- Email: [email protected]
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Virus assembly Lars Liljas Virus assembly is a term describing several areas of current research: protein–RNA recognition; the control of the formation of large complexes; and mechanisms of particle maturation. Our understanding of these processes is increasing as a result of the efforts of numerous studies. Crystal structures have recently been solved for relatively complex assembly intermediates. Addresses Department of Molecular Biology, Uppsala University, Box 590, 751 24 Uppsala, Sweden; e-mail: [email protected] Current Opinion in Structural Biology 1999, 9:129–134 http://biomednet.com/elecref/0959440X00900129
number of particles. The best characterized protein–nucleic acid recognition interaction is the binding of coat protein dimers of phage MS2 to a stem–loop in the single-stranded viral RNA [15]. In these small viruses, assembly is initiated by the formation of this complex. A complex of recombinant MS2 protein capsids with this stem–loop has been studied previously by X-ray crystallography and recent studies show interesting details concerning the determinants of the affinity and specificity of this complex [16•,17]. A number of recent studies have been carried out in order both to identify the packing signals in viral nucleic acids [18–23] and to determine which part of the protein is responsible for the specific recognition interaction [24,25•].
© Elsevier Science Ltd ISSN 0959-440X
Introduction Viruses form infectious virions in or when leaving their host cells. The assembly of a virus particle is, even for small, simple viruses, a relatively complex process that involves a large number of protein subunits interacting with each other and with the viral nucleic acid. For large viruses, the assembly process involves many components and proceeds in several steps. There are some steps in the assembly process that are common to all (or most) viruses, although the mechanisms might be widely different. The viral nucleic acid is specifically recognized in a way that avoids the packaging of unrelated nucleic acids. The size of the particle is regulated in some way, at least in nonhelical viruses with icosahedral symmetry. Finally, in many viruses, the particle undergoes rearrangements to make it suitably metastable, allowing the viral nucleic acid to be released upon infection. In this review, which covers results published in 1997 and 1998, some interesting results in these areas will be discussed. Structural studies of viruses have been reviewed recently [1], as has the assembly of specific groups of viruses [2–6]. A number of new crystal structures add to the already large database of detailed structural information on viral capsids and structural proteins [7•,8•,9–11,12••,13]. It is interesting to note that particles formed from the same protein molecules with indistinguishable capsid conformations might differ significantly in their properties, depending on their nucleic acid content [14•].
Nucleic acid recognition It is obvious that it will be advantageous if the majority of new virus particles contain viral nucleic acid molecule(s) rather than host nucleic acid. This is normally achieved through a specific interaction between the viral protein components and some sort of packing signal within the nucleic acids. In viruses with multiple segments of nucleic acids, there is the additional problem of ensuring that all genome segments are present in each particle or, at least, in a limited
Details concerning the conformation of the viral nucleic acid in icosahedral particles are unknown in most cases but, in a few cases, a significant part of the nucleic acid molecule follows the symmetry of the protein capsid. Since the base sequence does not follow this symmetry, only the interactions of the protein with the nucleic acid backbone can be studied [26•].
Pathways The coat proteins of many simple viruses can form virus-like particles when they are produced in a suitable expression system. These protein components seem to have all the properties that are needed for the formation of large particles. This is not true for more the complex viruses; for example, DNA phages with several types of proteins in their shells. These viruses follow assembly pathways that involve viral scaffolding proteins or other viral [27•] or host [28] components that are necessary for the formation of the virus particles, but that are not present in the mature virions. Mutations of various components involved in such pathways will block the completion of viral assembly and lead to the production of intermediates that can be identified. For simple viruses, it is difficult to define the assembly pathway, since after the initiation step, the process normally proceeds without the accumulation of intermediates. One approach is to try to estimate the energy of the various interactions in the final particle and, from that, try to find the most likely pathway by looking for energetically favored intermediates [29•]. This procedure has been used to find the assembly pathways of three viruses, two of which are icosahedral viruses with quasi-equivalent [30] packing of identical subunits. The third virus is rhinovirus, which has a shell composed of 60 copies of three distinct, but similar proteins. The procedure predicts a pathway for rhinovirus assembly that is supported by experimental evidence. In this case, there is no quasi-symmetry and the subunit–subunit interfaces are optimized for efficient, correct assembly, which is reflected in the calculated interaction energies.
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Figure 1
5
Apical domains
Carapace domain
2 Carapace domain
Dimerization domain
3
Schematic drawing [54] of the bluetongue virus VP3 (Protein Data Bank entry code 2btv). The two subunits making up the icosahedral asymmetric unit and the approximate positions of the icosahedral symmetry axes are shown. Each protein subunit has three domains. The central, large domain is labeled the carapace domain and contains many helices and a number of sheets. The other two domains are insertions into the sequence of the central domain; the apical domain, close to the fivefold axis, is mainly helical and the dimerization domain consists of two sheets and a number of helices.
Dimerization domain
Current Opinion in Structural Biology
The encapsidation of the viral genome in small icosahedral viruses seems to occur by the condensation of the protein coat around the nucleic acid molecule. In the complex phages, DNA is instead inserted into a preformed protein shell by a DNA packaging complex at one of the fivefold positions of the icosahedral head. In phage Φ29, this packaging involves a portal or connector complex, consisting of 12 subunits of the portal protein and an oligomer of the RNA molecule, the packaging RNA or pRNA [31]. Cryoelectron microscopy and image reconstruction of different forms of Φ29 have shown the general arrangement of the components [32••], but no high resolution structure is available yet. The connector complex has also been studied using atomic force microscopy [33•], revealing the 12-fold symmetry of the object.
Quasi-equivalence and switches Many icosahedral viruses have multiples of 60 identical protein subunits in their shells. The arrangement of these subunits usually follows the rules of quasi-equivalent packing that were suggested by Caspar and Klug [30] long before any high resolution virus structures were available. These rules predict that only certain multiples of 60 identical subunits will occur and these are denoted by the T number. With the first crystal structure determinations of quasi-equivalent viruses, it became clear that the problem of packing identical subunits in slightly different environments was solved by using segments of the polypeptide chain as position-sensitive switches. In some small plant
and insect RNA viruses with T = 3 quasi-symmetry, these switches are N-terminal segments that are ordered only in one of the three independent subunits, whereas in polyoma virus (T = 7), segments at the C terminus of the coat protein are either disordered or form different interactions depending on their position [34•]. There are other viruses in which the switching appears to be more subtle, involving small conformational changes that are regulated by the binding of metal ions and nucleic acids [3]. The observation of various types of switches in quasi-equivalent particles has led to the hypothesis that some sort of switching mechanism is required for correct virus assembly. In wildtype Flock House virus, an insect T = 3 virus, a segment of the coat protein, as well as a piece of ordered RNA, stabilize certain contact interfaces in a way that is similar to that observed in the plant viruses. In order to test this switching mechanism, the N-terminal segment involved in order/disorder switching was deleted, but particles with the same triangulation number were still produced [35•]. This suggests that the order/disorder switching mechanism is not required for correct assembly, although it might still be important and evolutionarily conserved because of its role in making the process efficient. Experience from studies of the small RNA phages indicates that a quasi-equivalent protein shell can be formed without any switching mechanisms. Such phages have a T = 3 capsid formed by 90 very similar coat protein dimers.
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The subunits have a conformation that is different from the β-sandwich topology found in most other simple icosahedral viruses. The only conformational difference between the subunits is found in a long loop forming fivefold and quasi-sixfold contacts. In a coat protein mutant in which a large portion of this loop is removed, the quasiequivalent packing is still the same [36•]. It appears that the protein packing in these viruses depends on the complementarity of the interacting surfaces of the dimers, rather than on any obvious switching mechanism [8•]. Bluetongue virus is a member of the Reoviridae family. The unique property of these viruses is that they have a core that functions as replicating machinery. The complete virion contains an outer layer of loosely bound protein molecules involved in attachment to the host cell. The core contains one copy of each of the approximately 10 viral double-stranded RNA molecules, which code for the viral proteins. The core also contains RNA polymerases, which produce the mRNA molecules used by the host cell to produce the viral proteins. Two layers of protein enclose the RNA and polymerase complex: one layer of 120 copies of viral protein 3 (VP3) and one layer of 780 copies of viral protein 7 (VP7). The crystal structure of bluetongue virus core has been determined at 3.5 Å resolution [12••]. This is the largest structure that has been solved by X-ray crystallography to date. Both the VP3 and the VP7 protein layers have icosahedral symmetry; the outer VP7 layer has a T = 13 arrangement, whereas the inner layer of 120 VP3 subunits does not follow the rules of the quasi-equivalent arrangement of subunits. The conformation of the VP3 subunit is shown in Figure 1. It has a long, thin triangular wedge shape and consists of three domains. Each of the three domains has a topology that is not observed in other proteins. Of the two independent subunits of VP3 in the icosahedral lattice, one forms the contacts at the fivefold and twofold axes. The other subunit shows a substantial difference in the relative orientation of one of its domains, and five copies of this VP3 subunit fit in between the five VP3 subunits forming the fivefold contact to form a decamer. Contacts between these decamers occur as twofold, quasi-twofold and threefold contacts. The contacts between the two independent VP3 subunits are not quasi-equivalent, but the elongated shape and the flexibility of the protein allow it to occupy roughly two halves of the repeating unit of the icosahedron. The VP7 layer has T = 13 symmetry and is arranged as 260 trimers — four and a third trimers in each icosahedral asymmetric unit. There are no extended arms acting as switches to regulate the packing of the trimers. Instead, the conformations of the trimers are very similar, as are the contacts between them. The structure of the VP7 layer therefore corresponds closely to the quasi-equivalent packing of subunits predicted by Caspar and Klug [30]. This is possible because the contacts occur only in a thin layer consisting of hydrophobic sidechains. The number of subunits and the curvature of the VP7 layer are determined by the size of the
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inner layer of VP3 subunits, which explains the lack of controlling switches in VP7. The contacts between the VP7 trimers and the VP3 layer are all different and suggest the ordered binding of trimers to the inner layer.
Scaffolding proteins The crystal structure of the viral procapsid of the icosahedral bacteriophage ΦX-174 [37••] gives some insight into the mechanisms involving scaffolding proteins. This virus belongs to the Microviridae family. The mature particle consists of four types of protein (F, G, H and J), of which F is the major capsid protein, present in 60 copies. The G protein is a spike protein, also present in 60 copies. The position of the 12 copies of the H protein is unknown, whereas the J protein is associated with the single-stranded viral DNA molecule. The assembly of this particle proceeds via a prohead consisting of proteins F and G, together with two scaffolding proteins, B and D, in 60 and 240 copies, respectively. The DNA molecule enters the prohead together with the J protein, while the B scaffolding protein leaves. Finally, the D scaffolding protein is released to produce the mature capsid. Structural studies of the assembly intermediates are complicated because of their metastable nature. In the current study [37••], the procapsid alters its conformation upon crystallization, closing the holes at the threefold axes that were observed in electron micrographs of the procapsids. The DNA molecule probably enters through one of these holes and the closed procapsid might therefore not represent a true intermediate of assembly. The crystal structure still shows the conformation and arrangement of the D scaffolding protein. The protein is mainly helical and the four D subunits in the icosahedral asymmetric unit differ significantly in conformation and are arranged in a surprisingly asymmetric manner, which probably reflects the fact that four D protein molecules (two dimers) bind differently on the surface of a single F protein. Pentamers of F and G proteins can form, but the twofold contacts between F proteins in the pentamers are weak (Figure 2). The function of the D protein seems to be that two D dimers bind to each F subunit on the surface of the pentamers and interact with two D dimers bound to another pentamer in order to allow the formation of the correct twofold contact. The B protein is located on the inside of the shell, but is mainly disordered. The scaffolding protein of the much larger phage P22 has been shown to influence the capsid size. A mixture of particles with T numbers 4 and 7 are produced in the absence of the scaffolding protein [38•], while almost only T = 7 particles are formed in the presence of the scaffolding protein. In this and other large phages, the scaffolding protein forms an inner scaffold, which is released when the DNA molecule enters the particle through the portal complex. This scaffold does not appear to have icosahedral symmetry. The conformation of the P22 scaffolding protein has been studied by spectroscopic methods [39–41], but no high resolution structure or strong evidence of a mechanism of action are available.
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Figure 2
Schematic drawing [54] of bacteriophage ΦX-174 protein subunits (Protein Data Bank entry code 1al0). (a) Arrangement of the F and G subunits of the prohead. The view is down the twofold axis and includes two icosahedral asymmetric units. The approximate positions
of the two-, three- and fivefold axes are indicated. (b) The same view showing the four dimers of the D scaffolding protein related by the twofold axis. The F and G subunits below the scaffolding proteins are shown in light gray.
Capsid maturation
the membrane, while the nucleocapsid and capsid proteins, together with the RNA, form an unusual conical structure. The mechanisms of this drastic rearrangement are not known and, although the structures of most of the domains of the Gag polyprotein are known, their method of association before and after the maturation step is not known. It has been suggested that proteolytic cleavage leads to significant conformational changes and the creation of new interfaces in the capsid protein, which in turn leads to association into the conical structure [49•,50].
After the assembly of the components of the capsids, they normally undergo some sort of maturation process. The reason for this is that the virus particle has to be suitably stable for the safe transport of the nucleic acid to a new host cell, but it might also have to have properties that allow the release of the nucleic acid at the right moment. The maturation process often involves the proteolytic cleavage of protein components [42•], but the binding of metal ions [9,43,44] or other compounds [45] and the formation of disulfide bonds [46–48] or other covalent links [4] are also observed.
Conclusions An unusual and striking form of maturation occurs in retroviruses. In HIV-1, the Gag polyprotein is initially associated with the membrane envelope. The Gag polyprotein is then cleaved into a number of components. One of them, the matrix protein, remains associated with
Although the conserved topology found in the coat proteins of many viruses is evidence of a common ancestry, the details of the mechanisms used in the various steps in the viral life cycle are often very different. In small icosahedral viruses, the steps in the assembly process are still poorly
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understood in spite of the detailed knowledge of the conformation of the mature particles. More is known about the assembly pathways of complex bacteriophages, but there are still no high resolution structures of such particles and therefore no detailed mechanisms of the individual steps. The crystal structure of the first member of the Reoviridae family shows the details of a very interesting protein arrangement. It appears that we are relatively close to the size limit for X-ray crystallograpic studies of virus particles, at least with existing methods, but for larger objects or more unstable intermediates, the improvements in cryoelectron microscopy are promising [32••,51–53].
References and recommended reading
14. Bothner B, Schneemann A, Marshall D, Reddy V, Johnson JE, • Siuzdak G: Crystallographically identical virus capsids display different properties in solution. Nat Struct Biol 1999, in press. Crystal structures of intact Flock House virus virions and virus-like particles formed by coat proteins from an expression system show no significant differences. This paper shows that such particles have widely different properties regarding susceptibility to proteases, which demonstrates that the crystal structures might have little relevance for understanding the flexibility of viral capsids. 15. Witherell GW, Gott JM, Uhlenbeck OC: Specific interaction between RNA phage coat proteins and RNA. Prog Nucleic Acid Res Mol Biol 1991, 40:185-220. 16. Valegård K, Murray JB, Stonehouse NJ, van den Worm S, • Stockley PG, Liljas L: The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions. J Mol Biol 1997, 270:724-738. The crystal structures of two complexes between protein capsids and specifically bound RNA stem–loops show that the higher binding affinity is due to an internal hydrogen bond within the RNA molecule. 17.
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Chandrasekar V, Johnson JE: The structure of tobacco ringspot virus: a link in the evolution of icosahedral capsids in the picornavirus superfamily. Structure 1998, 6:157-171. This paper describes the crystal structure of a representative of a new group of plant viruses that are related to comoviruses and belong to the picornavirus family. The 60 coat protein molecules each have three β-sandwichtype domains connected by linker segments. The similar coat protein molecules of picornaviruses, like poliovirus, are synthesized as polyproteins, which are cleaved by a viral protease before assembly, but in the tobacco ringspot virus, no cleavage occurs. 8. •
Tars K, Bundule M, Fridborg K, Liljas L: The crystal structure of bacteriophage GA and a comparison of bacteriophages belonging to the major groups of Escherichia coli leviviruses. J Mol Biol 1997, 271:759-773. The authors report the structure of an RNA phage and compare related phage structures in a search for the features that determine subunit packing in these viruses. 9.
Lentz KN, Smith AD, Geisler SC, Cox S, Buontempo P, Skelton A, DeMartino J, Rozhon E, Schwartz J, Girijavallabhan V et al.: Structure of poliovirus type 2 Lansing complexed with antiviral agent SCH48973: comparison of the structural and biological properties of three poliovirus serotypes. Structure 1997, 5:961-978.
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van den Worm S, Stonehouse NJ, Valegård K, Murray JB, Walton C, Stockley PG, Liljas L: Crystal structures of MS2 coat protein mutant in complex with wild-type RNA operator fragments. Nucleic Acids Res 1998, 26:1345-1351.
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10. Chen XS, Stehle T, Harrison SC: Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry. EMBO J 1998, 17:3233-3240.
26. Larson SB, Day J, Greenwood A, McPherson A: Refined structure of • satellite tobacco mosaic virus at 1.8 Å resolution. J Mol Biol 1998, 277:37-59. The 1.8 Å resolution structure of satellite tobacco mosaic virus, a small T = 1 virus, shows the arrangement of a large proportion of the viral RNA as double-stranded segments interacting with the protein at all equivalent positions of the icosahedral capsid.
11. Choi HK, Lu G, Lee S, Wengler G, Rossmann MG: Structure of Semliki Forest virus core protein. Proteins 1997, 27:345-359.
27. •
12. Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, Zientara S, •• Mertens PP, Stuart DI: The atomic structure of the bluetongue virus core. Nature 1998, 395:470-478. The crystal structure at 3.5 Å resolution of bluetongue virus cores. The core structure comprises the inner two protein layers of the three layers in the intact virions. This is the largest object (the protein shell has 900 protein subunits with a total molecular weight of 42 MDa) to have been solved to a resolution allowing an atomic model to be built. 13. Tao Y, Strelkov SV, Mesyanzhinov VV, Rossmann MG: Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain. Structure 1997, 5:789-798.
Hunt JF, van der Vies SM, Henry L, Deisenhofer J: Structural adaptations in the specialized bacteriophage T4 cochaperonin gp31 expand the size of the Anfinsen cage. Cell 1997, 90:361-371. The folding of the capsid protein of bacteriophage T4 requires the presence of the gp31 protein, which replaces bacterial GroES in the complex with chaperonin GroEL. The crystal structure shows that gp31 is similar to GroES, but the gp31–GroEL complex can accommodate the T4 coat protein, which is larger than the proteins that are folded with the assistance of the GroEL–GroES bacterial complex. 28. Hu J, Toft DO, Seeger C: Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. EMBO J 1997, 16:59-68.
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29. Reddy VS, Giesing HA, Morton RT, Kumar A, Post CB, Brooks CL III, • Johnson JE: Energetics of quasiequivalence: computational analysis of protein–protein interactions in icosahedral viruses. Biophys J 1998, 74:546-558. Association energies calculated using CHARMM were used to find likely assembly pathways for the black beetle virus, southern bean mosaic virus and human rhinovirus 14. The contributions of individual residues were calculated, allowing the possibility of testing the prediction by mutagenesis. 30. Caspar DLD, Klug A: Physical principles in the construction of regular viruses. Cold Spring Harb Symp Quant Biol 1962, 27:1-24. 31. Hendrix RW: Bacteriophage DNA packaging: RNA gears in a DNA transport machine. Cell 1998, 94:147-150. 32. Tao Y, Olson NH, Xu W, Anderson DL, Rossmann MG, Baker TS: •• Assembly of a tailed bacterial virus and its genome release studied in three dimensions. Cell 1998, 95:431-437. Three-dimensional reconstructions of phage F29 from electron micrographs of isometric heads with icosahedral symmetry, and prolate heads and complete virions with lower symmetry. 33. Müller DJ, Engel A, Carrascosa JL, Velez M: The bacteriophage F29 • head-tail connector imaged at high resolution with the atomic force microscope in buffer solution. EMBO J 1997, 16:2547-2553. In this paper, the new atomic force microscopy technique is applied to the study of viral components. Two-dimensional crystals of phage Φ29 connectors are shown to possess the expected 12-fold symmetry. 34. Stehle T, Harrison SC: High-resolution structure of a polyomavirus • VP1-oligosaccharide complex: implications for assembly and receptor binding. EMBO J 1997, 16:5139-5148. The crystal structure of polyomavirus VP1 pentamers shows the arrangement of the segments involved in the switching mechanism when the viral capsids form. 35. Dong XF, Natarajan P, Tihova M, Johnson JE, Schneemann A: Particle • polymorphism caused by deletion of a peptide molecular switch in a quasiequivalent icosahedral virus. J Virol 1998, 72:6024-6033. The removal of a peptide segment that is ordered in the ‘flat’ subunit contact leads to the production of normal T = 3 particles (and irregular structures), indicating that this segment is not required for correct subunit packing. 36. Axblom C, Tars K, Fridborg K, Orna L, Bundule M, Liljas L: Structure • of phage fr capsids with a deletion in the FG loop: implications for viral assembly. Virology 1998, 249:80-88. The small (T = 3) RNA phages have no obvious order/disorder switches to control their assembly. A loop forming contacts at fivefold and quasi-sixfold axes has widely different conformations that are dependent upon its position. This paper describes the crystal structure of a mutant of a small RNA phage, in which a significant portion of this loop has been removed without any effects on the subunit packing, suggesting that this loop is not crucial for assembly. 37. ••
Dokland T, McKenna R, Ilag LL, Bowman BR, Incardona NL, Fane BA, Rossmann MG: Structure of a viral procapsid with molecular scaffolding. Nature 1997, 389:308-313. The crystal structure of a procapsid-like particle of the DNA bacteriophage ΦX-174 showing, for the first time, the conformation and arrangement of scaffolding proteins in atomic detail.
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40. Parker MH, Stafford WF III, Prevelige PE Jr: Bacteriophage P22 scaffolding protein forms oligomers in solution. J Mol Biol 1997, 268:655-665. 41. Parker MH, Casjens S, Prevelige PE Jr: Functional domains of bacteriophage P22 scaffolding protein. J Mol Biol 1998, 281:69-79. 42. Curry S, Fry E, Blakemore W, Abu-Ghazaleh R, Jackson T, King A, • Lea S, Newman J, Stuart D: Dissecting the roles of VP0 cleavage and RNA packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus. J Virol 1997, 71:9743-9752. Crystal structures of empty capsids of a foot-and-mouth disease virus strain suggest that the mechanism leading to the cleavage of VP0 into VP4 and VP2 is similar to that suggested for poliovirus. 43. Zhao R, Hadfield AT, Kremer MJ, Rossmann MG: Cations in human rhinoviruses. Virology 1997, 227:13-23. 44. Hadfield AT, Lee W, Zhao R, Oliveira MA, Minor I, Rueckert RR, Rossmann MG: The refined structure of human rhinovirus 16 at 2.15 Å resolution: implications for the viral life cycle. Structure 1997, 5:427-441. 45. Lewis JK, Bothner B, Smith TJ, Siuzdak G: Antiviral agent blocks breathing of the common cold virus. Proc Natl Acad Sci USA 1998, 95:6774-6778. 46. Li M, Beard P, Estes PA, Lyon MK, Garcea RL: Intercapsomeric disulfide bonds in papillomavirus assembly and disassembly. J Virol 1998, 72:2160-2167. 47.
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Virus assembly Lars Liljas Virus assembly is a term describing several areas of current research: protein–RNA recognition; the control of the formation of large complexes; and mechanisms of particle maturation. Our understanding of these processes is increasing as a result of the efforts of numerous studies. Crystal structures have recently been solved for relatively complex assembly intermediates. Addresses Department of Molecular Biology, Uppsala University, Box 590, 751 24 Uppsala, Sweden; e-mail: [email protected] Current Opinion in Structural Biology 1999, 9:129–134 http://biomednet.com/elecref/0959440X00900129
number of particles. The best characterized protein–nucleic acid recognition interaction is the binding of coat protein dimers of phage MS2 to a stem–loop in the single-stranded viral RNA [15]. In these small viruses, assembly is initiated by the formation of this complex. A complex of recombinant MS2 protein capsids with this stem–loop has been studied previously by X-ray crystallography and recent studies show interesting details concerning the determinants of the affinity and specificity of this complex [16•,17]. A number of recent studies have been carried out in order both to identify the packing signals in viral nucleic acids [18–23] and to determine which part of the protein is responsible for the specific recognition interaction [24,25•].
© Elsevier Science Ltd ISSN 0959-440X
Introduction Viruses form infectious virions in or when leaving their host cells. The assembly of a virus particle is, even for small, simple viruses, a relatively complex process that involves a large number of protein subunits interacting with each other and with the viral nucleic acid. For large viruses, the assembly process involves many components and proceeds in several steps. There are some steps in the assembly process that are common to all (or most) viruses, although the mechanisms might be widely different. The viral nucleic acid is specifically recognized in a way that avoids the packaging of unrelated nucleic acids. The size of the particle is regulated in some way, at least in nonhelical viruses with icosahedral symmetry. Finally, in many viruses, the particle undergoes rearrangements to make it suitably metastable, allowing the viral nucleic acid to be released upon infection. In this review, which covers results published in 1997 and 1998, some interesting results in these areas will be discussed. Structural studies of viruses have been reviewed recently [1], as has the assembly of specific groups of viruses [2–6]. A number of new crystal structures add to the already large database of detailed structural information on viral capsids and structural proteins [7•,8•,9–11,12••,13]. It is interesting to note that particles formed from the same protein molecules with indistinguishable capsid conformations might differ significantly in their properties, depending on their nucleic acid content [14•].
Nucleic acid recognition It is obvious that it will be advantageous if the majority of new virus particles contain viral nucleic acid molecule(s) rather than host nucleic acid. This is normally achieved through a specific interaction between the viral protein components and some sort of packing signal within the nucleic acids. In viruses with multiple segments of nucleic acids, there is the additional problem of ensuring that all genome segments are present in each particle or, at least, in a limited
Details concerning the conformation of the viral nucleic acid in icosahedral particles are unknown in most cases but, in a few cases, a significant part of the nucleic acid molecule follows the symmetry of the protein capsid. Since the base sequence does not follow this symmetry, only the interactions of the protein with the nucleic acid backbone can be studied [26•].
Pathways The coat proteins of many simple viruses can form virus-like particles when they are produced in a suitable expression system. These protein components seem to have all the properties that are needed for the formation of large particles. This is not true for more the complex viruses; for example, DNA phages with several types of proteins in their shells. These viruses follow assembly pathways that involve viral scaffolding proteins or other viral [27•] or host [28] components that are necessary for the formation of the virus particles, but that are not present in the mature virions. Mutations of various components involved in such pathways will block the completion of viral assembly and lead to the production of intermediates that can be identified. For simple viruses, it is difficult to define the assembly pathway, since after the initiation step, the process normally proceeds without the accumulation of intermediates. One approach is to try to estimate the energy of the various interactions in the final particle and, from that, try to find the most likely pathway by looking for energetically favored intermediates [29•]. This procedure has been used to find the assembly pathways of three viruses, two of which are icosahedral viruses with quasi-equivalent [30] packing of identical subunits. The third virus is rhinovirus, which has a shell composed of 60 copies of three distinct, but similar proteins. The procedure predicts a pathway for rhinovirus assembly that is supported by experimental evidence. In this case, there is no quasi-symmetry and the subunit–subunit interfaces are optimized for efficient, correct assembly, which is reflected in the calculated interaction energies.
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Figure 1
5
Apical domains
Carapace domain
2 Carapace domain
Dimerization domain
3
Schematic drawing [54] of the bluetongue virus VP3 (Protein Data Bank entry code 2btv). The two subunits making up the icosahedral asymmetric unit and the approximate positions of the icosahedral symmetry axes are shown. Each protein subunit has three domains. The central, large domain is labeled the carapace domain and contains many helices and a number of sheets. The other two domains are insertions into the sequence of the central domain; the apical domain, close to the fivefold axis, is mainly helical and the dimerization domain consists of two sheets and a number of helices.
Dimerization domain
Current Opinion in Structural Biology
The encapsidation of the viral genome in small icosahedral viruses seems to occur by the condensation of the protein coat around the nucleic acid molecule. In the complex phages, DNA is instead inserted into a preformed protein shell by a DNA packaging complex at one of the fivefold positions of the icosahedral head. In phage Φ29, this packaging involves a portal or connector complex, consisting of 12 subunits of the portal protein and an oligomer of the RNA molecule, the packaging RNA or pRNA [31]. Cryoelectron microscopy and image reconstruction of different forms of Φ29 have shown the general arrangement of the components [32••], but no high resolution structure is available yet. The connector complex has also been studied using atomic force microscopy [33•], revealing the 12-fold symmetry of the object.
Quasi-equivalence and switches Many icosahedral viruses have multiples of 60 identical protein subunits in their shells. The arrangement of these subunits usually follows the rules of quasi-equivalent packing that were suggested by Caspar and Klug [30] long before any high resolution virus structures were available. These rules predict that only certain multiples of 60 identical subunits will occur and these are denoted by the T number. With the first crystal structure determinations of quasi-equivalent viruses, it became clear that the problem of packing identical subunits in slightly different environments was solved by using segments of the polypeptide chain as position-sensitive switches. In some small plant
and insect RNA viruses with T = 3 quasi-symmetry, these switches are N-terminal segments that are ordered only in one of the three independent subunits, whereas in polyoma virus (T = 7), segments at the C terminus of the coat protein are either disordered or form different interactions depending on their position [34•]. There are other viruses in which the switching appears to be more subtle, involving small conformational changes that are regulated by the binding of metal ions and nucleic acids [3]. The observation of various types of switches in quasi-equivalent particles has led to the hypothesis that some sort of switching mechanism is required for correct virus assembly. In wildtype Flock House virus, an insect T = 3 virus, a segment of the coat protein, as well as a piece of ordered RNA, stabilize certain contact interfaces in a way that is similar to that observed in the plant viruses. In order to test this switching mechanism, the N-terminal segment involved in order/disorder switching was deleted, but particles with the same triangulation number were still produced [35•]. This suggests that the order/disorder switching mechanism is not required for correct assembly, although it might still be important and evolutionarily conserved because of its role in making the process efficient. Experience from studies of the small RNA phages indicates that a quasi-equivalent protein shell can be formed without any switching mechanisms. Such phages have a T = 3 capsid formed by 90 very similar coat protein dimers.
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The subunits have a conformation that is different from the β-sandwich topology found in most other simple icosahedral viruses. The only conformational difference between the subunits is found in a long loop forming fivefold and quasi-sixfold contacts. In a coat protein mutant in which a large portion of this loop is removed, the quasiequivalent packing is still the same [36•]. It appears that the protein packing in these viruses depends on the complementarity of the interacting surfaces of the dimers, rather than on any obvious switching mechanism [8•]. Bluetongue virus is a member of the Reoviridae family. The unique property of these viruses is that they have a core that functions as replicating machinery. The complete virion contains an outer layer of loosely bound protein molecules involved in attachment to the host cell. The core contains one copy of each of the approximately 10 viral double-stranded RNA molecules, which code for the viral proteins. The core also contains RNA polymerases, which produce the mRNA molecules used by the host cell to produce the viral proteins. Two layers of protein enclose the RNA and polymerase complex: one layer of 120 copies of viral protein 3 (VP3) and one layer of 780 copies of viral protein 7 (VP7). The crystal structure of bluetongue virus core has been determined at 3.5 Å resolution [12••]. This is the largest structure that has been solved by X-ray crystallography to date. Both the VP3 and the VP7 protein layers have icosahedral symmetry; the outer VP7 layer has a T = 13 arrangement, whereas the inner layer of 120 VP3 subunits does not follow the rules of the quasi-equivalent arrangement of subunits. The conformation of the VP3 subunit is shown in Figure 1. It has a long, thin triangular wedge shape and consists of three domains. Each of the three domains has a topology that is not observed in other proteins. Of the two independent subunits of VP3 in the icosahedral lattice, one forms the contacts at the fivefold and twofold axes. The other subunit shows a substantial difference in the relative orientation of one of its domains, and five copies of this VP3 subunit fit in between the five VP3 subunits forming the fivefold contact to form a decamer. Contacts between these decamers occur as twofold, quasi-twofold and threefold contacts. The contacts between the two independent VP3 subunits are not quasi-equivalent, but the elongated shape and the flexibility of the protein allow it to occupy roughly two halves of the repeating unit of the icosahedron. The VP7 layer has T = 13 symmetry and is arranged as 260 trimers — four and a third trimers in each icosahedral asymmetric unit. There are no extended arms acting as switches to regulate the packing of the trimers. Instead, the conformations of the trimers are very similar, as are the contacts between them. The structure of the VP7 layer therefore corresponds closely to the quasi-equivalent packing of subunits predicted by Caspar and Klug [30]. This is possible because the contacts occur only in a thin layer consisting of hydrophobic sidechains. The number of subunits and the curvature of the VP7 layer are determined by the size of the
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inner layer of VP3 subunits, which explains the lack of controlling switches in VP7. The contacts between the VP7 trimers and the VP3 layer are all different and suggest the ordered binding of trimers to the inner layer.
Scaffolding proteins The crystal structure of the viral procapsid of the icosahedral bacteriophage ΦX-174 [37••] gives some insight into the mechanisms involving scaffolding proteins. This virus belongs to the Microviridae family. The mature particle consists of four types of protein (F, G, H and J), of which F is the major capsid protein, present in 60 copies. The G protein is a spike protein, also present in 60 copies. The position of the 12 copies of the H protein is unknown, whereas the J protein is associated with the single-stranded viral DNA molecule. The assembly of this particle proceeds via a prohead consisting of proteins F and G, together with two scaffolding proteins, B and D, in 60 and 240 copies, respectively. The DNA molecule enters the prohead together with the J protein, while the B scaffolding protein leaves. Finally, the D scaffolding protein is released to produce the mature capsid. Structural studies of the assembly intermediates are complicated because of their metastable nature. In the current study [37••], the procapsid alters its conformation upon crystallization, closing the holes at the threefold axes that were observed in electron micrographs of the procapsids. The DNA molecule probably enters through one of these holes and the closed procapsid might therefore not represent a true intermediate of assembly. The crystal structure still shows the conformation and arrangement of the D scaffolding protein. The protein is mainly helical and the four D subunits in the icosahedral asymmetric unit differ significantly in conformation and are arranged in a surprisingly asymmetric manner, which probably reflects the fact that four D protein molecules (two dimers) bind differently on the surface of a single F protein. Pentamers of F and G proteins can form, but the twofold contacts between F proteins in the pentamers are weak (Figure 2). The function of the D protein seems to be that two D dimers bind to each F subunit on the surface of the pentamers and interact with two D dimers bound to another pentamer in order to allow the formation of the correct twofold contact. The B protein is located on the inside of the shell, but is mainly disordered. The scaffolding protein of the much larger phage P22 has been shown to influence the capsid size. A mixture of particles with T numbers 4 and 7 are produced in the absence of the scaffolding protein [38•], while almost only T = 7 particles are formed in the presence of the scaffolding protein. In this and other large phages, the scaffolding protein forms an inner scaffold, which is released when the DNA molecule enters the particle through the portal complex. This scaffold does not appear to have icosahedral symmetry. The conformation of the P22 scaffolding protein has been studied by spectroscopic methods [39–41], but no high resolution structure or strong evidence of a mechanism of action are available.
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Figure 2
Schematic drawing [54] of bacteriophage ΦX-174 protein subunits (Protein Data Bank entry code 1al0). (a) Arrangement of the F and G subunits of the prohead. The view is down the twofold axis and includes two icosahedral asymmetric units. The approximate positions
of the two-, three- and fivefold axes are indicated. (b) The same view showing the four dimers of the D scaffolding protein related by the twofold axis. The F and G subunits below the scaffolding proteins are shown in light gray.
Capsid maturation
the membrane, while the nucleocapsid and capsid proteins, together with the RNA, form an unusual conical structure. The mechanisms of this drastic rearrangement are not known and, although the structures of most of the domains of the Gag polyprotein are known, their method of association before and after the maturation step is not known. It has been suggested that proteolytic cleavage leads to significant conformational changes and the creation of new interfaces in the capsid protein, which in turn leads to association into the conical structure [49•,50].
After the assembly of the components of the capsids, they normally undergo some sort of maturation process. The reason for this is that the virus particle has to be suitably stable for the safe transport of the nucleic acid to a new host cell, but it might also have to have properties that allow the release of the nucleic acid at the right moment. The maturation process often involves the proteolytic cleavage of protein components [42•], but the binding of metal ions [9,43,44] or other compounds [45] and the formation of disulfide bonds [46–48] or other covalent links [4] are also observed.
Conclusions An unusual and striking form of maturation occurs in retroviruses. In HIV-1, the Gag polyprotein is initially associated with the membrane envelope. The Gag polyprotein is then cleaved into a number of components. One of them, the matrix protein, remains associated with
Although the conserved topology found in the coat proteins of many viruses is evidence of a common ancestry, the details of the mechanisms used in the various steps in the viral life cycle are often very different. In small icosahedral viruses, the steps in the assembly process are still poorly
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understood in spite of the detailed knowledge of the conformation of the mature particles. More is known about the assembly pathways of complex bacteriophages, but there are still no high resolution structures of such particles and therefore no detailed mechanisms of the individual steps. The crystal structure of the first member of the Reoviridae family shows the details of a very interesting protein arrangement. It appears that we are relatively close to the size limit for X-ray crystallograpic studies of virus particles, at least with existing methods, but for larger objects or more unstable intermediates, the improvements in cryoelectron microscopy are promising [32••,51–53].
References and recommended reading
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Tars K, Bundule M, Fridborg K, Liljas L: The crystal structure of bacteriophage GA and a comparison of bacteriophages belonging to the major groups of Escherichia coli leviviruses. J Mol Biol 1997, 271:759-773. The authors report the structure of an RNA phage and compare related phage structures in a search for the features that determine subunit packing in these viruses. 9.
Lentz KN, Smith AD, Geisler SC, Cox S, Buontempo P, Skelton A, DeMartino J, Rozhon E, Schwartz J, Girijavallabhan V et al.: Structure of poliovirus type 2 Lansing complexed with antiviral agent SCH48973: comparison of the structural and biological properties of three poliovirus serotypes. Structure 1997, 5:961-978.
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van den Worm S, Stonehouse NJ, Valegård K, Murray JB, Walton C, Stockley PG, Liljas L: Crystal structures of MS2 coat protein mutant in complex with wild-type RNA operator fragments. Nucleic Acids Res 1998, 26:1345-1351.
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10. Chen XS, Stehle T, Harrison SC: Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry. EMBO J 1998, 17:3233-3240.
26. Larson SB, Day J, Greenwood A, McPherson A: Refined structure of • satellite tobacco mosaic virus at 1.8 Å resolution. J Mol Biol 1998, 277:37-59. The 1.8 Å resolution structure of satellite tobacco mosaic virus, a small T = 1 virus, shows the arrangement of a large proportion of the viral RNA as double-stranded segments interacting with the protein at all equivalent positions of the icosahedral capsid.
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27. •
12. Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, Zientara S, •• Mertens PP, Stuart DI: The atomic structure of the bluetongue virus core. Nature 1998, 395:470-478. The crystal structure at 3.5 Å resolution of bluetongue virus cores. The core structure comprises the inner two protein layers of the three layers in the intact virions. This is the largest object (the protein shell has 900 protein subunits with a total molecular weight of 42 MDa) to have been solved to a resolution allowing an atomic model to be built. 13. Tao Y, Strelkov SV, Mesyanzhinov VV, Rossmann MG: Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain. Structure 1997, 5:789-798.
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