muthukumar (2005) demonstrated a direct correlation between the net charge on the R domain (the capsid peptide arm) and the genome length (Figure 3.21). As noted earlier, viral capsids have to be sufficiently stable to protect the viral genome while outside the cell but be labile enough to enable the genome to be released into a newly infected cell. Various chemical interactions stabilize isometric viruses and these interlink with the processes of particle assembly. Assembly of virus capsids from CPs (reviewed by Reddy and Johnson, 2005) is a coordinated thermodynamic process for most, if not all, viruses and is dependent upon factors, such as ionic strength, pH, and temperature; nucleation is the underlying mechanism of the process (Zandi et al., 2006). For assembly there are specific interactions between viral RNA and the CP which enable the preferential encapsidation of the viral RNA in cells where there is a multitude of cellular RNAs (reviewed by Bink and Pleij, 2002; Rao, 2006). Specificity of encapsidation is also enhanced by coordination of packaging with viral replication (Chapter 16) and compartmentalization. As noted above, although icosahedra have the maximum enclosed volume for a shell comprising a given subunit size, there are limitations on their capacity. Thus, in several groups of viruses, the multicomponent viruses, their genomes are divided between two or more particles
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(A)
(Appendix C). Furthermore, these capacity constraints mean that the secondary and tertiary structure of the RNA in particles usually differs from that in the cell where translation, replication, and cell-to-cell movement take place.
(B)
A A
A
C H HOOC
E F
B. Architecture and Assembly of Small Icosahedral Viruses
GD I B
NH2
At present, we can distinguish seven kinds of structure among the protein shells of small icosahedral or icosahedral-based plant viruses whose architecture has been studied in sufficient detail. These are T = 1 particles, bacilliform particles based on T = 1, geminate particles based on T = 1, T = 3 particles, bacilliform particles based on T = 3, pseudo T = 3 particles, and T = 7 particles.
(C) HOOC
F
G
H
I
E
D
C
B H 2N
FIGURE 3.20 (A) The icosahedral capsid contains 60 identical copies of the protein subunit (blue) labeled A. These are related by fivefold (yellow pentagons at vertices), threefold (yellow triangles in faces), and twofold (yellow ellipses at edges) symmetry elements. For a given sized subunit, this point group symmetry generates the largest possible assembly (60 subunits) in which every protein lies in an identical environment. (B) A schematic representation of the subunit building block found in many RNA, and some DNA, viral structures. Such subunits have complementary interfacial surfaces which, when they repeatedly interact, lead to the symmetry of the icosahedron. The tertiary structure of the subunit is an eight-stranded β-barrel with the topology of the jellyroll (see C). β-strand and helix coloring is identical to (C). Subunit sizes generally range between 20 and 40 kDa with variation between different viruses occurring at the N- and C-termini and in the size of insertions between strands of β-sheet. These insertions generally do not occur at the narrow end of the wedge (B-C, H-I, D-E and F-G turns). (C) The topology of viral β-barrel showing the connections between strands of the sheets (represented by yellow or red arrows) and positions of the insertions between strands. The green cylinders represent helices that are usually conserved. The C-D, E-F, and G-H loops often contain large insertions From Johnson and Spier (1999) with permission of the publishers.
8000
Genome size per capsid
7000 6000 5000 4000 3000 2000 1000 0 0
1000
2000
3000
4000
5000
6000
Net charge on R domain
FIGURE 3.21 Correlation between the net charge on the R domain of viral capsid proteins and the genome length. Data from Belyi and Muthukumar (2005).
1. T = 1 Particles a. Satellite Viruses The satellite viruses are the smallest known plant viruses having a particle diameter of about 17 nm and capsids made up of 17- to 21-kDa polypeptides (see Chapter 5, Section II, A for satellite viruses). The structure of STNV was the first to be solved and shown to be made of 60 protein subunits of 21.3-kDa arranged in a T = 1 icosahedral surface lattice. The structure of the protein subunit has been determined crystallographically with refinement to 2.5 Å resolution (Jones and Liljàs, 1984). The general topology of the polypeptide chain is like that of the S domains of TBSV and SBMV (Figure 3.22) but the packing of the subunits in the T = 1 icosahedral structure is clearly different (Rossmann et al., 1983) and there is no P domain. In the amino terminus of STNV CP, there are only 11 disordered residues followed by an ordered helical section (residues 12–22) buried in the RNA. Three different sets of metal ion binding sites (Ca2+) have been located (Lane et al., 2011);. These link the protein subunits together. A more detailed structure for the protein shell has been proposed by Montelius et al. (1988). Using capsids reassembled from recombinant STNV CP and RNA, Lane et al. (2011) and Bunka et al. (2011) explored the detailed structure of STNV particles and encapsidated RNA. Figure 3.23A shows how the CP subunits fit together in the capsid with the N-terminal region on the inside. RNA could be detected with these capsids by neutron diffraction in H2O/D2O (Bentley et al., 1987) and low-resolution X-ray crystal structure determination (Lane et al., 2011) (Figure 3.23B). The two approaches agreed with each other and indicated that RNA distribution is associated with the capsid structure. Basic amino acids are well placed to make contact with the RNA. RNA fragments
Chapter | 3 Architecture and Assembly of Virus Particles
99
soon after replication. This has led to a model for virus assembly (Figure 3.24B). At higher pHs (about pH 8) STMV particles swell by about 5 Å (about 3%) (Figure 3.24A). Atomic force microscopy showed that, on swelling, both the protein and RNA stem loops move radially, the protein as dimer units (Kuznetsov et al., 2001). SPMV, with a diameter of 160 Å, is the smallest encapsidated virus currently known. Its structure has been solved at 1.9 Å resolution (Ban and McPherson, 1995) and have been compared to those of STNV and STMV (Ban et al., 1995; Makino et al., 2006). In spite of all having the β-barrel structure in the subunit that the fivefold contacts at the narrow end, the three viruses are remarkably different in: (i) the arrangements of the secondary structural elements; (ii) the fivefold protein interactions are organized by Ca2+ in STNV, an anion in STMV and apparently neither of these in SPMV; (iii) nucleic acid is visible in electron density maps of STMV and SPMV but shows different distributions in the two viruses.
2. Bacilliform Particles Based on T = 1 Symmetry As noted in Section IV, G, it has been proposed that bacilliform particles are based on icosahedral symmetry. FIGURE 3.22 Diagrammatic representation of the backbone folding of the coat protein of (A) TBSV, (B) SBMV, and (C) STNV shown in roughly comparable orientations. From Rossmann et al. (1983) with permission of the publishers.
that bind to the CP were selected and sequenced; 30 of these fragments formed stem loops containing the motif ACCA (Bunka et al., 2011); this suggests that the stem loops displaying the motif AxxA are involved in virion assembly. The proposed interaction with these stem loops with the CP N-termini is shown in Figure 3.23C (Lane et al., 2011) indicating that the RNA associated with the fivefold symmetry axis. From an analysis of chloride ion distribution Larsson and van der Spoel (2012) also predict a hot spot for RNA binding at the fivefold symmetry axis of STNV and STMV particles. The structure of STMV has been resolved to 1.8 Å (Larson et al., 1998) and showed double-helical segments of RNA centered on all the dyad (dimmer) axes of the virion (Figure 3.24A). Nearly 80% of the RNA forms the stem-loop elements (Larson and McPherson, 2001). It is considered that RNA replication and assembly are closely coordinated and thus, the new RNA must form the required secondary structures
a. Alfamovirus and Ilarvirus Genera i. Particle Structure Purified preparations of AMV contain four nucleoprotein components present in major amounts (bottom, B; middle, M; top b, Tb; and top a, Ta). They all contain an RNA species of definite length; the genome is split between the B, M, and top b RNAs. Three of the four major components are bacilliform particles, 18 nm in diameter and the fourth (Ta) is normally spheroidal with a diameter slightly larger than 18 nm (Figure 3.25A). However, two forms of Ta component have been recognized (Heijtink and Jaspars, 1976). Tat is spheroidal and soluble in 0.3 M MgSO4. Tab is a rodlet and insoluble in 0.3 M MgSO4. These two particles appear identical in other properties. The Ta components have 120 protein subunits, and Cusack et al. (1983) raised the possibility that these may have a non-icosahedral structure. From a careful study of the MWs of the RNAs, the protein subunit, and the virus particles, Heijtink et al. (1977) concluded that the number of CP monomers in the four major components is equal to 60+(N×18), N being 10, 7, 5, or 4. As noted above (Section IV, G) it has been proposed that such particles are based on icosahedra cut across various axes with the tubular portion made up of hexamer subunits. From optical diffraction studies on electron micrographs of negatively stained AMV particles,
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FIGURE 3.23 Architecture of STNV. Panel (A) Left hand side: Structural elements of a single subunit of STNV (Protein Data Bank ID 2BUK) with antiparallel β-sheets shown in magenta (BIDG) and green (CHEF) and the helical regions shown in blue. The three classes of associated calcium ions are shown as yellow spheres. Right hand side: The X-ray structure of STNV T = 1 structure with an icosahedron as a guide to its symmetry. From Bunka et al. (2011) with permission of the publishers. Panel (B) Subpanel (a) Positive neutron scattering density map of native STNV calculated by Bentley et al (1987) with contrast matched to show only nucleic acid density at 18 Å resolution. The view is along the twofold axis from the center of the virus. Strong cylinders of density run between the fivefold axes across the twofolds and the Cα traces of N-terminal helix clusters are shown at the threefolds in between. A plausible interpretation of an RNA duplex is shown along the central cylinder. From Bentley et al (1987) with permission of the publishers. Subpanel (b) RNA fragments (red sticks) found in an X-ray map, superimposed on the neutron scattering density. The helix axes run across rather than along the cylinders of neutron density, but the fit is good since the very low resolution of the neutron study allows the density from adjacent RNA fragments to merge together across the twofold axes. Panel (C) Subpanel (a) CP subunit with its associated RNA fragment. Three basic residues on the N-terminal helix (Arg14, Lys17, and Arg18) can make good hydrogen bonds to the three phosphate groups of the RNA duplex in a 1:1 relationship. Three basic residues on the internal surface of a neighboring subunit (Arg66, Arg9, 1 and Lys123) can make contact to phosphates in a loop linking the two strands of the duplex. The neighboring subunit is related by a fivefold axis and is shown with its associated RNA fragment. Subpanel (b) View of the inner surface of the capsid looking along a fivefold axis from the center of the particle. RNA fragments form a ring around the fivefold, with each associated with one N-terminal helix cluster. From Lane et al. (2011) with permission of the publishers.
Chapter | 3 Architecture and Assembly of Virus Particles
101
FIGURE 3.23 (Continued)
Hull et al. (1969) suggested that the tubular structure of the bacilliform particles was based on a T = 1 icosahedron cut across its threefold axis (in accord with the minimal energy analysis of Luque et al., 2010, described above) with rings of three hexamers (18 CP monomers) forming the tubular portion (Figure 3.25B).
Proton magnetic resonance studies provide a lowresolution model in which the CP consists of a rigid core and a flexible amino-terminal part of about 36 amino acid residues (Kan et al., 1982). The protein behaves as a water-soluble dimer stabilized by hydrophobic interactions between the two molecules. This dimer is the
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FIGURE 3.24 Structural transitions of STMV particles. (A) Central cross section of STMV based on the model refined at 1.8 Å resolution by X-ray crystallography (Larson et al., 1998). This model is derived from the orthorhombic crystal form which yields a 17-nm particle by AFM and quasi elastic light scattering. The protein is shown in ribbon format in red, and the RNA as all atoms in white. Tabulated below are relevant values for the native and swollen particles. From Kuznetsov et al. (2001) with permission of the publishers. (B) Schematic diagram illustrating a path of assembly consistent with the structural model. RNA strands emerging from the replicative complex exhibit local, otherwise transient, stem-loop structures. (a) Although metastable stem-loop structures might rapidly unravel in the absence of protein, permitting restructuring to the physiological conformation, they are secured in the presence of protein; thus, the production of new double-stranded replicative form RNA is terminated. (b) Coat protein dimers proceed to bind contiguously along the RNA molecule. (c) When a sufficient number of stem-loops and coat protein subunits have been formed, the complexes coalesce into a fluid but disordered aggregate. (d) The complexes of RNA and coat protein sort themselves out through cooperative intramolecular and intermolecular interactions, and the pentameric nucleus of a virion forms. (e) Additional RNA-protein subcomplexes add to the nucleus until the entire RNA-protein mass condenses into the icosahedral virion, with protein on the outside and RNA on the inside. From Larson and McPherson (2001) with permission of the publishers.
morphological unit out of which the viral shells are constructed. Under appropriate conditions of ionic strength, ionic species, pH, temperature, and protein concentration, the protein dimer forms a T = 1 icosahedral structure built from 30 dimers (Driedonks et al., 1977). This structure has been confirmed by X-ray crystallographic analysis (Fukuyama et al., 1983) at 4.5 Å resolution that was further refined to 4.0 Å resolution together with cryoelectron microscopy and image reconstruction (Kumar et al., 1997). This showed
that the subunit structure and dimer association is structurally similar to CCMV. Large holes are observed at the pentamer axes giving a porous particle structure. AMV is unstable with regard to high ionic strength and SDS, and is sensitive to RNase, which might be explained by the holes in the protein coat. Conformational changes occur at mildly alkaline pH (Verhagen et al., 1976). AMV nucleoproteins are readily dissociated into protein and RNA at high salt concentrations and bacilliform particles can be reformed under appropriate conditions. Thus, the
Chapter | 3 Architecture and Assembly of Virus Particles
103
FIGURE 3.24 (Continued)
virus is mainly stabilized by protein:RNA interactions (van Vloten-Doting and Jaspars, 1977). Under a wide range of solvent conditions, AMV particles do not show the phenomenon of swelling described below for bromoviruses (Oostergetel et al., 1981). Compared with the small isometric viruses, AMV may be regarded as being in a permanently swollen state and thus resemble CMV. Purified preparations of some AMV strains can be fractionated by polyacrylamide gel electrophoresis to reveal the presence of at least 17 nucleoprotein components (Bol and Lak-Kaashoek, 1974) each of which is made up from the single viral CP species (van Beynum et al., 1977). Besides the four major RNAs, there are at least 10 minor RNA species of different lengths. The nucleoproteins occurring in minor amounts contain both major and minor species of RNA. Particles of somewhat different size may contain the same RNA, while particles of the same size may contain different RNAs. Thus, a small variation is possible in the amount of RNA encapsulated by a given amount of viral protein (Bol and Lak-Kaashoek, 1974). Although AMV
is labile in gradients of CsCl it is stable and bands isopycnically in gradients of Cs2SO4 and metrizamide (Hull, 1976b). In these gradients it forms two bands at very similar densities, the major band containing mainly bottom component and the minor band predominantly the other components. This indicates that the components have very similar, but not identical, protein:RNA ratios. Some AMV strains, e.g., 15/64 and VRU, form unusually long particles (Figure 3.26) (Hull, 1970a; Heijtink and Jaspars, 1974) with particles up to 1 μm in length; these particles do not contain any abnormally long RNA molecules and most likely contain several molecules of the genome segments. Reconstitution experiments indicated that the tendency to form long particles is CP-directed (Hull, 1970a). In a comparison of the CPs of normal length particle strains with those of long particle strains, Thole et al. (1998) identified two amino acid substitutions, Ser66 and Leu175, which were associated with the long particles. When these amino acid alterations were introduced into a normal length particle strain long particles were formed.
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(A) 19
19
19
19
19
36 48
58
Top a (Ta) Top b (Tb) Middle (M)
Bottom (B)
6.5 nm 9.4 nm
(B)
(a)
(b)
(c) (d)
(e)
FIGURE 3.25 Structure of AMV particles. (A) Top: Sizes (in nm) of the four main classes of particle. Bottom: A schematic representation of the distribution of protein and RNA in AMV bottom component. RNA is indicated by the hatched area and the protein molecules are represented by ellipsoids. The model is derived from the analysis of both the 30 S and the bottom component by small-angle neutron scattering. Bottom part reprinted from Cusack et al. (1981) with permission of the publishers. (B) Geodestix models showing the proposed structure of the components of AMV. (a) Topz, (53 S) component; (b) topa component; (c) topb component; (d) middle component; (e) bottom component. From Hull et al. (1969) with permission of the publishers.
Ilarviruses such as TSV have quasi-isometric or occasionally bacilliform particles of four different size classes (van Vloten-Doting, 1975) that appear to share many properties in common with AMV (van Vloten-Doting, 1976). The top component of TSV has been crystallized to give a hexagonal
space group, but the crystals were not amenable to X-ray diffraction as they were disordered (Senke and Johnson, 1993). ii. Particle Assembly The CP of AMV assembles into bacilliform particles in the presence of nucleic acids or
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105
FIGURE 3.26 Electron micrograph of unfractionated preparation of VRU strain of AMV negatively stained in saturated uranyl acetate. Magnification marker = 100 nm. From Hull (1970a) with permission of the publishers.
into T = 1 icosahedral particles in the absence of nucleic acid (Bol and Kruseman, 1969; Hull, 1970b; Driedonks et al., 1977). Both AMV and ilarviruses (TSV and PNRSV) bind RNA, a process important in viral replication (Box 7.3) as well as virus assembly. Driedonks et al. (1980) followed polymerization in the analytical ultracentrifuge. They postulated four stages in assembly of the virus: (i) an initiation stage; (ii) initial cap formation; (iii) cylindrical elongation; and (iv) a cap formation or closure stage. The dimer of the CP is a very stable configuration in solution (Driedonks et al., 1977) and is likely to be involved in virus assembly. X-ray crystallography of the T = 1 particle indicates that dimer is formed by the C-terminal arm of one subunit hooking around the N-terminal arm of an adjacent subunit (Kumar et al., 1997). The ability to form dimers is controlled by the C-terminus of the CP (Choi and Loesch-Fries, 1999). b. Ourmiaviruses Members of the Ourmiavirus genus have bacilliform virions of 18 nm diameter and of three lengths, 30, 37, and 45.5 nm (Lisa et al., 1988). The protein subunits cluster into dimers or trimers. The pointed ends may be formed from icosahedra cut through threefold axes for a dimer or twofold axes for a trimer. The tubular body does not form a continuous geometrical net as with AMV but contains discontinuities marked by fissures between double disks of the protein (Figure 3.27). Particles consisting of 2, 3, 4,
FIGURE 3.27 Structure of Ourmia melon mosaic virus. Top: “Averaged” images of negatively stained particles. Each image was built up photographically by equal superimposed exposures of 10 original particle images. The top row has two double disks and the bottom row has three. Bottom: Sketches showing the suggested arrangement of double disks in particles of different length. Particles of type D have not yet been observed. (Courtesy of R.G. Milne).
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or 6 double disks have been observed, with 4- and 6-disk particles being rare. Figure 3.27 illustrates these structures diagrammatically.
3. Other Particles Based on T = 1 Symmetry a. Geminivirus Structure Geminiviruses contain ssDNA and one type of coat polypeptide. Particles in purified preparations consist of twinned or geminate icosahedra (Figure 3.28A). From a study of negatively stained particles together with models of possible structures Francki et al. (1980) suggested that particles of CSMV consists of two T = 1 icosahedra joined together at a site where one morphological subunit is missing from each, giving a total of 22 morphological units in the geminate particle. This structure received support from a study of the size of the DNA and protein subunit of this virus, indicating that each geminate particle shell, consisting of 110 polypeptides of 28-kDa arranged in 22 morphological units, contains one molecule of ssDNA with MW of 7.1 × 105 (about 2.1 kb). The fine structure of MSV particles was determined by cryoelectron microscopy and 3D image reconstruction (Zhang et al., 2001) (Figure 3.28B and C) and confirmed the model suggested by Francki et al. (1980). The structure of the CP of MSV has been deduced by modeling it on the atomic coordinates of STNV CP as a template motif (Zhang et al., 2001) (Figure. 3.28D and E). The overall structures of ACMV particles and subunit proteins (Figure 3.28F) are similar to that of MSV (Böttcher et al., 2004) but with some important differences. The capsomeres of ACMV particles protrude less than those of MSV which can be explained an additional 14 residues in the MSV βF/βG loop; it is suggested that this may be related to the leafhopper transmission of MSV (Chapter 12, Section III, E, 2, a). ACMV has a six residue surface loop (βD/βE) (Figure 3.28E) not present in MSV; this loop has a sequence identified as essential for whitefly transmission (Chapter 12, Section III, G, 1). A region of the CP of TYLCV between amino acids 129 and 134 has been shown to be essential for both the correct assembly of the virions and for whitefly transmission (Noris et al., 1998).
4. T = 3 Particles a. Tymovirus Genus The particles of tymoviruses are about 30 nm diameter and made up of subunits of about 20-kDa. i. Classes of Particle Purified TYMV preparations can be fractionated on CsCl density gradients into several components. There are three classes of particle: The empty protein shell. About one-third to one-fifth of the particles found in a TYMV preparation isolated
from infected leaves are empty protein shells (the top or T component). These contain no RNA but otherwise are identical in structure to the protein shell of the infectious virus (B1 component). Full particles can be converted to empty ones by freezethaw or by alkali treatment. When TYMV B1 is taken to pH 11.6 in 1 M KCl, the particles swell from 14.6 to 15.2 nm radius within 30 s (Keeling et al., 1979). RNA escapes from these particles in 3–10 min in a partially degraded state. In addition, an amount of protein is lost within 1–3 min that is equivalent to the loss of one pentamer or hexamer of subunits from each particle. No such loss of protein occurs with the minor nucleoproteins (Keeling and Matthews, 1982). On return to pH 7.0 the radius of the resultant empty shells returns to normal. The nucleoproteins containing less than the full genome RNA do not swell at pH 11.6 under the same conditions and their RNA does not escape, although it is also degraded within the particle. Infectious virus nucleoprotein (B or bottom components and particles derived from them). The infectious virus fractionates to form two density classes in CsCl gradients (B1a and B1b) that are equally infectious (Matthews, 1974). A third B1 fraction, B1c, denser than B1b, has been characterized. B1b and B1c both contain copies of the CP mRNA as well as a molecule of genome RNA. These B1 components can be converted in strong solutions of CsCl to a B2 series with higher densities, especially if the pH>6.5. These are designated B2a, B2b, and B2c. Their formation is prevented by the presence of 0.1 M MgCl2 in the CsCl. Nucleoprotein particles containing subgenomic RNAs and having densities in CsCl intermediate between that of the T and B components. Mellema et al. (1979) and Keeling et al. (1979) isolated a series of eight minor components. The coat mRNA and a series of eight other subgenomic RNAs of discrete size have been isolated from these particles. These eight subgenomic RNAs have not yet been firmly allocated to particular nucleoprotein species. If the coat mRNA and the eight others are encapsidated in various combinations, and numbers of copies per particle, there may in fact be a very large number of particles of slightly differing density. Some properties of the minor noninfective nucleoprotein fractions were determined by Mellema et al. (1979) and Keeling et al. (1979). Their RNA content ranges from about 5% for fraction 1 to 28% for fraction 8. The proportion of total minor nucleoproteins relative to the infectious nucleoproteins is about 5% on a particle number basis. The CP cistron is found in most of the minor nucleoproteins along with other subgenomic RNAs (Pleij et al., 1977; Higgins et al., 1978).
Chapter | 3 Architecture and Assembly of Virus Particles
(A)
(B)
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(D) A
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B
(E)
FIGURE 3.28 Structure of geminivirus particles. (A). Purified geminivirus from Digitaria negatively stained in 2% uranyl acetate. Bar = 50 nm. From Dollet et al. (1986) with permission of the publishers. (B). Fit of 110 copies of MSV-N pseudo-atomic model, shown as Cα tracings, the two apical capsomeres (green), 10 peripentonal capsomeres (red), and 10 equatorial capsomeres (blue).The box and right-hand double-headed arrow indicate details shown in the original figure from Zhang et al. (2001). Panel (C) shows the equatorial capsomere interactions (position of left hand double-headed arrow in panel B). (D). Ribbon drawings of polypeptide chains of STNV coat protein (left hand drawing) and MSV-N coat protein (right hand drawing). In these drawings the β-strands are labelled according to convention. From Zhang et al. (2001) with permission of the publishers. (E). Model of a single ACMV coat protein for comparison with that of MSV (right hand model in panel D). The βD/βE loop is shown in red, with the six residue ACMV insertion in violet. The βF/βG loop is depicted in blue, with the four-residue ACMV insertion in cyan. From Böttcher et al., 2004 with permission of the publishers.
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ii. The Protein Shell Using negative-staining procedures, Huxley and Zubay (1960) and Nixon and Gibbs (1960) showed that the protein shell of TYMV is made up of 32 protuberances, occupying two structurally distinct sites in the shell. Klug and colleagues used TYMV extensively in developing X-ray diffraction and electron microscopy as tools for the study of smaller isometric viruses. Klug et al. (1966) and Finch and Klug (1966) concluded that the protein shell has 180 scattering centers lying at a radius of about 14.5 nm. These points were identified with protuberances of the protein structure units at the surface of the particle. A higher resolution X-ray analysis to 3.2 Å was made by Canady et al. (1995, 1996). Each individual protein subunit is somewhat banana shaped. Within the intact virus, each protein subunit is made up of about 9% α-helix and 43% β-sheet and about 48% of the polypeptide is in an irregular conformation (Hartman et al., 1978). This conclusion was broadly confirmed by Tamburro et al. (1978). The polypeptide chain forms an eight-stranded antiparallel β sandwich (β barrel) (Figure 3.29A) (Canady et al., 1996). The X-ray data on the virus gave good agreement for a model of the protein shell with 32 scattering centers lying at a radius of about 121 Å and extending to a radius of 159 Å from the center of the particle. Figure 3.29B summarizes the knowledge of the external arrangement of the protein subunits, while Figure 3.29C shows views of these particles in three different orientations obtained by the 3D image reconstruction technique. Thus, the surface of the particle has the subunits in a T = 3 arrangement with the pentameric and hexameric protein aggregates protruding from the surface and forming deep valleys at the quasi threefold axes. The N-terminal 26 residues of the A-subunit are disordered, whereas those of the B- and C-subunits interact around the interior of the quasi sixfold cluster where they form an annulus (Figure 3.29D) There are extensive internal contacts between the A-, B- and C-subunits (Figure 3.29E). The three histidine residues of each protein subunit are positioned to the interior and are accessible for interaction with the RNA genome. The appearance of the interior surface of the virus capsid suggests that a pentameric subunit is lost during virus disassembly. X-ray analysis of crystals of artificial empty TYMV capsids show that there are clear differences in the conformation of a loop in the A subunit located at the opening of the pentamer when compared with “full” particles (van Roon et al., 2004). It is suggested that these differences may confirm evidence for interactions between the CP and viral RNA at the N terminus at the icosahedral fivefold axis, depending on the pH and the state of the virus. It is proposed that this RNA–protein interaction plays a role in encapsidation or decapsidation of the virus, as it has been
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described that the virus on decapsidation loses at least one pentamer through which the viral RNA can escape. The structure of the tymovirus PhyMV has been resolved to 3.0 Å (Krishna et al. 1999). The basic structure is similar to that of TYMV but there are some differences. The N-terminal 17 residues of the A subunits making up the 12 pentamers show order that is not seen in TYMV and have a conformation that is very different from that observed in the B and C subunits constituting the hexameric capsomeres. Analysis of interfacial contacts indicates that hexamers are held together more strongly than pentamers and that hexamer–hexamer contacts are stronger than pentamer–hexamer contacts. These observations suggested an explanation for the formation of empty capsids which might be initiated by a change in the conformation of the A subunit N-terminal arm. The folding of the polypeptide backbone and the arrangement of subunits within the viral capsid of DYMoV is very similar to that of TYMV (Larson et al., 2000). However, there is a major difference in the position of the N-terminal regions within the particles. The N-termini of B and C subunits (see Figure 3.19 for nomenclature of subunits) of TYMV comprising the hexameric capsomere form an annulus around the interior of the capsomere; the corresponding N-termini of the pentameric capsomere are not visible in electron density maps. In DYMoV the N-termini from the A and B subunits form the annuli, thereby resulting in a much strengthened association between the two types of capsomere and giving a more stable capsid (Larson et al., 2000). iii. Location of the RNA Finch and Klug (1966) considered that folds of the RNA in TYMV were intimately associated with each of the 32 morphological protein units. They thought that it was the presence of this RNA in and around these positions that enhanced the appearance of the 32 morphological subunits seen in electron micrographs of this virus, as compared to empty protein shells. However, neutron small-angle scattering shows that there is very little penetration of the RNA into the protein shell and that the protein subunits are densely packed. Comparison of cryoelectron microscopy images of full and empty capsids of TYMV identified strong inner features around the threefold axes of the full, but not the empty, particles (Böttcher and Crowther, 1996). This suggested that substantial parts of the RNA are icosahedrally ordered. By comparing electron density maps from crystals of B particles and empty T particles, prepared by freeze-thawing, Larson et al. (2005) showed that a large proportion of the encapsidated RNA has icosahedral distribution. Four unique segments of base-paired double-helical RNA lie between 33 Å and 101 Å radius and are arranged around either twofold or five icosahedral axes as well as single-stranded loops of RNA associating with pentamereric and hexameric capsomeres where they contact the inner surface (Figure 3.29F). It was
Chapter | 3 Architecture and Assembly of Virus Particles
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(B)
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c (F)
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Cytoplasm Quasi sixfold contacts
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Quasi threefold contacts
Salt bridges
(G)
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Chloroplast Replicase and negative sense strand RNA
FIGURE 3.29 Structure of TYMV. Panel (A) Ribbon diagram of the TYMV coat protein with orientations of the quasi sixfold, quasi threefold, and quasi twofold axes indicated. The B-subunit is shown; the A- and C-subunits are virtually the same except that the N-terminal 26 residues are disordered in the A-subunit. The eight strands of the β-barrel are labeled, along with the N- and C-termini. Helix CD, which is a regular α-helix, is indicated. The EF helix, appearing behind, is irregular, with some qualities of a 310-helix. Two small segments of β-sheet are also formed by residues 6–8 and 140–142. Panel (B) Drawing of the outside of particle showing clustering of protein subunits into groups of five and six. Panel (C) Views of the reconstructed density distribution plotted section by section onto photographic plates, which have been stacked to form a glass block. Only the top three-quarters of the model is shown so that the top morphological units are not superimposed on other units at the bottom of the model. The views are approximately down a twofold axis in subpanel (a), a threefold axis in subpanel (b), and a fivefold axis in subpanel (c). The central morphological units in subpanels (b) and (c) are clearly a hexamer and a pentamer, respectively. The morphological units viewed sideways on are seen to be slightly conical, spraying out at their bases to form connections with neighboring units. Panel (D) Image of the annuli made by the N-termini of the B-(blue) and C-(green) subunits interior to the loops between β-strands F and G (FG loops), viewed along a quasi sixfold axis. Residues 1–10 of each subunit are colored orange (B) and red (C). The N-terminal peptide, which is not seen in the A-subunit, crosses under each subunit and contacts a neighbor near residue 5. Panel (E) Hydrogen bonding, intersubunit contacts, and accessibility diagram for the B-subunit of TYMV.
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considered that the remaining RNA, which was not seen in the electron density maps, lies between the secondary structure elements and is not icosahedrally organized. Low pK proton-donating groups are involved in TYMV RNA-capsid interactions suggesting that nonbase-paired nucleotides, mainly cytosine are involved (Bink et al., 2004). Unlike many other RNA viruses, the CP N-terminus of TYMV and PhyMV is not involved in interactions with the RNA (Sastri et al., 1999), but histidine(s) on the inner side of the capsid are implicated in CP-RNA binding but not in capsid assembly (Bink et al. 2004; Sastri et al., 1999). The RNA within TYMV may be stabilized by two kinds of interactions: first by glutamyl or aspartyl side chains hydrogen bonding to cytosine phosphate residues as suggested by Kaper (1972), and second by the spermine and spermidine found in this virus which could neutralize a significant proportion of the charged RNA, together with divalent cations. It is probable that all tymoviruses are stabilized in part by polyamines, but some members of the group may lose these components quite readily during isolation. Thus, BeMV isolated in the absence of CsCl contained about 100–200 polyamine molecules and 500–900 Ca2+ ions per virus particle. The polyamines could readily be exchanged with other cations such as Cs, leading to a loss of particle stability (Savithri et al., 1987). iv. Assembly of Tymoviruses Little is known about the assembly processes of TYMV particles. There is some information on the interactions between the CP and RNA. It is suggested that TYMV His68 and His180 are both instrumental during the process of encapsidation by interaction of the positively charged histidines at low pH with the RNA phosphates and during the decapsidation process through deprotonation of the histidines when the virus enters the cell in a somewhat more neutral environment (pH 7 or higher). This neutralization of the histidines may induce changes in the RNA configuration, followed by a destabilization of the virion (Bink et al. 2004). Deletion mutation analysis of TYMV RNA showed that
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mutants lacking the protease/helicase (pro/hel) region (see Appendix A Profile 48 for genome map of TYMV) did not encapsidate the viral RNA. However, mutations in the pro/ hel region which abolished replication of the RNA did not affect packaging (Shin et al., 2010). TYMV particles have not yet been reassembled in vitro from RNA and protein subunits. Figure 3.29G illustrates a possible model for the assembly of TYMV particles in vivo. Matthews (1981) discusses this in more detail. The model predicts that there is an accumulation of CP just before virus assembly begins, and that, unlike TMV replication, there would be no accumulation of complete uncoated viral genomes. As the pentamer and hexamer clusters are depleted in the membrane, others would replace them from the electron-lucent layer until the supply was exhausted. Empty protein shells presumably represent errors in virus assembly, which take place in the absence of RNA. They can form because of the strong protein–protein interactions in the shell of this virus. The model readily explains the effect of 2-thiouracil on TYMV replication. When genome RNA synthesis in the vesicles is blocked by the analogue, empty protein shells are made in increased amounts from the accumulated CP and from further protein being synthesized on preexisting coat mRNA. The model also explains the apparent requirement of illumination for virus assembly (Rohozinski and Hancock, 1996). It is suggested that the light-induced generation of low pH drives TYMV assembly. b. Bromovirus Genus The protein shell of these viruses is 25–28 nm in diameter, made up of 180 protein subunits of 19.4 kDa. The three genomic RNAs (RNA 1, 2, and 3) are packaged separately; the subgenomic RNA for CP (RNA4) is packaged with RNA3. i. Stability of the Virus The stability of bromovirus particles is related to the pH. As the pH is raised between 6.0 and 7.0, particles of BMV and CCMV swell about 10%
FIGURE 3.29 (Continued) Atoms were assumed in contact if they were 4.11 Å apart for van der Waals interactions, 3.3 Å for hydrogen bonds, and 3.8 Å for salt bridges. Residues were considered inaccessible if they had less than 5 Å2 accessible surface area. Panel (F) The distribution of secondary structural elements within the TYMV capsid. Associated with each of the 60 icosahedral operators are two identical helical segments perpendicular to and intersecting dyad axes (green and blue) and two lying parallel with (purple) and perpendicular (magenta) to the fivefold axes, and two clusters of single loops, hexameric (yellow) and pentameric (orange), occupying the capsomere cavities. It is not immediately apparent from the difference in Fourier maps how the remaining nucleotides link these elements together. In (a) are all the secondary structural elements lying in a plane through the coplanar two-, three-, and fivefold axes. In (b) are all the elements in the entire virus. Panel (G) Model for the assembly of TYMV. (i) Pentamer and hexamer clusters of CP subunits are synthesized by the ER and accumulate in the cytoplasm overlying clustered vesicles in the chloroplast. (ii) These become inserted into the outer chloroplast membrane in an orientated fashion—that is, with the hydrophobic sides that are normally buried in the complete protein shell lying within the lipid bilayer, with the end of the cluster that is normally inside the virus particle at the membrane surface, (iii) An RNA strand synthesized or being synthesized within a vesicle begins to emerge through the vesicle neck. (iv) At this site a specific nucleotide sequence in the RNA recognizes and binds a surface feature of a pentamer cluster lying in the outer chloroplast membrane near the vesicle neck, thus initiating virus assembly. (v) Assembly proceeds by the addition of pentamers and hexamers from the uniformly orientated supply in the membrane. (vi) The completed virus particle is released into the cytoplasm. Panels A, D, and E are from Canady et al. (1996), panel B from Finch and Klug (1966), panel C from Mellema and Amos (1972), panel F from Larson et al. (2005), panel G from Matthews (1991) with permissions from the relevant publishers. A more detailed version of this figure can be found on http://booksite.elsevier.com/9780123848710
Chapter | 3 Architecture and Assembly of Virus Particles
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FIGURE 3.30 Backbone model of the CCMV capsid in the native (A) and swollen (B) state with calcium ions shown in red. Modeling calcium cavity residues in the native (C) and swollen (D) form of the CCMV virus. The most important distances from calcium ions (yellow spheres) are shown (in Å). From Konecny et al. (2006) with permission of the publishers.
(Zulauf, 1977; Speir et al., 1995). The consequences of this swelling depend on many factors and particularly on the ionic conditions. The particles are readily disrupted in 1 M NaCl at pH 7.0. When swollen virus is dissociated, the protein subunits can reassemble under a variety of conditions to form a range of products, described by Bancroft and Horne (1977) (Section V, B, 4, b, iv). Swelling of CCMV particles is the result of an expansion of the virion capsid at the pseudo-threefold axes due to electrostatic repulsion at the carboxylate cages while the contacts within the hexameric and pentameric morphological units are conserved (Konecny et al., 2006) (Figure 3.30A and B). Although the swelling creates large openings in the capsid at the pseudo-threefold axes, the viral RNA is not spontaneously released, rendering the swelling reversible. The capsid dimer subunit interactions are retained, but shifted in their geometry. The expanded state is stabilized by dimer and hexamer inter-subunit contacts, mainly interwoven C-termini, and, predominantly, by RNA-protein interactions. Empty capsids cannot swell without dissociating due to the absence of RNA-capsid stabilizing interaction
Three binding sites for the calcium ions in one unit of CCMV and BMV virions have been proposed. They are situated at the interface of the subunits A–B, B–C, and C–A (Figure 3.30C and D) (Konecny et al., 2006). Konecny et al. (2006) analyzed the electrostatic properties of CCMV and showed that strong electrostatic repulsion at the pseudo-threefold axis sites of virus particles is the most likely driving force for capsid swelling. Strong positive electrostatic potential on the inside, inner capsid shell suggests that the electrostatic interactions between enveloped, negatively-charged RNA and the inner capsid shell are dominant. As the capsid swells, patches of negative electrostatic potential on the capsid surface are exposed, which destabilizes RNA-capsid interactions and may aid the RNA release process. It is unlikely that the RNA could escape from inside the capsid (at neutral pH) through channels formed by swelling due to electrostatic repulsion. ii. Particle Structure (reviewed by Kao et al., 2011) Finch et al. (1967b) found that the morphological units of BBMV protrude at least 1.5 nm from the body of the particle. The negative stain appeared to penetrate into the center
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FIGURE 3.31 Structure of bromoviruses. Panels (A) and (B) Crystallographic structure of BMV, panel (A) pentameric view, panel (B) hexameric view. The pentameric A subunits are shown as yellow ribbons along with a central cation bound by carboxylate groups. The B and C hexameric subunits are cyan and green, respectively: the cations on the quasi-threefold axes are also present; in panel (B) the β-annulus at the center of the capsomere forms a pore of about 6 Å diameter. Ions on the quasi-threefold axes are also shown. Panel (C) Diagram of BMV capsid showing contacting surface areas for the symmetry-related protein subunits. Along each unique interface the surface area buried by that interface is noted. The values in parentheses are those for the closely related CCMV structure. This provides some measure of the degree of interaction of the two subunits across each interface. Exact rotation axes are identified by filled symbols and quasi rotation axes by open symbols. Panel (D) Structure of BMV coat protein monomer and asymmetric trimer. The models are generated from the structure of Lucas et al. (2002a,b) [protein data bank (pdb) 1JS9]. The monomer shows a surface rendering in which the positively charged residues are in blue and the negatively charged residues are in red. The N-and C-termini are denoted by the letters N and C, respectively. The trimer with the three conformers A, B and C, illustrates the locations of the N- and C-termini that form the capsid. The monomer is the C conformer. Panels (A), (B), and (C) from Lucas et al. (2002a,b), panel (D) from Kao et al. (2011) with permission from the relevant publishers.
of the virus particles, suggesting the presence of an appreciable central hole, which is not found in TYMV. The presence of this hole, about 5.5 nm in radius, was confirmed by X-ray diffraction studies (Finch et al., 1967b), and for BMV by small-angle neutron scattering. The structure of native and swollen particles of CCMV has been solved using X-ray crystallography (to 3.2 Å) and cryo-electron microscopy (Speir et al., 1993; 1995); that of native particles of BMV has been solved to 3.4 Å (Lucas et al., 2002a). The structures of the two viruses are very similar. The polypeptide chains of the CP subunits
are arranged in β barrels with the C-terminal regions of adjacent subunits being interwoven. The N-termini from six CP subunits at each threefold axis also interact with each other to give what is termed a “β-hexamer structure” (Willits et al., 2003). Additional particle stability is provided by contacts between metal ion (primarily Ca2+)mediated carboxyl cages on each subunit and by protein interactions with regions of ordered RNA. In the model of BMV (Figure 3.31) (Lucas et al., 2002a), the pentameric A subunits are composed of residues 41–189, the B and C subunits comprising the hexameric capsomeres
Chapter | 3 Architecture and Assembly of Virus Particles
of residues 25–189 and 1–189 (Figure 3.31A and B, respectively). Amino-terminal segments of the A and B subunits are not visible in electron density maps. The initial 25 amino acid residues at the amino-terminal ends of the C subunits were observed as continuous chains, contiguous with the remainder of the C subunits at amino acid 26, but they were somewhat disordered. The proposed contacting areas between the subunits of BMV are shown in Figure 3.31C. iii. Location of the RNA The CP N-terminal 25 amino acids for BMV and 26 amino acids for CCMV are rich in basic amino acids, and structure prediction methods indicate that this sequence may interact in a helical form with the RNA (Argos, 1981; Zhao et al., 1995) thus forming an R domain. There are further RNA-interacting domains in BMV, two in the N-terminal part of the CP, amino acids 30–49 and 80–89 and therefore downstream of the R domain, and two in the C-terminal part of the CP (amino acids 140–153 and 178–183) (Calhoun and Rao, 2008). These individually play some role in virus stabilization. Modeling of RNA in CCMV particles indicates that it forms a shell close to the capsid with the highest densities associated with the capsid dimers. These high-density regions are connected to each other in a continuous net of triangles. The overall icosahedral shape of the net overlaps with the capsid subunit icosahedral organization. Medium density of RNA is found under the pentamers of the capsid (Zhang et al., 2004). In a cryoelectron microscopy study of CCMV, Fox et al. (1998) concluded that RNAs 1, 2 and 3 + 4 were packaged in a similar manner against the interior surface of the virion shell. The viral RNA appeared to have an ordered conformation at each of the quasi-threefold axes. iv. Assembly of Bromoviruses (reviewed by Kao et al., 2011) The pioneering work of Bancroft and colleagues showed that the protein subunits of several bromoviruses could be reassembled in vitro to give a variety of structures (Bancroft and Horne, 1977). In the presence of viral RNA, the protein subunits could reassemble to form particles indistinguishable from native virus and, especially CCMV, has constituted a model system for viral assembly studies (Zlotnick et al., 2000; Lavelle et al., 2007). In in vitro assembly experiments there is a lack of specificity within the bromoviruses for heterologous encapsidation of RNA. Hiebert et al. (1968) described the formation of “hybrids” of RNA and capsid between BMV, BBMV, and CCMV. However, the competition experiments of Cuillel et al. (1979) with various foreign RNAs showed that under appropriate conditions BMV protein can recognize its own RNA molecules to some extent. The RNA sequences that mediate in vitro virus assembly differ between BMV and CCMV. The genomic and subgenomic RNAs of BMV have a 3′-terminal tRNA-like
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structure (TLS) (Chapter 6, Section I, D, 2) which mediates assembly of that virus (Choi et al., 2002). Although CCMV RNA has a 3′-TLS, this is not required either in cis or in trans for packaging (Annamalai and Rao, 2005); CCMV CP could package BMV RNA lacking the 3′-TLS. Bromovirus RNAs are packaged into three morphologically indistinguishable particles. Genomic RNAs 1 and 2 are encapsidated in separate particles whereas genomic RNA3 is co-packaged with sub-genomic RNA4 into a single particle (Appendix A, Profile 53). The packaging of BMV RNA3 is mediated by a bipartite signal, the TLS described above and a cis-acting element of 187 nucleotides of the nonstructural movement protein gene (Choi and Rao, 2003). In plants, only replicated BMV RNAs are suitable for encapsidation and efficient packaging of RNA4 is coupled not only to its transcription, but also to the translation of the CP from RNA4 (Annamalai and Rao, 2006); this indicates that some requirements for RNA packaging will not be detected in in vitro assembly reactions. The BMV virion forms a pseudo T = 2 particle when BMV RNA is absent, suggesting that the particles have a mechanism to form preassembled immature particles until RNA can be encapsidated (Sullivan and Ahlquist, 1999). BMV RNA3 can be encapsidated without RNA4, indicating a mechanism to bring RNA4 into a particle that is at least partially assembled (Choi and Rao, 2003; Annamalai et al., 2008). In a study of the self-assembly of CCMV CP with RNA molecules ranging in length from 140 to 12,000 nt, Cadena-Nava et al. (2012) show that RNA is encapsidated if and only if the protein/RNA mass ratio is sufficiently high and corresponds to equal RNA and CP N-terminal charges in the assembly mix. Depurination of A771 (within ORF3) and A1006 (in the intergenic region) of BMV RNA3 inhibits its packaging into virus particles (Karran and Hudak, 2011). It is suggested that the base removal results in decreased thermodynamic stability of the local RNA structure required for packaging which enables the CP to recognize and discard damaged RNA. There are several models for the assembly of CCMV. The atomic resolution structure showed an interleaving of subunits around the quasi-sixfold vertices which suggested that capsid assembly was initiated by a hexamer of dimers (Speir et al., 1995). Alternatively, Zlotnick et al. (2000) suggested that capsid assembly is nucleated by a pentamer of dimers and proceeds by the cooperative addition of further dimers (Figure 3.32A). The assembly of CCMV particles is hypothesized to be a three-step process: specific binding of a few copies of CP, followed by RNA:RNA folding: then cooperative binding of CP to the nucleoprotein complex (Johnson et al., 2004) (Figure 3.32B). Initially, in the in vitro assembly of CCMV, CP dimers bind RNA with low cooperativity and form deformed
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(A) Dimer
Pentamer of dimers
Pseudo-T = 2 capsids by association of pentamers T = 3 capsids by cooperative addition of dimers
(B)
Low cooperativity
“Folding”
C1
High cooperativity
FIGURE 3.32 Models for the assembly of CCMV. (A) Assembly of capsid protein (CP) begins with the formation of a pentamer of dimers. From this starting point, assembly may proceed by cooperative addition of dimers to yield T = 3 capsids or by association of pentamers to form pseudo T = 2 particles. Oligomers of CCMV coordinates (Spier et al., 1995) were generated by VIPER (Natarajan et al., 2005) and displayed using WEBVIEWER Lite. From Zlotnick et al. (2000) with permission of the publishers. (B) Proposed assembly path of CCMV. (i) CP initially binds excess RNA with low cooperativity. Because there is excess RNA, little will be encapsidated. This is the ideal time for searching for high-affinity CP binding sites. (ii) CP and RNA slowly fold to form the C1 nucleoprotein complex. This step is not required for RNA packaging. About ten CP molecules per RNA molecule are required to get folding; however, the actual stoichiometry or structure of the C1 complex is not known. (iii) Addition of CP to C1 is highly cooperative, to yield complete virus particles. The surface representation of CCMV is from VIPER. From Johnson et al. (2004) with permission of the publishers.
virus-like particles of 90 CP dimers and one copy of RNA. At a stoichiometry of about 10 CP dimers per RNA, the CP slowly folds the RNA into compact structures that can be bound with high cooperativity by additional CP dimers. This folding process is exclusively a function of CP quaternary structure being independent of RNA sequence. The β-hexamer structure appears not to play a significant part in particle assembly but is likely to contribute to particle stability (Willits et al., 2003). These different ideas about particle assembly may reflect differences between using in vitro and in vivo systems. c. Cucumovirus Genus Purified preparations of cucumoviruses contain four RNA species housed in three particles of 30 nm diameter in the same arrangement as the bromoviruses. Some isolates of cucumoviruses have associated with them a small satellite RNA (Chapter 5, Section II, B, 2, a). Using electron microscope methods similar to those employed in their study BBMV, Finch et al. (1967a), showed that CMV resembled bromoviruses, both in surface structure and in the fact that there is a central hole in the particle. There is substantial penetration of the RNA into the shell of protein (Jacrot et al., 1977). The packing of the protein subunits is such that about 15% of the surface (at a radius of 11.7 nm) could be made up of holes, which would expose the RNA to inactivating agents and could explain the sensitivity of this virus to RNase.
Cryoelectron microscopy and reconstruction to 23 Å resolution shows that CMV is structurally similar to CCMV (Wikoff et al., 1997). The CMV structure was confirmed by X-ray crystallography which also showed that the CP subunits have a β-barrel structure (Smith et al., 2000). Thus, CMV and CCMV particle structures are similar in (i) particle morphology, (ii) size and orientation of their β-barrels, (iii) stabilizing interactions for hexamer formation, and (iv) subunit primary sequence. CMV differs from CCMV in that it does not have proposed metal binding sites. The CMV structure clearly demonstrated that the residues important for aphid transmission lie at the outermost portion of the βH-βI loop (Chapter 12, Section III, A, 8, a) (Smith et al., 2000). The structure of TAV, solved by X-ray crystallography (Lucas et al., 2002b) shows that its structure is very similar to that of CMV. However, there are some differences in the outer electrostatic distribution on the particles which probably explain differences in particle stability (Pacios and García-Arenal (2006). Modeling the CP structure demonstrated that the structures of various strains of CMV and PSV are similar though there are some differences which possibly related to differences in function (Gellért et al., 2006; ObrepalskaSteplowska et al., 2008). CMV has relatively unstable particles when compared to those of bromoviruses. CMV particles do not swell at pH 7.0 under conditions where those of bromoviruses do—or in reality, they do not “shrink” at pHs below 7.
Chapter | 3 Architecture and Assembly of Virus Particles
Thus, they behave essentially as swollen particles stabilized primarily by protein:RNA interactions. The difference in behavior of of cucumviruses and bromoviruses may be partly due to the fact that the B and C subunits of CMV combine to form a unique bundle of six amphipathic helices orientated down into the virion core at the threefold axes and the lack of metal binding sites (Smith et al., 2000). d. Tombusviridae Family The 30 nm diameter particles of most members of the Tombusviridae are composed of subunits of 38–43 kDa encapsidating a single species of genomic RNA; the genomes of those in the genus Dianthovirus are divided into two segments (Appendix A, Profile 72). i. Structure of Members of the Tombusviridae The structure of two members of the genus Tombusvirus, TBSV and CNV (Harrison et al., 1978; Olson et al., 1983; Katpally et al., 2007), five members of the genus Carmovirus, CarMV, TCV, MNSV CPMoV, and HCRSV (Hogle et al., 1986; Morgunova et al., 1994; Doan et al., 2003; Ke et al., 2004; Wada et al., 2008; Cheng et al., 2009), and one member each of the genera Necrovirus (TNV) and Dianthovirus (RCNMV) (Oda et al., 2000; Sherman et al., 2006) have been determined crystallographically. Each virus contains 180 protein subunits arranged to form a T = 3 icosahedral surface lattice, with prominent dimer clustering at the outside of the particle, the clusters extending to a radius of about 17 nm. All the members of the Tombusviridae that have been studied, except TNV, have very similar basic particle structures even though there are differences in the amino acid sequences of the CPs. Features of the structure are exemplified by TBSV (Figure 3.33). The capsid is formed by 180 chemically identical CP subunits in three quasi-equivalent conformations (A, B, and C). Each CP subunit is composed of three distinct structural domains, which include the RNA-interacting (R), shell (S), and protruding (P) domains. The S domains build the spherical shell while the P domains associate into 90 dimers (30 C-C and 60 A-B dimers). The P and S domains are connected by a flexible hinge, conformational differences that distinguish the A, B, and C subunits being localized within this hinge region. These hinges point either down (in A-B dimers) or up (in C-C dimers). In addition, the loop that connects the R and S domains (the arm) is ordered in C subunits but disordered in A and B subunits. Mutations of the hinge of TCV do not affect movement of the virus through the plant but some alter the symptoms induced by the virus (Lin and Heaton, 1999). Each P domain forms one-half of the dimer-clustered protrusions on the surface of the particle occupying approximately one-third of one icosahedral triangular face. The P
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domain of MNSV is involved in compatibility with, and transmission by, its fungal vector (Chapter 12, Section V) (Ohki et al., 2010). The S domain forms part of the icosahedral shell which is about 3 nm thick and from it protrudes the 90 dimer clusters formed by the P domain pairs. In addition to domains P and S, each protein subunit has a flexibly linked N-terminal arm containing 102 amino acid residues comprising the R domain and the connecting arm called “arm a” (Figure 3.33 panel A, subpanel b). The CP assumes two different large-scale conformational states in the shell that differ in the angle between domains P and S by about 20° (Figure 3.33 panel A, subpanel d). The conformation taken up depends on whether the subunit is near a quasi dyad or a true dyad in the T = 3 surface lattice. The state of the flexible N-terminal arm also depends on the symmetry position. The N-terminal arms originating near a strict dyad follow each other in the cleft between two adjacent S domains. On reaching a threefold axis, such an arm winds around the axis in an anticlockwise fashion (viewed from outside the particle). Two other N-terminal arms originating at neighboring strict dyads will be at each threefold axis. The three polypeptides overlap with each other to form a circular structure called the β-annulus, made up of 19 amino acids from each arm, around the threefold axis (Figure 3.33 panel B). Thus 60 of the 180 N-terminal arms create an interlocking network that, in principle, could form an open T = 1 structure without the other 120 subunits. The R domain and the “a” connecting arm arising from the 120 subunits at quasi-dyad positions hang down into the interior of the particle in an irregular way so that their detailed position cannot be derived by X-ray analysis. The RNA of TBSV is tightly packed within the particle and these N-terminal polypeptide arms very probably interact with the RNA. The T = 3 particles of CNV have an articulated internal structure with two major internal shells that are absent in artificial T = 1 particles (Katpally et al., 2007). Mutagenesis analysis suggests that the R-domain forms an internal scaffold that may play a role in T = 3 capsid assembly. Each CP subunit of CNV has a 34 amino acid arm, comprising an 18 amino acid “β” region and a 16 amino acid “ε” region. The former region contributes to the β-annular structure involved in particle structure and the latter to quasi-equivalence and RNA binding (Hui and Rochon, 2006). TBSV particles swell by about 12% (in radius) at pH>7 and in the presence of metal chelating agents such as EDTA (Robinson and Harrison, 1982). They can be recompacted in the presence of calcium ion at pH>7 or in EDTA-containing buffers at pH≤6 indicating that they are stabilized by both ion-dependent and divalent cation-mediated bonds (Krüse et al., 1982). On swelling, the S and P domains undergo rigid body movements preserving many subunit interactions but leading to the formation of openings at the quasi-threefold axes (Robinson and Harrison, 1982).
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(A)
a b
d
c (B)
FIGURE 3.33 Architecture of TBSV particle. Panel (A) Subpanel (a) Order of domains in polypeptide chain from N terminus to C terminus. The number of residues in each segment is indicated below the line. The letters indicate R domain (possible RNA binding region), arm a (the connector that forms the β-annulus and extended arm structure on C subunits and that remains disordered on A and B), S domain, hinge, and P domain. Subpanel (b) Schematic view of folded polypeptide chain, showing P, S, and R domains. Subpanel (c) Arrangement of subunits in particle. A, B, and C denote distinct packing environments for the subunit. S domains of A subunits pack around fivefold axes; S domains of B and C alternate around threefold axes. The differences in local curvature can be seen at the two places where the shell has been cut away to reveal S domain packing near strict (top) and quasi (bottom) dyads.
Chapter | 3 Architecture and Assembly of Virus Particles
Two divalent cation binding sites are a feature of each of the three trimer contacts between the S domains. Mutation of the calcium-binding sites of TCV can affect cell-to-cell or long distance movement or induce delayed mild systemic symptoms (Laasko and Heaton, 1993; Lin and Heaton, 1999). Although the basic structures of particles of members of the Tombusviridae (except TNV—see below) are similar there are some differences in detail. For instance, a deletion in the βC strand in the S domain relative to that of TBSV is considered to be distinctive of the genus Carmovirus (Ke et al., 2004). Furthermore, the C-terminus of carmovirus CPMoV is in a different position to that of TBSV (Figure 3.34A). MNSV is classified as a carmovirus based on genome organization and probable translation strategy yet its structure is more similar to that of TBSV than to other carmoviruses (Wada et al., 2008). The R-domain and arm regions of the dianthovirus, RCNMV, are 50 residues shorter than those of TBSV (3.34B) (Sherman et al., 2006). The crystal structure of TNV has been resolved to 2.25 Å (Oda et al., 2000). Some basic features are similar to other T = 3 icosahedral viruses but the subunits lack a P domain which makes it more similar to sobemoviruses than to tombusviruses. ii. Assembly of Tombusviruses As with bromo-, alfamo-, and cucumo-viruses, dissociation and reassociation of virus particles has been shown with tombusviruses. TCV particles dissociate at high pH and ionic strength giving rise to CP dimers and a ribonucleoprotein complex made up of the genomic RNA, six CP subunits, and an 80-kDa protein that is a covalent CP dimer. TCV particles can be reassembled in vitro using CP and either the free RNA or the ribonucleoprotein complex. For both forms of the RNA, the process is selective for viral RNA, and proceeds by continuous growth of a shell from an initiating structure (Sorger et al., 1986). Analysis of the encapsidation of various mutant viral RNAs showed that a 186-nucleotide region at the 3′ end of the CP gene, with a bulging hairpin loop of 28 nucleotides is indispensable for TCV RNA encapsidation (Qu and Morris, 1997). As noted above, dianthoviruses differ from all the other Tombusviridae genera in that the genome is divided between two segments (RNA 1 and RNA2). The origin of
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assembly (OAS) of RCNMV is delimited to a 34 nt transactivator segment of RNA2; RNA 1 does not have an OAS (Basnayake et al., 2008). e. Sobemovirus Genus i. Structure of Sobemoviruses The particles of sobemoviruses are 30 nm diameter and are made up of subunits of about 30 kDa surrounding a single species of RNA. The virus particle shows little surface detail in negatively stained preparations. The structure of the particles of various sobemoviruses (SBMV, SCPMV, SeMV, RYMV, CoMV, and RGMoV) have been determined (Abad-Zapatero et al., 1980; Silva and Rossmann, 1987; Bhuvaneshwari et al., 1995; Opalka et al., 2000; Qu et al., 2000; Tars et al., 2003; Plevka et al., 2007). All these sobemoviruses have the same basic structure with the particles comprising 180 CP subunits which have the canonical β-barrel structure (Figure 3.22). They resemble tombusviruses (except TNV—see above) in the subunits having an S domain and an N-terminal R-domain but differ in lacking a P domain. This lack of the P domain accounts for the smaller subunit (about 260 amino acids compared with about 380), and the smaller radius and smoother outside appearance of virus particles in electron micrographs. The arrangement of the three quasi-equivalent subunits (A, B, and C) is very similar to that in TBSV. As with TBSV, part of the R domain of the C subunits is in an ordered state forming a β-annulus around each icosahedral threefold axis. The R domain is shorter than that in TBSV. The first 64 residues (the R domain) of the A and B subunits and the first 38 residues of the C subunit are disordered and associated with the RNA. Cation binding sites (Ca2+) are near the external surface of the shell, the viruses being strongly dependent on Mg2+ and Ca2+ for its structural integrity. About 200 of these ions are bound firmly to each virus particle and their removal with EDTA causes a reversible destabilization of the structure. Treatment with trypsin removes the N-terminal 61 amino acids from the isolated protein subunit. The resulting 22-kDa fragment can assemble into a 17.5-nm-diameter T = 1 particle (Erickson and Rossmann, 1982). Rossmann (1985) gives a detailed account of SBMV structure. There are some differences in the details of the structures of various sobemoviruses (Opalka et al.,
FIGURE 3.33 (Continued) Subpanel (d) The two states of the TBSV subunit found in this structure, viewed as dimers about the strict (s2) and local (q2) twofold axes. Subunits in C positions have the interdomain hinge “up” and a cleft between twofold-related S domains into which fold parts of the N-terminal arms. Subunits in the quasi-twofold-related A and B positions have hinge “down,” S domains abutting, and a disordered arm. Panel (B) Packing of subunits in the icosahedral asymmetric unit of TBSV and notation for interfaces. The different kinds of subunit contacts are labeled D (dimer), T (trimer), P (pentamer), and H (hexamer), with a subscript showing the types of subunit interacting across the contact in question. The positions of Ca2+ at the T interfaces are shown by small circles. A fivefold axis is indicated at the top of the diagram. The β-annulus with threefold symmetry is shown twice in the lower part of the diagram. Subpanels (a), (b), and (c) of panel (A) and panel (B) from Olson et al. (1983); panel (A) subpanel (d) from Harrison et al. (1978) with permission of the relevant publishers.
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CPMoV TBSV
(A) C-Terminus
C-Terminus
Interior (B)
FIGURE 3.34 Structure of tombusviruses. (A) Stereo figure comparing ribbon diagrams of coat proteins of CPMoV (red) and TBSV (green) viewed looking into the P-domain contact interface with the RNA interior towards the bottom of the figure. From Ke et al. (2004) with permission of the publisher. (B) Amino acid sequence alignment of RCNMV and TBSV CPs. The overall identity and similarity between the two proteins are 26% and 39%, respectively. The S domains are the most closely related (35% identical, 48% similar) and the P domains are more diverse (27% identical and 44% similar. There is no significant identity or similarity between the R domains and the arm regions. Arrows delimit the structural domains. The sequence numbering and domain assignments are based on TBSV X-ray structure (Olson et al., 1983). Identical residues are shaded gray, whereas similar residues are boxed. Brackets mark conserved residues that provide the Ca2+ ligands in TBSV. From Sherman et al. (2006) with permission of the publishers.
2000; Qu et al., 2000; Tars et al., 2003; Plevka et al., 2007). For example, Opalka et al. (2000) showed that SCPMV has an exterior face of deep valleys along the twofold axes and protrusions at the quasi-threefold axes whereas the surface of RYMV is comparatively smooth. Particles of both viruses display two concentric shells of density beneath the capsid layer which are interpreted as ordered layers of genomic RNA. SeMV also has some differences when compared with SBMV (Bhuvaneshwari
et al., 1995). The polar interactions at the quasi-threefold axes are substantially less in SeMV and the positively charged residues on the RNA-facing side of the subunits and in the N-terminal arm (R domain) are not well conserved suggesting that the protein:RNA interactions differ between the two viruses. Circular dichroism studies on TRosV suggest that the RNA within the particle has considerable base-pairing (Denloye et al., 1978).
Chapter | 3 Architecture and Assembly of Virus Particles
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Relaxed dimer β-annulus not ordered
(A)
Pentamer of dimers
CP N∆65
CP R28-36E
T=1 No RNA T=3 Compact capsids
With RNA CP N∆36 with RNA Assembly intermediate β-annulus gets ordered in C-C dimers
Pseudo T=2 unstable
T=1 Swollen T=3 capsids Ca2+
Compact capsids T=3
FIGURE 3.35 Assembly and structure of SeMV. Panel (A) Model of SeMV assembly. The assembly of SeMV T = 3 capsids begins with the relaxed dimers interacting with each other to form a pentamer of dimers or a 10-mer intermediate. The 10-mer could serve as a common intermediate for the formation of wild-type T = 3 capsids as well as for all the mutant capsids, such as pseudo T = 2 and T = 1 capsids. As the assembly of 10-mers proceeds in the presence of RNA, the gaps in the incomplete icosahedral structure could be filled by addition of relaxed dimers whose β-annulus gets ordered (tensed C-C dimers) to complete the T = 3 shell. The capsids are stabilized by the encapsidation of RNA and binding of calcium ions. The A, B and C subunits that form the icosahedral shell are shown in green, red and blue, respectively. Panel (B) Structure of SeMV CP showing pentameric and hexameric capsomeres. Subpanel (a) A type of subunits that form pentamers at the icosahedral fivefold axes are shown in green. Subpanel (b) Hexamers of B and C type subunits present at the quasi sixfold axes are shown in blue and red, respectively. Subpanel (c) The β-annulus structure formed by the N terminus of three C type subunits at the quasi sixfold axes are represented in yellow. Subpanel (d) The residues 48–58 that form the β-annulus are shown as wire-frame model. The black dots represent hydrogen bonds between the amino acid residues. From Satheshkumar et al. (2005) with permission of the publishers.
ii. Assembly of Sobemoviruses SBMV particles can be assembled in vitro at low ionic strength from isolated RNA and CP. The components assembled into T = 1 or T = 3 particles depending on the size of the viral RNA used and the pH (Savithri and Erickson, 1983). SCPMV CP binds to specific sites on the viral RNA (Hacker, 1995). This region, mapped to nucleotides 1410–1436, is predicted to fold into a hairpin with a 4-base loop and a duplex stem of 24 nucleotides.
Sobemovirus particles are stabilized by protein–protein, protein–RNA and calcium-mediated protein–protein interactions and presumably each of these plays a role in particle assembly. Some of the most detailed studies on assembly of this genus of viruses have been done on SeMV (Satheshkumar et al., 2005; Savithri and Murthy, 2010). By analysis of CP mutants, a model for SeMV has been proposed (Figure 3.35A). The N-terminal R-domain
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FIGURE 3.35 (Continued)
which controls RNA encapsidation and particle size has two sub-domains, the arginine-rich motif (ARM) and the β-annulus structure (Figure 3.35 panel B, subpanel c). A minimum of three arginines is required for RNA encapsidation; mutation of all the arginines almost completely abolished RNA encapsidation but did not affect T = 3 capsid formation though these were less stable than RNAcontaining capsids. This suggested that capsid assembly is entirely mediated by CP-dependent protein–protein intersubunit interactions and encapsidation of the genomic RNA enhances the stability of the capsids. The formation of the β-annulus structure at the icosahedral threefold axes is not the switch that determines the pentameric and hexameric clustering of CP subunits but is thought to optimize inter-subunit interactions (Satheshkumar et al., 2005) as does calcium binding (Savithri and Murthy 2010). Water molecules also play a role in capsid structure and integrity and presumably also in assembly (Sangita et al., 2005).
The model suggests that pentamers of A/B dimers are formed in which the β-annuli are disordered; these initiate the assembly of capsids at the fivefold axes. Further assembly proceeds in the presence of RNA. Interaction of the ARM with RNA imposes order in the N-terminal segments of dimers added to the 10-mer complex leading to the formation of the β-annulus and C/C dimers. Subsequent addition of dimers leads to the formation of swollen T = 3 particles which become compact after the addition of calcium ions at the inter-subunit interfaces. One further observation which may be relevant to in vivo particle assembly is that interaction with anionic lipids causes the R-domain of SCPMV to assume an α-helical structure (Lee et al., 2001). f. Potato Leafroll Virus PLRV is the type species of the genus Polerovirus in the family Luteoviridae (Appendix A, Profile 61). It has
Chapter | 3 Architecture and Assembly of Virus Particles
isometric particles about 25–30 nm diameter considered to be T = 3 composed of CP subunits of about 23 kDa. The CP has 33% sequence similarity to that of RYMV. Terradot et al. (2001) developed a 3D structure for PLRV based on modeling its CP compared with that of RYMV; this is consistent with immunological and site-directed mutagenesis data. g. Pea Enation Mosaic Pea enation mosaic disease is caused by a complex of two viruses, PEMV-1 and PEMV-2, the genomes of which are encapsidated in isometric particles composed of the same CP, encoded by PEMV-1. The complex has two particle sizes, that encapsidating RNA-1 being about 28 nm diameter and that containing RNA-2 about 25 nm diameter. PEMV-1 is stable in CsCl but PEMV-2 is labile. Both the particles band at the same density in Cs2SO4 and in sucrose made up in D2O (Hull and Lane, 1973; Hull, 1976b) indicating that they have very similar nucleic acid content. Based on estimates of the molecular weight of the particles and the CP, Hull and Lane (1973) suggested that PEMV-1 particles had 180 subunits consistent with a T = 3 icosahedral structure but that PEMV-2 particles had 150 subunits. A quasi-icosahedral model has been proposed for PEMV-2 (Hull, 1977). Particles of PEMV-1 and PEMV-2 can be separated on electrophoresis in polyacrylamide gels (Hull and Lane, 1973). Some viral strains that have lost their aphid transmissibility form two homogeneous bands in gels but, in others that are aphid transmitted, the band formed by PEMV-1 is very heterogeneous (Hull, 1977). The reason for this heterogeneity is unknown.
5. Bacilliform Based on T = 3 Symmetry As noted in Section IV, G, it has been suggested that the structure of bacilliform particles is based on icosahedral symmetry. RTBV has bacilliform particles of 130 × 30 nm. From the size of the CP and the diameter of the particle it was proposed that these particles could be based on T = 3 icosahedral symmetry (Hull, 1996). Optical diffraction of electron micrographs of RTBV particles indicated that the size of the morphological subunit was 100 Å and the structure was based on a T = 3 icosahedron cut across its threefold axis.
6. Pseudo T = 3 Symmetry Members of the family Comoviridae have icosahedral particles formed of one, two, or three CP species. The structure resembles that of picornaviruses in that, if all the CP species are considered as one protein, the symmetry would appear to be T = 1. However, the larger polypeptides of viruses with one or two protein species form to give “pseudo-molecules” and for each virus the structure can
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be considered to be made up of effectively three “species.” This then gives a pseudo T = 3 symmetry. a. Comovirus Genus i. Structure of Comoviruses CPMV has a diameter of about 28 nm and an icosahedral structure with an unusual arrangement of subunits. The virus shell contains two proteins, a large (L) and small (S) one (42- and 22-kDa) with different amino acid compositions (reviewed by Sainsbury et al., 2010) (Appendix A, Profile 29). Purified preparations of CPMV contain three classes of particle (B component containing the larger RNA, M component with the smaller RNA and T component being empty particles), with identical protein shells, that can be separated by centrifugation. Early studies on this virus were complicated by the essentially trivial fact that the smaller protein is susceptible in situ to attack by proteolytic enzymes both in the host plant and after isolation of the virus. CPMV contains about 200 spermidine molecules and a trace of spermine per particle. CPMV may be similar to TYMV with respect to neutralization of charged phosphates, since particles containing RNA also contain polyamines, and there is no evidence for a mobile protein arm (Virudachalum et al., 1985). From the sizes of the protein and the virus shell, Geelen et al. (1972) calculated that there should be 60 of each of the two structural proteins in the shell. The structure of CPMV has been solved by X-ray crystallography (Lin et al., 1999; Lin and Johnson, 2003) and shows that the two CPs produce three distinct β-barrel domains in the icosahedral asymmetric unit (Figure 3.36A). The S CP forms the A-domain and the L CP forms the B- and C-domains. Although two of these β-barrel domains are covalently linked, their relationship is essentially like the β-barrels found in the three major separate CPs of the picornaviruses. The basic structures BPMV and RCMV are similar to that of CPMV but there are differences in detail (Lin and Johnson 2003). Nearly 20% of the RNA in BPMV particles M component (containing RNA 2) binds to the interior of the protein shell in a manner displaying icosahedral symmetry. The RNA that binds is single-stranded, and interactions with the protein are dominated by non-bonding forces with few specific contacts (Chen et al., 1989). From resolving the protein:RNA interactions in BPMV at 3.0 Å resolution Chen et al. (1989) suggested that seven ribonucleotides that can be seen as ordered in the structure lie in a shallow pocket on the inner surface of the protein shell formed by the two covalently linked domains of the large CP. Using refined structures of M and B components (containing about 33% RNA) Lin et al., (2003) showed that RNA in B component was ordered in the same location as in M component (Figure 3.37). Although the ordered RNA density in both nucleoprotein particles is the average of the contents of 60 icosahedral
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(A) a
b
A
B C
B domain
A domain
B domain
C domain C
B
L
A domain
C domain A
S
C
B
L
A
S
FIGURE 3.36 Panel (A) The structures of viral capsid and icosahedral asymmetric unit of RCMV. Subpanel (a) Stereoview of a space-filling drawing of the RCMV capsid. Two proteins (S and L subunits) are in the RCMV capsid. The S subunit forms the A domain (in blue), while the L subunit forms the B (red) and C (green) domains. All atoms are shown as spheres corresponding to a diameter of 1.8 Å. The pentameric S subunits form the protrusion. Subpanel (b) At the top, the icosahedral asymmetric unit of the capsid is color coded in the schematic presentation of the CPMV capsid. The S subunit occupies the A position, forming the A domain around the fivefold axis; the two domains of the L subunit occupy the B and C positions. Positions A, B, and C are quasi-equivalent positions of identical gene products on a T = 3 surface lattice. At the bottom, a stereoview of a ribbon diagram of the icosahedral asymmetric unit is shown. All three domains are variants of the jelly-roll β-sandwich structure. The schematic presentation of composite proteins, L and S subunits, is also shown. From Lin et al. (2000) with permission of the publishers. Panel (B) Structure of the TRSV capsid, capsid protein and the three domains in the capsid protein.
Chapter | 3 Architecture and Assembly of Virus Particles
(B)
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(a)
(b)
(c)
DE loop
BC loop
A domain
βF
βG
αA βC’ βI
αB
βE βC
βI
GH loop
βC’’ C-B domain-linking polypeptide
βI βB
GH loop
αB
βD βG
βE
βF
βH
HI loop
βC
βD αA
GH loop
EF loop
βB
B-A domain-linking polypeptide
βH
βD βI
βG
DE loops
B domain
HI loop N terminus
βC αA’
αA”
βB
C domain
Structure
FIGURE 3.36 (Continued) Subpanel (a) A CPK model of the pseudo T = 3 TRSV capsid showing the prominent surface features. The C and B domains (in green and red, respectively) are clustered around the threefold axis, while the A domains (cyan) are clustered around the fivefold axes. Subpanel (b) an enlarged view of one of the 60 copies of the capsid protein in the TRSV capsid represented as a CPK model. Subpanel (c) A cartoon representation of the tertiary structure of the C, B, and A domains in the capsid protein. The secondary structure features in the three domains, the two domain-linking polypeptides and the N and C termini of the capsid protein are indicated. From Chandrasekar and Johnson (1998) with permission of the publishers.
asymmetric units, both nucleoprotein components show that the base density for the first two nucleotides is predominantly purine, while the next five are predominantly pyrimidine. The presence of a dominant nucleotide sequence visible in the electron density suggests that there is a
consensus sequence distributed in the viral genome and that this repeating sequence may serve as the encapsidation signal for RNA packaging and virus assembly. The nucleotide strand is helical with base stacking despite being singlestranded. The empty capsid (T component) demonstrates
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FIGURE 3.37 RNA in the capsid of BPMV. Trefoils of the RNA superimposed on the electron density viewed from the exterior. The electron density in gray scale and semitransparent is calculated to 20 Å. RNA tri mers are drawn as CPK models. The carbon atoms are in black; oxygen and phosphate atoms are in red, and nitrogen atoms are in blue. The ribbon diagram of one of the protein trimers is also shown. Also shown on left of particle is a ribbon diagram of a portion of the capsid (A domain, blue; B domain, red; C domain, green). From Lin et al. (2003) with permission of the publishers.
that RNA dictates the order of the N-terminal 19 residues of the L CP subunit because these residues are invisible in the T component. ii. Assembly of Comoviruses OkMV produces large quantities of empty viral protein shells in inoculated cucumber cotyledons (Marshall and Matthews, 1981). At 1 day after inoculation, about one-half the total viral protein shells were found in the nucleus. This accumulation occurred in the presence of virus particles that were found exclusively in the cytoplasm. The most likely explanation for this active and preferential accumulation is that viral CP enters the nucleus in the form of monomers or pentamers and hexamers and is assembled into shells once inside. Coexpression of the regions of CPMV RNA2 that encode the large and small CPs in Spodoptera frugiperda (sf21) insect cells using baculovirus vectors led to the formation of virus-like particles that had the sedimentation characteristics of empty particles (Shanks and Lomonossoff, 2000; Saunders et al., 2009) (Chapter 15, Section IV, F, 1, b for use of these particles in nanotechnology). However, no particles were formed when either of the proteins was expressed individually. Similarly, CPMV proteins expressed in protoplasts (Wellink et al., 1996) and ArMV proteins expressed in transgenic plants and insect cells (Bertioli et al., 1991) form empty virus-like particles. Proteolysis of the small CP of CPMV results in the loss of the C-terminal 24 amino acids (Taylor et al., 1999).
FIGURE 3.38 A proposed mode for the encapsidation of BPMV RNA. Panel (A) (1) The encapsidation signals are distributed throughout the genome. (2–3) For the encapsidation, the RNA would partially fold with interacting segments compatible with the icosahedral capsid. (4–8) The capsid protein assemble around the RNA by recognition of the specific segment to form virus particles. Based on the intermediates found in Poliovirus (Watanabe et al., 1965), pentameric capsid proteins are suggested to be the assembly unit. For clarity, only the icosahedral surface lattice and RNA in the front are schematically shown in the assembled virus particle in 8. Panel (B) Opening of the capsid showing a thread of RNA through the capsid that is consistent with the encapsidation model. From Lin et al. (2003) with permission of the publishers.
Infections with mutants of cDNA of CPMV RNA2 from which the 24 C-terminal amino acids had been deleted were debilitated in virus accumulation with a much increased proportion (73%) of top component particles not containing RNA. It is suggested that the C-terminal region of the small CP is involved with RNA packaging. Based on the structure of RNA within the capsid described above a model for RNA packaging during BPMV assembly has been proposed (Lin et al., 2003) (Figure 3.38). b. Nepovirus Genus The genome organization of nepoviruses is similar to that of comoviruses (Appendix A, Profile 31). The capsid comprises 60 subunits of a single polypeptide species of 52–60 kDa folded into three trapezoid β-barrel domains (named C, B and A from the N-terminus) covalently linked together
Chapter | 3 Architecture and Assembly of Virus Particles
as “a string of beads” (Figure 3.36B) (Chandrasekar and Johnson, 1998). The structure of TRSV particles is similar to that of CPMV, being pseudo T = 3 made up of the three β-barrel domains. 3D homology structure models of the virions and CP subunits of GFLV and ArMV constructed based on the crystal structure of TRSV showed that these two nepoviruses have the same basic pseudo T = 3 structure (Schellenberger et al., 2010); there are some specific differences, one of which could be related to nematode transmission (Chapter 12, Section IV, A). c. Other Possible Pseudo-T = 3 Symmetry Plant Viruses Sadwaviruses resemble comoviruses in having two CP subunits, a large one of 40–45 kDa and a small one of 21–29 kDa which are cleaved from polyproteins. Although no structural studies have been made on this virus it is thought that the particle structure resembles that of comoviruses. Members of the family Sequiviridae and genus Cheravirus have isometric particles about 30 nm in diameter on which no obvious structural features can be seen by electron microscopy. They have three CP species which are processed from a polyprotein (Turnbull-Ross et al., 1993; Zhang et al., 1993; James and Upton, 2002) (see Appendix A, Profiles 35 and 32, respectively) and those of RTSV are present in the capsid in equimolar amounts (Druka et al., 1996). In this they resemble animal picornaviruses, and it is likely that they have a pseudoT = 3 symmetry.
7. T = 7 Particles a. Structure of Caulimoviruses CaMV has a very stable isometric particle about 50 nm in diameter containing dsDNA. The circular dsDNA is encapsidated in subunits of a protein processed from a 56-kDa precursor to several products, the major ones being approximately 44, 39, and 37 kDa; the product of ORF III (for genome organization of CaMV see Appendix A Profile 7) is also found in CaMV capsids. Electron microscopy shows a relatively smooth protein shell with no structural features. Neutron diffraction studies revealed a central empty cavity, about 25 nm diameter (Chauvin et al., 1979; Krüse et al., 1987) and suggested a four concentric shell structure. The particle consists of an outer protein shell within which is a zone where both DNA and protein are present. Most of the DNA is in a zone with very little protein, while the central region of the particle (one-eighth of the volume) is occupied only by solvent. The DNA does not appear to be associated with any significant amount of histone-like protein (Al Ani et al., 1979b). Exposure of the virus to pH 11.25 leads to the release of DNA tails without total disruption of the protein shell (Al Ani et al., 1979a). Calculations from MW of the CP and the amount of protein in the virus suggested that it may have a T = 7
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icosahedral structure (Krüse et al., 1987). Using cryoelectron microscopy and image reconstruction procedures, the structure of CaMV was solved to about 3 nm (Cheng et al., 1992). These studies showed that CaMV particles are composed of three concentric layers of solvent-excluding density surrounding a large (c27 nm diameter) solvent-filled cavity (Figure 3.39A). The outer layer (I) comprises 12 pentameric units and 60 hexameric units arranged in a T = 7 icosahedral symmetry, this being the first example of a T = 7 virus that obeys the icosahedral rules. The dsDNA genome is distributed in layers II and III together with some of the capsid protein. The ORF III product (P3) (also termed the virionassociated protein—VAP) has two N-terminal functional domains covering the first 60 amino acids, one involved with self-organization and one with interactions with P1 and P2 (involved with cell-to-cell movement and aphid transmission, respectively (Chapter 10, Section IV, B, 2, a and Chapter 12, Section III, B) and two structural C-terminal domains (from amino acid 61 to 129) which are involved with binding P3 to the capsid and nonspecific DNA binding (Plisson et al., 2005 for references). CaMV P3 binds to CaMV particles between capsomeres traversing the outer layer and penetrating the intermediate layer giving a lateral network of digitations (Plisson et al., 2005) (Figure 3.39B) Although caulimovirus P3 molecules (also in badnaviruses the analogous P2 molecules which bind to CP) assemble as tetramers in solution (Stavolone et al., 2001), Plisson et al. (2005) suggest that they are dimers when they bind to virus particles. It is suggested that this change in conformation might control the interactions with P1 and P2. b. Assembly of Caulimoviruses Little is known about the assembly of caulimoviruses as they are not amenable to the disassembly and reassembly processes described above. CaMV CP interacts with the viral pregenomic RNA leader (Guerra-Peraza et al, 2000) with the zinc finger motif (C-X2-C-X4-H-X4-C) playing a major role. Although CaMV encapsidates the DNA phase of the reverse transcription replication cycle (see Chapter 7, Section VII for CaMV replication) this association of CP with RNA may lead to the formation of the replication complex. As noted above, the precapsid protein (P56) is processed to P44, P39, and P37. P44 is phosphorylated whereas P39 and P37 are not suggesting that P39 is derived from P44 by N-terminal processing (Chapdelaine et al., 2002). Furthermore, P39 contains the zinc finger motif whereas P37 does not indicating that P37 is derived from P39 by C-terminal processing. Chapdelaine et al. (2002) suggest that this processing leads to the formation of the virion-like replication complex and the final stabilization of the virus particles.
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(A)
(B)
D
AD
P3
Outermost layer Intermediate layer Innermost layer
FIGURE 3.39 Structure of CaMV particles. Panel (A). Left: Recon struction of CaMV surface structure showing T = 7 symmetry. Right: Cutaway surface reconstruction showing multiplayer structure. From Cheng et al. (1992) with permission of the publishers. Panel (B) Interaction model of P3 with CaMV capsid. P3 is present as a dimer when binding the CaMV capsid by interaction of N-terminal sequences, forming digitation (D) domain. The anchoring domain (AD domain) consists of the association of three P3 monomers. The C-terminal part of P3 penetrates the intermediate layer of the shell and then interacts with both the CaMV capsid and the nucleic acid. From Plisson et al. (2005) with permission of the publishers.
VI. MORE COMPLEX ISOMETRIC VIRUSES A. Phytoreovirus Phytoreoviruses have distinctly angular particles about 65–70 nm diameter that contain 12 pieces of dsRNA. There are seven different proteins in the particle of the type species, WTV (Reddy and MacLeod, 1976) and RDV. Unlike most animal reoviruses which have triple-shelled capsids, the particles of phytoreoviruses consist of an outer shell of protein and an inner core containing protein and the 12 species of dsRNA. However, there is no protein in close association with the RNA. The particles are readily disrupted during isolation, by various agents. Under suitable conditions, subviral particles can be produced, which lack the outer envelope and which reveal the presence of 12 projections at the fivefold vertices of an icosahedron. The structure of RDV has been resolved to atomic level (Lu et al., 1998; Wu et al., 2000; Zhou et al., 2001; Nakagawa et al., 2003). The virus particles have two distinct icosahedral shells, a T = 13 outer shell, 700 Å diameter, composed of 260 trimeric clusters of P8 (46-kDa) and an inner T = 1 shell, 567 Å diameter and 25 Å thick, made up of 60 dimers of P3 (114-kDa); the outer shell was shown to be left handed (T = 13l). P8 forming the outer
shell has two significant domain structures, I being entirely α-helical and II which is mainly β-barrel structure; domain II is on the virus surface. The protein subunits interact electrostatically side-by-side to form the trimers. In the particle there are five kinds of trimers, T, S, R, Q, and P, which are icosahedrally independent (Figure 3.40 panel A) Subunits of P3 forming the core capsid resemble crescent-shaped plates. There are two icosahedrally independent forms of P3, P3A, and P3B with different shapes. The N-terminal segment of P3B is well ordered and interacts with P3A; the N-terminal segment of P3A is completely disordered. Dimer formation changes the conformation of each P3 type and is thought to initiate structural assembly of the core particle (Figure 3.40 panel B). In the core particle the disordered P3A N-terminal arms penetrate the inner space of the shell and might interact with P7 and/or nucleic acids in the core. In the complete particle the P8 T-trimer of the outer capsid binds more tightly than the other P8 trimers to P3 of the inner capsid thus acting as an anchor for binding of the other P8 trimers. The interaction is between positively charged patches on the inner surface of the T trimers with concentrated negative charges on the outer surface of P3 subunits located along the threefold axes of the core particles. This explains how the T = 13 icosahedron of the outer shell interrelates to the T = 3 icosahedron of the inner capsid. Based on the structures and interactions described above, Nakagawa et al., (2003) proposed a model for the assembly of RDV particles (Figure 3.40 panel B). Combining the results of the structures of RDV intact particles, empty particles lacking the 12 dsRNA segments and virus-like particles composed of the P3 core and P8 outer capsid generated with a baculovirus gene-expression system with those of biochemical analyses, Miyazaki et al. (2010) assigned proteins of the transcriptional machinery and dsRNA to clusters around the fivefold axes and along the radial concentric layers. A component of the transcriptional machinery, P7, was assigned to the outermost region of the density clusters implying an interaction between the dsRNA and P7 and explaining the spiral organization of dsRNA around the fivefold axis.
B. Fijiviruses Fijiviruses have 10 pieces of RNA in spherical particles 65–70 nm diameter. They resemble phytoreoviruses in having double-shelled particles but differ in having spikes (also called turrets) both on the outer shell (“A”-type spikes) and on the inner shell (“B”-type spikes). MRDV is one of the best characterized members of the Fijivirus genus (Milne and Lovisolo, 1977). Milne et al. (1973) detected 12 “A”-type spikes projecting from the surface of intact MRDV particles, one at each fivefold symmetry axis. These spikes are about 11 nm long.
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FIGURE 3.40 Structure and assembly of RDV. Panel (A) Subpanel (a) Cα-trace of the core structure. The icosahedrally independent molecules P3A and P3B are colored in light blue and pink, respectively. Notations of individual P3 proteins are also shown (the first capital letter denotes the icosahedrally independent molecules, A and B). Subpanel (b) Cα-trace of the outer shell of RDV. The icosahedral asymmetric unit contains 13 copies of P8 proteins, designated P (red), Q (orange), R (green), S (yellow), and T (blue). Panel (B) Proposed hierarchy for the assembly of RDV. On the basis of the strength of interactions between the various subunits the following sequence of events is proposed. (1) Insertion of the N-terminal arm of P3B into P3A initiates the assembly of a P3 dimer which (2) acts as a unit piece in the jigsaw puzzle. (3) A pentameric structure of dimers of P3 protein forms around an icosahedral fivefold axis, and then (4) this pentameric structure assembles (5) to form the core structure of the RDV particle. (6) The trimeric structure of P8 proteins acts as a unit piece of the assemblies and these trimers attach to the icosahedral threefold axis at the T-site first. Orientation of the T-trimer on the surface of the core at the icosahedral threefold axis is defined by electrostatic complementarities. (7) R-trimers then attach via interactions with the inner shell and with the T-trimers. (8) Q-trimers and s-trimers attach to the core surface and, at the final stage of viral assembly, (9) P-trimers attach at the icosahedral fivefold axes to form the complete virus particle. From Nakagawa et al. (2003) with permission of the publishers.
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Beneath each “A”-type spike is a “B”-type spike about 8 nm long, revealed when the outer coat is removed. The “B”-type spikes are associated with some differentiated structure in the inner core, which is termed a base plate. Detached “B”-type spikes can be seen to be made up of five morphological units surrounding a central hole. Cores without spikes (smooth cores) contain 136and 126-kDa polypeptides. The spiked cores contain, in addition, a 123-kDa polypeptide that is, therefore, probably located in the “B”-type spikes (Boccardo and Milne, 1975). None of the other polypeptides have been unequivocally associated with particular structures seen in negatively stained images of particles (Boccardo and Milne, 1975; Luisoni et al., 1975). FDV has a very similar structure to that of MRDV, as revealed by electron microscopy.
C. Oryzaviruses Members of the Oryzavirus genus have particle of 57–65 nm diameter that contain 10 pieces of dsRNA. The particle structure differs significantly from those of the other two plant reovirus genera in that oryzaviruses appear to lack an outer capsid and thus, to consist of the inner core. There are 12 “B”-type spikes, 8–10 nm in height, 23–26 nm wide at the base and 14–17 nm at the top (Figure 3.41 panel A). RRSV has an icosahedral capsid of approximately 700 Å in diameter, which consists of a polyhedral core particle of about 500 Å in diameter to which spikes of approximately 200 Å in diameter and 100 Å in height are attached. RRSV contains at least six structural proteins, namely P1, P2, P3, P4A (RdRp), P8B, and P9, with molecular weights of 138 K, 133 K, 131 K, 141 K, 42 K, and 39 K, respectively, that are encoded by 10 segments of the dsRNA genome. The capsid of RRSV, encapsidating 10 segments of dsRNA, appears to consist of at least four kinds of protein: P2, a capping enzyme; P3, a capsid shell protein; P8, a major capsid protein that is cleaved to yield P8A and P8B; and P9, a spike protein involved in transmission via the insect vector. The spike structures are pentameric each being surrounded by five peripheral trimers sitting at the Q position (Figure 3.40B) of the T = 13l (left) icosahedral symmetry; thus, there are 60 copies of the peripheral trimer (Miyazaki et al., 2008). This has led to a model for RRSV as compared to other reoviruses (Figure 3.41 panel B).
D. RNA Selection During Assembly of Plant Reoviruses Every WTV particle appears to contain one copy of each genome segment because (i) RNA isolated from virus has equimolar amounts of each segment (Reddy and Black, 1973) and (ii) an infection can be initiated by a single particle (Kimura and Black, 1972). Thus, there is a significant
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problem with this kind of virus. What are the macromolecular recognition signals that allow one, and one only, of each of 10 or 12 genome segments to appear in each particle during virus assembly? For example, the packaging of the 12 segments of WTV presumably involves 12 different and specific protein–RNA and/or RNA–RNA interactions. The first evidence that may be relevant to this problem comes from the work of Anzola et al. (1987) with WTV. They established the structure of a defective (DI) genomic segment 5, which was only one-fifth the length of the functional S5 RNA because of a large internal deletion. However, this DI RNA was packaged at one copy per particle, as for the normal sequence. Thus, they established that the sequence(s) involved in packaging must reside within 319 base pairs of the 5′ end of the plus strand and 205 from the 3′ end. Reddy and Black (1974, 1977) showed that an increase in the DI RNA content in a virus population led to a corresponding decrease in the molar proportion of the normal fragment, that is, the DI fragment competes only with its parent molecule. Thus, there must be two recognition signals, one that specifies a genome segment as viral rather than host, and the other that specifies each of the 12 segments. Anzola et al. (1987) suggested that a fully conserved hexanucleotide sequence at the 5′ terminus and a fully conserved tetranucleotide sequence at the 3′ terminus might form the recognition signals for viral as opposed to host RNA. They also found segment-specific inverted repeats of variable length just inside the conserved segments, indicating that the specific recognition sequence for each individual genome segment. A similar inverted repeat was found for segment 9 of RDV (Uyeda et al., 1989). As with animal reoviruses, other members of the plant reoviruses have conserved 5′ and 3′ sequences between each of the genomic RNAs. These similarities strengthen the idea that the 5′- and 3′-terminal sequences have a role in the packaging of these segmented RNA genomes but do not explain how 12 specific sites are constructed out of the three proteins known to be in the nucleoprotein viral core. RDV P7 core protein, P1 (polymerase) and P5 (guanyl transferase) bind specifically and with high affinity to all the 12 viral dsRNA segments and P7 forms complexes with P1 and P3 (the major core protein) suggesting that the core proteins interact as a unit (Zhong et al., 2004). This is in accord with the suggestion that reovirus dsRNA is formed within the nascent cores of developing virus particles, and that the dsRNA remains within these particles. It implies that the mechanism that leads to selection of a correct set of 12 or 10 genomic RNAs involves the ss plus strand. Thus, the base-paired inverted repeats could be the recognition signals. It may be that other virus-coded “scaffold” proteins transiently present in the developing core are involved in RNA recognition rather than, or as well as, the three proteins found in mature particles.
Chapter | 3 Architecture and Assembly of Virus Particles
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(A) (a)
270 Å
(b) 90 Å
720 Å
150 Å
245 Å
315 Å 290 Å
355 Å
(B)
FIGURE 3.41 Structure of plant reoviruses (structure of phytoreovirus shown in Figure 3.40). Panel (A) Structure of oryzavirus, RRSV. Subpanel (a) Surface representation of RRSV. Subpanel (b) Central cross-section (40 Å thick). The map is colored according to the distance from the center of the virus particle, for which color coding is indicated. Local structures are visible as follows: core capsid, yellow; clamp protein, green; long turret, blue structures located at the fivefold axes; and peripheral trimers, blue trimers located around turrets. Panel (B) Relationships among turreted reoviruses. Schematic representation of the virions of RRSV, cytoplasmic polyhedrosis virus (CPV), the Orthoreovirus core and the Orthoreovirus virion. The RdRp (red), capsid (yellow), clamp (green), trimeric outer capsid (sky blue), turret, (blue), and spike (blue-purple) proteins are shown. From Miyazaki et al. (2008) with permission of the publishers.
VII. ENVELOPED VIRUSES A. Rhabdoviridae Rhabdoviridae is a family of viruses whose members infect vertebrates, invertebrates, and plants (see Jackson et al., 2005 for review of plant rhabdoviruses). Most and perhaps all plant rhabdoviruses are rounded at both ends to give a bacilliform shape. Electron microscopy on thin
sections and negatively stained particles has been used to determine size and details of morphology (Figure 3.42). Particle size estimations range from 45 to 100 nm in width and 130–350 nm in length. These size measurements can be only approximate and are probably underestimates because of shrinkage taking place during dehydration for electron microscopy. Particles of these viruses readily deform and fragment in vitro unless pH and other
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(A) (a)
(b)
P protein
L protein
N protein plus RNA M protein
G protein
Individual unit of RNP – Protein
(B)
Matrix protein
Surface projections
Coil of RNP
Lipid layer
FIGURE 3.42 Structure of plant rhabdoviruses. Panel (A) Electron micrograph and diagram illustrating SYNV morphology. Subpanel (a) Transmission electron micrograph of a negatively stained virion showing the striated inner core, envelope and glycoprotein spikes. Subpanel (b) Depiction of the architecture of the virus particle. The nucleocapsid core is composed of the minus-sense genomic RNA, the nucleocapsid protein (N), the phosphoprotein (P), and the polymerase protein (L). The matrix protein is involved in coiling the nucleocapsid, attachment of the nucleocapsid core to the envelope, and associations with the G protein. The membrane lipids are host-derived and are interspersed with trimeric glycoprotein (G) spikes arranged as surface hexamers. The sc4 protein (not depicted) is believed to form a minor component of the envelope of purified SYNV particles. From Jackson et al. (2005) with permission of the publishers. Panel (B) Model for rhabdovirus structure showing the proposed 3D relationship between the three proteins and the lipid layer. From Cartwright et al. (1972) with permission of the publishers.
conditions are closely controlled (Francki and Randles, 1978). Rhabdoviruses have a complex structure being constructed on the basic plan shown in Figure 3.42 panel A subpanel b. They are constructed of three layers of
varying electron density. The outer layer contains spikelike projections composed of the glycoprotein (G) (for genome organization see Appendix A, Profiles 22 and 23) that extend 5–10 nm above the particle surface. The spikes appear to be arranged as surface hexamers and the
Chapter | 3 Architecture and Assembly of Virus Particles
G protein subunits are thought to associate as trimers. The middle layer of the particle consists of a host-derived lipid membrane penetrated by the G protein. This membrane surrounds an inner core made up of the M protein surrounding a helical ribonucleoprotein which is thought to consist of the N protein, the P protein, and the L (polymerase) protein. The detailed fine structure remains to be determined. In particular, it is not clear how the hexagonally arrayed M protein is related spatially to the helical RNP. Likewise the way the rounded ends are formed and the arrangement of RNP within them is not established. Cartwright et al. (1972) and Hull (1976a) suggested that the distribution of the G protein spikes is related to the structure of the M protein layer which is thought to be based on half icosahedral rounded ends as in other bacilliform particles. Orchid fleck virus (OFV) (an unclassified virus) has bacilliform particles containing a bipartite genome encoding six proteins (Kondo et al., 2006, 2009). The products from ORFs 1, 4, 5, and 6 have similarities to nuclear rhabdovirus N, M, G, and L proteins, respectively. However, OFV differs from rhabdoviruses in having a bipartite genome, and the virus particles being much smaller than those of rhabdoviruses and lacking a membrane. It is suggested that OFV particles resemble the rhabdovirus inner cores.
B. Tospoviruses 1. Tospovirus Structure TSWV is very unstable and difficult to purify (Joubert et al., 1974). Its structure has been studied in thin sections of infected cells, and in partially purified preparations. Isolated preparations contain many deformed and damaged particles. Tospovirus particles are spherical with a diameter of 80–110 nm and comprise a lipid envelope encompassing the genomic RNAs which are associated with the N protein as a nucleoprotein (RNP) complex. The viral polymerase is also contained within the particle. The lipid envelope contains two types of glycoproteins termed Gc (or G1 and Gn or G2 (Figure 3.43)
2. Tospovirus Assembly In plant cells, TSWV assembly involves enwrapment of a glycoproteins-containing Golgi stack around the RNPs, forming double-enveloped virus particles that become singly enveloped by fusion with other membranes and accumulate in large vesicles within the cytoplasm. The likely course of events is that the N protein interacts cooperatively with the viral RNA to form the RNP. The glycoproteins, primarily Gc, which retains in the ER then interacts with the N protein. Glycoprotein Gn has a cytoplasmic domain that effects Golgi localization and interaction
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FIGURE 3.43 Model showing structure of TSWV particle. The three nucleoprotein components are shown inside the particle with glycoprotein spikes (G1 and G2) extending through the lipid envelope. From Kormelink (2005) with permission of the publishers.
with Gc. It then “rescues” Gc and co-migrates with it to the Golgi where membrane envelopment of the Gc, Gn, and RNP complex occurs (Kainz et al., 2004; Snippe et al., 2007a,b; Ribeiro et al., 2009a,b).
VIII. DISCUSSION Packaging, or encapsidation, of the viral genome by structural protein components leading to the assembly of infectious progeny virions is an essential step in the infection cycle of viruses infecting plants and other organisms. A virion is a self-assembling macromolecular complex which gives a stable structure protecting the viral genome with the maximum genetic efficiency and requiring the minimum energy. The maximum genetic efficiency is effected by multiple copies of one or a few proteins formed to fit the basic shape of the particle (Reddy and Johnson, 2005). The two basic plant virus particle shapes are rods and isometric spheres; protein subunits of rod-shaped particles are essentially a 2D wedge (like a piece of cake) and those of isometric viruses are a 3D wedge, both of which are dictated by the folding in the secondary and tertiary structure of the polypeptide chain. The minimum energy requirement is given by quasi-equivalent interactions between the subunits and flexibility between domains within the subunits (Zandi et al., 2004). The flexibility is exemplified by “molecular switches” between the surface and internal structures which regulate quasi-equivalent interactions (also termed 3D domain swapping), different forms of which are shown for RYMV, CCMV, and TYMV (Spier et al., 1995; Canady et al., 1996; Qu et al., 2000).
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The concepts of the helical structure of rod-shaped viruses and the icosahedral structure of isometric viruses (Crick and Watson, 1956; Klug and Caspar, 1961) have contributed greatly to the understanding of virus structure. But more recent further application of mathematical theories to viral structures, such as the tiling theory for isometric viruses (Section IV, F) should encourage “thinking outside the box.” However, these new approaches also need to take into account differences in the surface and internal structures of isometric viruses which are of great importance to their dynamic functioning. The size of viral genome encapsidated depends upon the size of the virion. For rod-shaped viruses there is theoretically no limit to the size of the virion, but there must be some constraints, which have become apparent in the use of such viruses for producing recombinant proteins or nanotechnology (Chapter 15, Sections IV, B and C), and are currently not understood. For instance, why are most short rod-shaped particles rigid and most long particles flexuous? For isometric viruses there are constraints as discussed by Mannige and Brooks (2009). Often not considered in understanding virion shapes and sizes is that they are assembled in specific parts of the cellular environment which are poorly understood. As well as being stable and protecting the viral genome, virions must also be capable of contributing to the various stages of the virus infection cycle, e.g., vector transmission (Chapter 12), uncoating and expression of the genome (Chapter 6), in some cases replication of the genome (Chapter 7), positioning viral RNPs to the right place in the cell at the right time (Chapter 16), and moving the virus around the plant (Chapter 10). For the expression of the viral genome in a newly infected cell, the stable particle must disassemble in the cellular environment. Many of the stabilizing bonds, e.g., pH-dependent protein– protein bonds (Carillo-Tripp et al., 2008), divalent cation bonds, and protein–RNA bonds (Bink and Pleij, 2002) are amenable to relaxation under cellular conditions and the arrangement of the viral RNA within the particle can present the 5′ end to initiate initial translation. Movement between and within plants is often facilitated by surface features on the virion which, in many cases, interact with other viral nonstructural gene products and with host proteins and membranes. As described above, members of many groups of viruses each have the same basic virion structure but have variations which contribute to specific properties needed in their infection cycle. Assembly of viruses is a cooperative process usually initiated by a nucleation process (Zandi et al., 2006). This is taking place in cells in which there are many host RNAs. In most cases, virions contain just the viral nucleic acid which points to the importance of specificity of the nucleation process (Rao, 2006; Sun et al., 2010) and the close links between genome replication and virion assembly.
Plant Virology
Zlotnick et al. (2000) suggest that there are a limited number of common assembly processes for isometric viruses with capsid assembly being initiated by preformed polymers (e.g., dimers, pentamers, or hexamers) of subunits. This also pertains to rod-shaped viruses as described above. Advances in the capacity, speed, and availability of computers, the use of non-crystallographic symmetry averaging, and developments in computer graphics have allowed resolution down to atomic detail for the proteins and protein coats of several geometric plant viruses, both rodshaped and isometric. X-ray crystallographic analysis can, of course, be applied only to viruses or virus CPs that can be obtained in crystalline form. For larger viruses that have not been crystallized, other techniques, especially electron microscopy, remain important for studying virus architecture. However, as pointed out by Witz and Brown (2001) these techniques give a static image of what in reality is a dynamic macromolecule. Further understanding of the functioning of viral capsids in the infection cycle will come from merging a wide range of techniques both on the virions themselves (Endres and Zlotnick, 2002; Egelman, 2008; Morton et al., 2008; Zink and Grubműller, 2009) and using modification approaches such as mutagenesis.
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Chapter | 3 Architecture and Assembly of Virus Particles
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