The structure of the adenovirus capsid

J. Mol. Biol. (1985) 185, 125-143

The Structure of the Adenovirus

Capsid

IIT. The Packing Symmetry of Hexon and its Implications for Viral Architecture Roger M. Burnett Department of Biochemistry and Molecular Biophysics College of Physicians and Surgeons Columbia University New York, N.Y. 10032, U.S.A. (Received 4 January

1985)

The orientation and location of the 240 hexons comprising the outer protein shell of adenovirus have been determined. Electron micrographs of the capsid and its fragments were inspected for the features of hexon known from the X-ray crystallographic model as described in the accompanying paper. A capsid model is proposed with each facet comprising a small p3 net of 12 hexons, arranged as a triangular sextet with three outer hexon pairs. The sextet is centrally placed about the icosahedral threefold axis, with its edges parallel to those of the facet. The outer pairs project over the facet edges on one side of the icosahedral twofold axes at each edge. The model capsid is defined by the underlying icosahedron, of edge 445 A, upon which hexons are arranged. The hexons are thus bounded by icosahedra with insphere radii of 336 A and 452 A. A quartet of hexons forms the asymmetric unit of an icosahedral hexon shell, which can be closed by the addition of pentons at the 12 vertices. Considering the hexon trimer as a complex structure unit, its interactions in the four topologically distinct environments are very similar, with conservation of at least two-thirds of the inter-hexon bonding. The crystal-like construction explains the flat facets and sharp edges characteristic of adenovirus. Larger “adenovirus-like” capsids of any size could be formed using only one additional topologically different environment. The construction of adenovirus illustrates how an impenetrable protein shell can be formed, with highly conserved intermolecular bonding, by using the geometry of an oligomeric structure unit and symmetry additional to that of the icosahedral point group. This contrasts with the manner suggested by Caspar & Klug (1962), in which the polypeptide is the structure unit, and for which the number of possible bonding configurations required of a structure unit tends to infinity as the continuously curved capsid increases in size. The known structures of poiyoma and the plant viruses with triangulation number equal to 3 are evaluated in terms of hexamer-pentamer packing, and evidence is presented for the existence of larger subunits than the polypeptide in both cases. It is suggested that spontaneous assembly can occur only when exact icosahedral symmetry relates structure units or sub-assemblies, which would themselves have been formed by self-limiting closed interactions. Without such symmetry, the presence of scaffolding proteins or nucleic acid is necessary to limit aggregation.

1. Introduction

distinctive icosahedral shape and projecting fibers (Fig. 1). The location and organization of the structural proteins are still relatively unclear, despite the extensive investigation of adenovirus as a model system in molecular biology (see review by Philipson, 1983). Human type 2 adenovirus has a particle &I, of 175x lo6 (Green, 1970), of which approximately 150 x lo6 is protein

The adenovirus family currently comprises 112 known species, which have been found in 18 different mammalian hosts (Wigand et al., 1982). The adenovirus virion is characterized by its

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Fiber A

Peripentonal

hexon

Group-of-nine hexons

Figure 1. A schematic view of the ieosahedral adenovirus capsid, showing the major external proteins. The penton complex is formed from the penton base and fIbre and each is surrounded by five peripentonal hexons. Dissociation of the virion progressively releases the pentons and peripentonal hexons and then the planar groups-of-nine hexons to leave the viral core. The GON is left-handed when viewed from outside the capsid. Illustration by John Mack from Burnett (1984).

distributed among 11 to 14 different polypeptides. The single copy of linear double-stranded DNA is complexed with viral-coded protein and enveloped in an icosahedral protein shell to form a core (Brown et al., 1975). The core is itself encapsulated in an outer shell, the main component of which is hexon, a trimeric molecule of M, 3 x 109,077 (Akusjarvi et al., 1984). Hexon carries the major antibody recognition sites for adenovirus, which provide the main distinction among the individual species within the family. The organization of hexons dictates the overall shape of the outer capsid, which consists of a shell of 240 hexons closed by the 12 pentons at the fivefold vertices. The pentons consist of a base, embedded within the outer shell, and a protruding fiber, which serves as the attachment site to receptors on the cellular surface. Recent reports that the penton base is a trimer (Devaux et al., 1982), and the fiber a dimer (Green et al., 1983; Devaux et aZ., 1984), are at variance with the pentameric and trimeric states long assumed (Philipson, 1983). The new assignment would create a symmetry mismatch at the vertex, between the penton base and the peripentonal hexons, in addition to that between the fiber and base. Early electron microscopic investigations of adenovirus revealed that the spherical shape first seen (Hilleman et aZ., 1955) reflected a polyhedral capsid (Valentine & Hopper, 1957; Tousimis & Hilleman, 1957). The advent of negative stain techniques allowed Horne et al. (1959) to show that the 252 capsomeres were arranged in the form of an icosahedron. The first attempt to explain this shape was made by Horne & Wildy (1961) who suggested that hexagons and pentagons were packed on the surface of an icosahedron. Upon the development of

a general theory for virus construction, which explained the features of many spherical viruses, the capsid was thought to consist of 1506 identical single polypeptide structure units arranged on an icosahedral lattice with triangulation number T = 25 (Caspar & Klug, 1962). The capsomeres were presumed to arise by clustering of the polypeptides into 240 hexamers about the local and 12 pentamers about the sixfold axes icosahedral fivefold axes. The Caspar-Klug model successfully explained the number of capsomeres in several viruses including adenovirus, but was found later to be incorrect both chemically and structurally for the latter. The capsomeres consist of two chemically distinct species (Valentine & Pereira, 1965; Maize1 et al., 1968), with “pentons” at the 12 vertex positions and “hexons” at the remaining 240 positions, the names reflecting the five neighbors of a penton and the six neighbors of the hexons (Ginsberg et al., 1966). Additionally, hexon is trimeric (Griitter & Franklin, 1974), and does not arise from six polypeptides with a particularly high degree of association. Electron micrographs of whole capsids sometimes show details of individual hexons (e.g. see Nermut, 1975; Adam & N&z, 1983), but their appearance is unfortunately not sufficiently reliable to provide conclusive evidence for the organization of hexons in the eapsid. More detailed investigation has been facilitated by the availability of dissociation fragments called the “groups-of-nine” hexons. These planar objects (Fig. 1) are freed from the facets of the capsid by the action of sodium dodecyl sulfate, heat, acetone, urea or pyridine (Smith et al., 1965; Russell et al., 1967; Laver et al., 1968; Maize1 et al., 1968; Prage et al., 1970). Hexons within a, planar GONt can be visualized with greater detail by the electron microscope than those within a virion. Crowther & Franklin (1972) analyzed electron micrographs of the GON and found that the component hexons are arranged on a small p3 net and have threefold rather than sixfold symmetry. The GONs have a characteristic hand, defined by the relationship of the outer duos to the inner trio in their propeller-like configuration. Negatively stained GONs reveal that a distinctive surface morphology is associated with each hand (Nermut, 1975). The GONs are left-handed when defined relative to a view from outside the capsid (Pereira &. Wrigley, 1974). GONs interact in various ways and can be re-assembled into partial capsids and empty shells lacking peripentonal hexons (Pereira & Wrigley, 1974). The chemical and structural results thus indicate that the capsid is not organized in accordance with Caspar-Klug principles. Progress in understanding the organization of the capsid has been limited by the resolution attainable in electron microscopy. Our determination of the structure of hexon by X-ray crystallography (Burnett et al., 1985) has t Abbreviation

used: GON(s), group(s)-of-nine

hexons.

Hexon Packing in Adenovirus allowed us to extend the information obtainable from electron microscopy. Hexon has two main characteristics that allow the determination of its orientation and location in all 240 positions within the viral capsid. The first is that the base of the molecule has a well-defined pseudo-hexagonal shape that limits to two the number of ways in which hexons may interact in a close-packed planar array. The second is that the top of the molecule has a triangular shape, more in accord with its trimeric nature, that can reveal its precise orientation from Since the morphological electron micrographs. features are known with great confidence, an unusual degree of information can be extracted from electron micrographs of capsids and capsid fragments. A brief report on hexon packing within the GON has appeared earlier (Burnett et al., 1981). The crystal structure determination of hexon is described in the accompanying paper (Burnett et al., 1985). A following paper will describe how polypeptide IX, a different structural protein, can associate with hexons in a manner that explains the of the capsid observed dissociation pattern (Burnett, unpublished results). Structural studies on hexon have been recently reviewed (Burnett, 1984). 2. The Capsid Model Two assumptions were made at the start of this investigation. The first was that the shape of soluble hexon, as determined from the crystal structure, was an accurate reflection of its shape when it was within a capsid or a GON. Two pieces of supportive evidence may be cited. The pseudohexagonal symmetry of hexon is ideally suited to its function as a packing unit. Furthermore, hexon is that denaturation 6 Mso resistant t’o guanidine. HCl (Horwitz et al., 1970), or 8 M-urea (Pettersson, 1970)) is required to separate its subunits. The second assumption was that all hexons in the capsid are identical. This ignores the greater ease of dissociation of peripentonal hexons from t’he capsid than those from within the GON. and also the non-random dissociation of the GON. However, it has become clear that the presence of polypeptide TX influences t’he dissociation of the GON and masks the underlying symmetry of its component hexons (Burnett. 1984, and unpublished results). Furt’hermore, the requirement that assembly should require the selection of the correct structural unit from different classes of hexon would seem an unnecessary complication. The derivation of the capsid model is described in this se&ion. The constraint imposed on modelbuilding was that the features of published micrographs of the GON should be interpretable in terms of the hexon molecule. A detailed comparison of the model with experimental data is presented in the following section. Ideally, the resultant packing model should explain not only how identical units are used to form the complicated

127

adenoviral capsid, but also show how the observed dissociation occurs. The nomenclature employed here to describe subassemblies of the adenovirus capsid is in accord with Burnett (1984). The term trimer will be reserved for the quaternary structure of hexon. Groups of hexons will be referred to as duos, trios, quartets, quintets, sextets, septets, octets and nonets. The well-established name of the group-ofnine (GON) will be kept for the particular nonet of hexons it describes. (a) Hexon The envelope model used to investigate hexonhexon interactions was derived from diffraction data for the native protein and three derivatives (Burnett et aZ., 1985). Although the phases were determined for the native reflections to the nominal resolution of 2.9 A, only reflections to 6 A were employed in calculating the electron density map used for the model. As a consequence, the phases are significantly better than if only 6 A resolution data had been used, and have a mean figure of merit of O-9. Since only those features involved in packing were of interest, a molecular envelope was drawn on each section separating the protein density from solvent. The envelopes on adjacent sections were averaged and transferred to wood, from which the shape representative of a 4 A slice through the molecule was cut. The first model was constructed to show the inner cavity within the molecule and was divided at the boundary between the top and base (Fig. 2(a)). Two additional models were constructed, but the central hole and the division were omitted in order to facilitate their assembly. Hexon comprises two distinct parts that are not domains in the usual sense, but are rather regions of distinctly different morphological symmetry. The upper 64 A of the molecule consists of three towers rising above a triangular section. Its symmetry is therefore consistent with that of the trimeric molecule. The lower 52 A of hexon forms a base, the greater part of which has a pronounced hexagonal character. The main features of the molecule and their dimensions are summarized in Table 4 of Burnett et al. (1985). Hexon may be symbolized conveniently by a triangle superimposed on a hexagon (Fig. 2(b)). The molecule has a slight asymmetry, since the apices of the triangular top are not precisely above the edge midpoints of the underlying hexagonal base. They are rotated approximately 10” counter-clockwise when the molecule is viewed from above. The hexon base has six vertical faces of approximate width 52 A and height 42 A, commencing 10 A above the bot’tom of the molecule. The hexon basal faces are of two chemically distinct types, and have been designated the A, for those below the abices, and the R faces (Burnett, 1984). Hexon-hexon interactions were initially studied by inspecting the wooden models and arranging planar configurations them in close-packed

128

R. M. Burnett

misleading impression could be gained if the GONs were not constrained to lie on the planar facets of a cardboard icosahedron of the correct size. (b) Placement Electron

of hexons within

micrographs

the group-of-nine

of the whole

capsid

do not

show the individual hexons in sufficient detail for their orientation to be determined. The GONs are planar

/

\ (b)

Figure 2. (a) An envelope model of the hexon trimer, derived from an electron density map at a nominal resolution of 6 A (Burnett et al., 19&j), in side view. When viewed from above, the upper and lower portions of the molecule have distinctly different morphological symmetry. The upper 64 A has a triangular shape, in accord with the molecular symmetry. The major part of the lower 52 A possesses pseudo-hexagonal symmetry, which allows hexons to form close-packed arrays. Hexon is orientated in the capsid such that the tops lie to the outside, giving the typical spiky appearance seen in their electron micrographs. The two morphological components do not correspond to individual domains in the molecule. (b) Hexon may be symbolized conveniently by a triangular top resting on a hexagonal pedestal. The top are rotated by vertices of the triangular

and

thus,

when

lying

on

the

electron

microscope grid, present an array of hexons whose threefold axes are aligned parallel to the axis of the microscope. In addition, the planarity ensures that there is uniformity at the lower surface between the protein and the grid and at the upper surface of the GON. Negative staining may therefore be expected to reveal the features of the individual hexons in a relatively consistent fashion. Our first efforts to correlate the hexon structure with electron micrographs of GON were made using those of Nermut (1975) and led to the assignment of the orientation of their component hexons (Burnett et al., 1981). The X-ray model of hexon has one feature that allows the determination of the orientation of all the hexons within the GON. The hexon triangular top is twisted relative to its hexagonal base. Thus, a p3 net of hexons (Crowther & Franklin, 1972) will display an alternating pattern

of large

and

small

cavities

between

the

tops. There are two possible arrangements of hexons to form a GON (Fig. 3). The first has the large cavity at the centre (Fig. 3(a)), whereas the

approximately 10” counter-clockwise from the midpoints of the underlying vertical contact faces of the base, when seen from above, or outside the capsid. The extensive contact faces, of approximate width 52 A and height 42 A, are denoted A and B, with the former below the apices of the triangular top.

representative of those seen in electron micrographs of GONs. The limited number of interactions in close-packed arrays were summarized by drawing or plotting symbolic hexons so that the possible A : B, A : A, or B : B interactions could be visualized quickly. Investigation of the non-planar interactions was

more difficult, as it proved surprisingly difficult to cut sufficiently accurate representations of GONs from plywood or polyurethane. The most accurate representation was achieved by gluing together hexagonal machine nuts. Even with the latter, a

(b)

Figure 3. A symbolic representation of GONs seen from above, or outside the capsid. The counter-clockwise offset of the apices of the triangular tops from the exact midpoints of the underlying hexagonal bases leads to an alternating pattern of large and small cavities between the tops. The two possible arrangements are shown for a left-handed GON. (a) The arrangement revealed by electron micrographs, with the larger cavity at the centre. (b) The other possible arrangement, with the smaller cavity at the centre.

Hexon Packing in Adenovirus second has the small cavity at this location (Fig. 3(b)). Investigations in this laboratory have confirmed the assignment. GONs were minimally stained with lo4 uranyl acetate in order to emphasize the morphology of the surface in contact with the electron microscope grid (J. van Oostrum, M. Mohraz, P. R. Smith & R. M. Burnett, unpublished results). The right-handed images show triangular hexon t’ops on a p3 net, and also the pattern of alternating large and small cavities with great c8larit.y (Fig. 4(a): (b)). The left-handed images reveal the large central hole in the hexon base (Fig. 4tt.1, (d)). The GON model is also consistent with the assignment of their absolute hand (Pereira & Wrigley. 1974). An array of hexon tops would have the spiky appearance characteristic of the virion surface. The GOSS seen edge-on in Pereira & \;Z’rigley (1974) show a hint of the smooth inner surface and the spiky outer surface of the facet’s, corresponding to arrays of hexon bases and tops.

129

(c) Orientation of the peripentonal

hexons

The orientation of hexons within the GON also determines their position within the capsid, as the sharp capsid facets seen in electron micrographs indicate that the GONs remain planar. The geometry then dictates how GONs interlock at t’he edges to form an incomplete outer capsid. The relationship of each GON to its neighbor gives additional significance to the pseudo-hexagonal of hexon. Due to the icosahedral symmetry geometry, hexagons can interdigitate at the capsid edge in a manner that maintains close-packing (Fig. 5). The packing leads to an impenetrable protein covering, not only within the facet’s, but also at the edges. Since the GONs account for only 1X0 of the capsid hexons, the problem remains of positioning the remaining 60 hexons. These are the peripentonal hexons, which were so named not only because of their location, but because the ease with which they dissociate suggested different bonding

(a)

Cd)

Figure 4. Electron micrographs of minimally

stained left- and right-handed GOKs. Pairs of unfiltered ((a). (c)) and rotationally fikered ((b). (d)) digitized images are shown. Note that only the morphology of the surface in contact with the grid is revealed by these images. The hexon tops are revealed by GONs that are inverted on the grid and so have right-handed images. (a), (b) A right-handed GON showing triangular objects corresponding to hexon tops lying on a p3 net. Note the large central cavity, surrounded by three others at the centers of the outer trios. The pattern determines the absolute arrangement of hexons within the GON, and therefore within the capsid, and corresponds to the symbolic view in Fig. 3(a). (c), (d) A left-handed GON consistent with an array of hexon bases.

130

R. M. Burnett

Figure 5. Models showing the organization of hexons within the adenovirus capsid. All hexons in each facet make asymmetric A : B interactions with their neighbors in the same facet. The mutual orientation of two adjacent facets is such that the close contacts occurring at the edge are all symmetric A : A or B : B interactions. (a) Two asymmetric unit,s. marked on the model capsid, are related by an exact twofold icosahedral axis to create a partial edge. Additional peripentonal hexons are required to complete the edge. (b) A different choice of asymmetric unit is shown on the model. Three of these planar quartets are related by the exact threefold icosahedral axis within a facet.

than that of other capsid hexons. Indeed, it was even suggested that peripentonal hexons form a distinct class of hexons, with those at the edges and in the facets forming two other classes, corresponding to three types of hexon reported in infected cells (Shortridge & Biddle, 1970). The formation of GONs was recently shown to be dependent on the presence of a different adenoviral structural protein, polypeptide IX (Colby & Shenk, 1981). Polypeptide IX is unnecessary for assembly,

but appears to serve as a capsid cement. Polypeptide IX can be positioned within the GON to explain why the GON does not dissociate in the manner expected of a p3 net (Burnett, 1984, and unpublished results). Since the different dissociation properties of the peripentonal hexons could also be revealing the presence of other proteins, attention was focused on building a model capsid from identical hexon molecules. Since hexons within a GON lie on a p3 net, their

Hexon Packing

interaction is asymmetric with dissimilar A and B faces in contact (Fig. 3). The asymmetric planar interact,ion between an A face and a B face involves two distinct sets of amino acid residues: those of A directed to B; and those of B directed to A. The t’wo will be described as AB and B,, and the general class of int’eractions between the A and B surfaces as A : B. The interactions at a capsid edge are symmetric, due to the icosahedral geometry, and fall into two classes A : A and B : B. Thus the two symmetric interactions are non-planar, and the asymmetric interaction is planar. There are two possible arrangements of peripentonal hexons around the vertex that maintain fivefold a arrangement with close-packed symmetry. Ths first is with each peripentonal hexon making asymmetric interact’ions (A : B) with each of its two neighbors in the GON, and A: A interactions within the ring of five peripentonal hexons about the vertex (Fig. 5). The second possibility is with symmetric interactions (A : A and B : B) between the peripentonal hexon and its neighbors in the GON, and B : B interactions within the ring. Note that a ring with fivefold symmet’ry cannot’ be constructed with A : B interactions between the hexons. The first peripentonal arrangement would continue the planarity of the GON into the peripentonal region, due to the planarity of the A : B interactions of the peripentonal hexon with the GOh’. The second arrangement, with symmetric interactions. would truncate the capsid at the vertices, providing small platforms from which the fiber would rise. Electron micrographs of the capsid show facets that are distinctly triangular and provide no evidence of truncation at the vertices. This evidence strongly suggests that the first alternative is correct, and we have sought its direct confirmation using electron microscopy. We have succeeded in producing “quarter-capsids”, which lie with their fivefold axis normal to the grid. These contain five facets novel capsid fragments surrounding a complete vertex with five peripentonal hexons around the penton base. Preliminary results from rotationally filtered images have confirmed that each facet is a small p3 net (Oostrum & Burnett, 1984). The quarter-capsids show the difference in orientation, of the two hexons along the edge on either side of the twofold (Fig. 5) that is a consequence of planar GONs arranged on an icosahedron but is not seen in electron micrographs of complete virions. (d) Overall symmetry

of the capsid hexons

Once all 240 hexons have been positioned in the capsid, it’ is possible to consider the symmetry of the whole assembly. It is immediately clear that there are four independent hexon molecules unrelated by icosahedral symmetry. Together, these form the asymmetric unit of an icosahedral shell of hexons with holes at the fivefold vertices. There are two ways of grouping the four

in Adenovirus

131

symmetry-independent hexons. The first is shown in Figure 5(a), giving an asymmetric unit that embraces a half-edge. Two of these quartets are related by an icosahedral twofold axis to form t,he major part of the edge. At each vertex, an additional peripentonal hexon is required to complete the edge. This is contributed by the adjacent edge, as each peripentonal hexon is part of two edges. The second choice of asymmetric unit is shown in Figure 5(b) and groups the same independent hexom in a different manner. Here, the quartet is planar and consists of an outer trio of a GON, together with the peripentonal hexon that it embraces. The peripentonal hexon is at the outer extreme of the p3 array of a capsid facet. The asymmetric unit is related to two others in t’he facet by the strict, threefold axis at the facet center. The choice of asymmetric unit depends on which feature should be emphasized. The first emphasizes the edge interactions, and the second the facial interactions. The second is somewhat easier to visualize due to its planarity. It should be noted that these alternative asymmetric units do not depend on the orientation assigned to the peripentonal hexon. The orientation chosen has additional translational symmetry that maximizes the number of similar contacts made by each of the four independent hexons. A det’ailed analysis of these contacts will be made in the next section. Early descriptions of adenovirus architecture assumed that the topologically distinct locations lay adjacent to the penton, at the edge, or in the facet. The unexpected result from our model is that each independent hexon participates in bot,h the edge and the facet. The interdigitation at the edge allows three hexons to reach into the facet to lie adjacent to the t,hreefold axis so that the central portion is covered, The earlier topological assignment was made assuming spherical hexons, which obscured differences due to their real shape. (e) Interactions

of individual

huxms

The interactions made by each of the topologically distinct hexons with their neighbors can now be assessed. The pseudo-hexagonal shape of hexon is consistent with close-packing not only in the facet’ but also at the edge. Although other proteins could lie between hexons. it is more probable that they serve t,o stabilize hexonhexon interactions rather than to separate hexons at the base. Additionally, the dimensions of a close-packed capsid, as derived in a subsequent’ section. are in good agreement with the measurements from electron microscopy. The analysis thus assumes that only hexons are involved in constructing the framework of the outer protein shell of the capsid. It is also assumed that the hexon trimer is the fundamental structure unit. The molecule is highly resistant to unfolding and is many times more stable than the structures of which it forms a part. It is thus t,he natural choice for a building block. This method of analysis is in distinction to that

132

R. M. Burnett

associated with the original paper by Caspar $ a single polypeptide is Klug (1962), in which assumed to be the structure unit. The four symmetry-independent hexons are marked in the three asymmetric units forming the facet shown in Figure 6. One of the asymmetric units is shown in enlarged view in Figure 7. The distinct hexons are numbered 1 to 4 (HZ, to Hz,) and, as a mnemonic? hexons 2 and 3 have been placed adjacent to the strict two- and threefold icosahedral axes. The enlarged view shows each hexon with the interactions made by its basal contact faces with their neighbors in the capsid. The capital letter denotes the face, the subscript the face that is contacted, and the superscript the sense of mutual rotation. It is convenient to resolve the rotation into two components, the first being a “t,wist” about the normal to the interface, and the second a “hinge” perpendicular to both the normal and the molecular axis. The direction of twist can differ and is taken as positive if the rotation of the second of a pair of hexons, arranged on an axis so that the first is at, the origin, is clockwise when looking along that axis. This is the same convention as that, for dihedral angles in a polypeptide chain (ILPAC-IUR Commission, 1969). The hinge motion is always in the same direction and gives t’he convex curvature of the capsid. The arrangement of t’he hexons on the underlying icosahedron is such that, the hexons form flat facets with sharp edges, as seen in electron micrographs of the virion. The A : A hexon-hexon interface at the strict twofold axis is normal to a line parallel to the edge (Fig. 8). This interface is made by Ai contacts, the relative motion of the two hexons being a pure twist about the interface normal without hinge motion. There are two other types of symmetric contact at the edge. The relative motions of these are more complex, as the faces involved are not normal to the capsid edge, so that each has an identical hinge motion but equal twists of opposite sign. The first complex symmetric cont,act, AA, is again made by a pair of A faces, but with an opposite twist to that at the twofold. The

Figure 6. The 12 hexons comprising a planar facet are shown. The symmetry-independent hexons are numbered counter-clockwise so that Hz, is adjacent to the exact twofold icosahedral axis. and Hx3 is adjacent to the exact threefold axis. Hz, is the peripentonal hexon and Hz, completes an edge between Hx, and Hx,.

Figure 7. The bonds of each hexon within the asymmetrir unit are indicated. The subscript denotes the contact surface bo which the bond is made and the superscript the relative twist away from the planar arrangement in which the hexon molecular axes are parallel. The contacts within this planar asymmetric unit. which corresponds to that of Fig. 5(b), are all eit’her A, or R*.

second, Hi, is formed from a pair of B faces. The Ai and Bi edge thus consists of alternating contacts on either side of a central Ai contact at t’he strict twofold axis. The nomenclature used for contacts symbolizes the array of bonds involved in each interface (i.e. the sum of the hydrogen bonds and van der Waals’ contacts at the particular relative orientation of the two hexons). The total bonding of each hexon can be evaluated by listing its six contacts as a linear set. To determine the degree of similar bonding for the different hexons, the sets are cyclically permuted to provide the alignment with the maximum homology. Table 1 shows the alignment. together with the consensus bonding pattern that is closest to that of all four hexons. The second part of Table 1 lists the agreement with the consensus pattern exhibited by each of the hexons. The degree of conservation of bonding can be assessed by considering the fraction of the total identical in the four contacts that are environments. Hx, is in a special position as it’ is next to the penton and thus automatically has different bonding at the B, face. Even here, no less than 6776 of its bonds are identical to the consensus pattern. Hx, and Hz, have 83% identical bonding, and Hx, is identical to the consensus set. Tf hexon were a hexamer, then the same arguments would apply for the derivation of the bonding scheme shown in Table 1. One face would then be required to form four different types of selfbonding (AA, AA, Ai, Ap). With a pseudohexagonal trimer, the B face makes contact with t’hree different faces (BA, Bi, BP). Only the A face makes more than one contact with the same face (AA : A;). A trimer leads to greater accuracy in forming the correct bond, since the bonding from each face to another is unique in the cases (As, B,, Hi, HP) and restricted to two for (AA, A:). The pseudo-hexagonal symmetry of the base thus

Hexon Packing

in Adenovirus

133

performed on the individual hexon polypeptides would not reveal a similar conservation of bonding. Thus, the whole hexon must be considered as the structure unit.

3. Comparison with Experimental Data Figure 8. An illustration of the mechanism. resembling a wood joint, by which the edge is constructed in adenovirus. The pivot is parallel to the horizontal zigzag line at the boundary between the upper and lower halves, and bisects a half-lap joint. The zigzag shape of the interface gives each protruding half the characteristics of a finger joint. As the lower portion moves down to make the correct dihedral angle bet)ween the facets, its forward vertices on t’he right rise up the vertical grooves in which they lie. Correspondingly. t,he symmetry-related vertices of the upper portion on the left rise up the grooves of the lower portion. The motion causes the A faces at the twofold to swivel about their contact plane t)o form the AR’ configuration. The motion at’ the other interfaces is more complicakd as the faces tilt away from each other. with rotations of opposite sign for Ai and BB+. The position of the hinge axis must lie above the bottom of the ;1 face so that maximum contact is maintained along the edge. Tf it were at the bottom. contact would cease when the facets are non-planar. Likewise, the hinge axis cannot be at the top. as hrxons would then push each other apart at the bottom. The axis thus lies between the top and bottom. These interactions are best visualized by reference

to Fig. 5.

permits close-packing to occur at the edge as well as the deviation from hexagonal the face, whilst symmetry restricts the set of different interactions required from each face when making both facial and edge cont,acts. Kate that the same analysis

Table 1 The bondA made by each independent asymmetric unit Bonds

Hrxon IIX, HJ, II.r, Ii.,,, (11.1,) (‘oI1seIIsus

Location

;r,

B,

A,

B,

A,

Bi

I’eripentonal

ila

H,, A,

B,

Ai

Bi

.Adjacent to Z-fold

LAB .-la 24, Aa

fc.4 B, BA B,

B, B, B, B,

A,

BB+ Adjacent to 3-fold BJ O?thin the edge B, Within p3 facet Bf

A-1, A, A, A,

A, de A,

Agreement with consensus bonding

Hrxon 1i.r , 11x, IIL, KY4 (ff.r,)

hexon in the

+ + + Planar: A,. B,

+ + + +

+ + + +

+ + + + +

Non-planar:

Fraction

+ f + +

+ + + + Ai,

identical 4/G 5/G 5/6 616 4/G

Ai,

Bi.

(BP)

The origin’ for each set, of six bonds, corresponding to the six vrrt,ical basal contact faces, has been chosen to maximize the similarity. The consensus hexon has bonds that are most similar to those of the four independent hexons.

(a) Electron

micrographs

of groups-of-nine

The X-ray crystallographic model of hexon provides an unusual opportunity for the evaluation of results previously obtained from electron as the molecular features are now microscopy, known with great confidence. Although several electron micrographs of the GON were published earlier, PlJermut (1975) was the first to present images that clearly showed different morphology in left- and right-handed GONs. These were utilized in our earlier studies on hexon arrangement (Burnett et al., 1981). The GON is left-handed when viewed from outside the capsid (Pereira & Wrigley, 1974). as shown in Figures 1 and 3(b). The hexon base, with its polygonal shape and large cavity. could clearly be correlated wit’h the image of a left-handed Nermut GON showing doughnut-shaped structures. However. since the symmetry of the base is pseudo-hexagonal, the image provides no indication of the orientation of the individual hexons. Two of the nine hexons are distinctly hexagonal in the unfiltered image, but, the threefold averaged hexons in the left-handed CONS show no enhancement of the hexagonal appearance but rat’her a ring-like shape with a large central hole. The right-handed Nermut GOK differs in showing a distinct clarification in the appearance of the individual hexons upon averaging. The triangular shape and small central hole of each hexon are more pronounced in two of the three independent groups that were averaged in the threefold rot)ational superposition. Hexons in these two groups appear to lie on a p3 net, consistent with the results of Crowther & Franklin (1972). The hexons in the third group, which consists of the outermost hexons in the GON, fail to indicate their orient)ation clearly. However, if the alternative arrangements of cavities are considered (Fig. 3), the electron micrograph of the right-handed GON reveals the overall patt’ern clearly. A cavity at the center of the GON is repeated at the centers of the three outer trios. Smaller cavities are seen at the three related locations adjacent to the central cavity. Although outer hexons are not resolved clearly enough by negative stain for their triangular shape to be discerned, the cavities are less sensitive to variability in stain penetration. Our results confirm the original finding of Crowther & Franklin (1972) t,hat hexons are arranged on a p3 net, but the hand of their averaged GON images is inconsistent with the molecular features of the X-ray model. The GONs originally analyzed were all right-handed, but they show ring-like structures similar t*o t)hose normally

134

R. M. Burnett

seen in left-handed GONs (for example Fig. 4(c), (d)). However, rotationally filtered images showed three peaks of density for the inner hexons, which would be consistent with right-handed GONs. Additionally, the three peaks are at first sight consistent with the three peaks of density seen in the triangular hexons in the right-hand Nermut GONs. One possible explanation is that) the Crowther and Franklin GONs were more heavily stained, and that the peaks of density represent a projection of the entire molecule. There is an alternative explanation that emphasizes the amount of information available when a model is known with great confidence. The appearance of the unfiltered Crowther & Franklin images suggests that the GONs are lefthanded rather than right-handed. The hand would then be consistent with the authors’ observation that the GONs lay in the preferred orientation. The percentage of GONs known to be lefthanded when lying on carbon film is 75:/b to 90%, the value depending somewhat on the pH employed (Nermut & Perkins, 1979). if it is assumed that the rot’ationally averaged images were accidentally inverted, the hand not being relevant leftto the original st,udy. then they are truly handed GONs. The images should therefore show hexon bases, but why then do they show triangles? Three small protrusions project downward from a more circular section in the lowest 4 to 6 A of the pseudo-hexagonal hexon base (Burnett et aZ., 1985). Thus, a low level of stain will reveal the true molecular threefold symmetry and, as the strain rises higher. will reveal the circular and finally the pseudo-hexagonal shape. The Crowther and Franklin images reveal outer hexons in the GON that are rings, and inner hexons t’hat have triplets of density. This difference in appearance was attributed by Crowt,her & Franklin to the lack of preservation of the specimens. It is more likely that stain differentially penet’rates the GON, coating t,he outer hexons to a greater height than those innermost. The angle made by the tops and “feet” relative to the axes of the p3 net can be measured. The feet are 30” counter-clockwise from the tops in the hexon model. Despite the distortion inherent in the images. t’he feet in inverted lefthanded model GONs are better matched than the tops to the posit,ions of the densit’y triplets in each molecular image. (b) Interactions

of groups-of-nine

Electron microscopy has revealed that GONs associate in pairs in two ways. The first type was observed by Smith et al. (1965), and later work showed a second (Pereira & Wrigley, 1974). The first type, X in the nomenclature of Pereira and Wrigley, was that seen in the capsid where two GONs form a non-planar pair with a dihedral angle of 41.81” (Fig. 9(a)). The second kind, Y, has more contacts at its interface and yet was found to be planar (Fig. 9(b)). Pereira and Wrigley assigned the dihedral angle on

00

41 81’ c1

\ C’

ib)

Figure 9. Pairs of GOrJs are shown interacting in the two alternative configurations observed in electron micrographs by Wrigley & Pereira (1974). The axis found in the capsid is marked c. (a) The configuration t,hat has t,he same interactions and dihedral angle (41.81”) as that within t,he capsid. The detailed interactions at the interface are also indicated and these include the twofold operation of t,he icosahedral capsid. The broken lines indicate the positions of peripentonal hexons when GOK’s form a capsid edge. (b) An alternative configuration with the bonding at the interface indicated for both the observed planar form (0”) and for a hypothetical capsidlike configurat)ion (41231”). The first of these has seven contacts between the hexon faces. The second has a set of five contacts of the same class as used in (a), but arranged differently and lacking symmetry. Two contacts in the planar arrangement, for the hexon marked X, a-ould be broken if the non-planar arrangement, were formed. Although the planar configuration has an array of (Iontarts that is not found in the capsid, it is more stable since it possesses a twofold symmetry axis. Any deviation from planarity would stretch or compress symmetryrelated bonds on either side of the irnerface. Thus a symmetrical set of forces will oppose any motion and restore t,he configuration to its equilibrium position. Rot,ation about the axis marked c’ would rreat,e a joint of the opposite “hand” to that seen in the capsid and yield non-planar arrangements that. although symmetrical. arc not observed.

the basis of observations showing that GONs in rings of five and GONs assembled into empty shells both have X interactions, but that rings of six GONs have Y interactions. On geometrical grounds X must be non-planar and Y planar. Thus both planar and non-planar forms of the symmetric A : A and B : B interactions at the interface can

Hexon Packing in Adenovirus occur. This observation presents the paradox t,hat X. which is non-planar, has three A : A and two R : R contacts, whereas Y has three A : A and four I? : B contacts but is planar. It therefore appears t,hat the addition of two B : B contacts induces planarity. Since the peripentonal hexons were not considered, as their orientation and even identity were unknown, the nature of the interface between t)wo GONs was unclear. The plausible assumption was therefore made that the edge maintains as many contacts as possible, lying between axes c and r’ in Figure 9(b). The paradox was resolved once the positions of the peripentonal hexons had been assigned, and complete facets rather than GONs were used to model the edge. It was then obvious that the capsid axis is that marked c in Figure 9, with the correct analysis for Y depicted in Figure 9(b). The interface is shown in both the planar (0”) and non-planar (41.81”) configurations. In t’he latter, when the groups are rotated about e the marked hexon lifts away from its former neighbors. In the hypothetical non-planar Y configuration, Y has five contacts whic,h are of the same kind as those seen in the X arrangement. but there is one fewer iii and once more Bi . Tn considering why the non-planar Y case does not occur, the relative strengths of Ai and Bi bonds have less relevance than the symmetry of the various arrangements. In non-planar X there is a central twofold axis, as within the capsid. A similar twofold does not occur when Y is non-planar, but a twofold axis of a different type occurs when it is planar. The contacts related by this symmetry axis are different to those found in the capsid, but are presumably facilitated by the extensive nature of the haxon basal faces. The surface contact area will be greatest when the arrangement is planar. Twofold symmetry, at an interface between two groups, imparts a powerful stabilizing influence on the configuration. Any attempt to increase or decrease the dihedral angle will be resisted by equal and opposite forces. Consider Y in the hypothetical non-planar configuration (Fig. 9(b)). Any tendency for t’he marked hexon to form bonds with its neigh hors and encourage’ formation of a planar state will be resisted by the existing bonds, but these are as>-mmetric and so the situation is inherently unst’able. When the planar Y state has been reached, the arrangement is symmetric and hence stable. Symmetry alone is insufficient for bonding to occur. For example. a rotation of 41.81” about axis c’ in Figure 9(b) gives a configuration analogous to that in the capsid, but’ with individual hexonhexon eont,acts at the interface in the opposite sense to their capsid counterparts. This configuration. although symmetric, does not give rise to favorable cont)acts and so is not observed. (c) Size of thr capsid The molecular dimensions of hexon and its known packing make it possible to calculate the size of the adenovirus capsid for comparison with

135

results from other techniques. Two factors must be considered in using the center-to-center distance of 89 L%for close-packed hexons. The first is the finite thickness of the capsid, corresponding to the height of hexon. The second is the choice of the appropriate spherical equivalent for the capsid. The “diameter” of an icosahedral capsid is often cited without an exact definition. There are three possible spheres that may be considered. The circumsphere touches all the vertices, the intersphere touches all the edges, and the insphere touches all the facets (a useful reference is Pugh, 1976). The respective radii differ markedly (0.951057e, 0.809017e and 0*755761e, where e is the edge length of the icosahedron). The most appropriate diameter would seem to be that, of the intersphere. Not. only is it the sphere of intermediate size, but presumably the capsid commonly rests on the electron microscope grid upon a facet. The perpendicular view would then be normal to lines joining the midpoints of opposing edges, approximately half-way up the icosahedron. Assuming that hexons are arranged on an icosahedron as in Figure 5, an additional half-hexon at each vertex is required to complete the edges. The length of the underlying icosahedron is therefore that of five hexons. Thus the edge is 445 a and the insphere radius to t’he facet is 336 8. The equivalent radius for the icosahedron that just touches the tops of the hexons requires the addition of the hexon height, to give 452 A. The radius to the division between top and base, the limit to which negative stain may penetrate, is 388 d. The edge lengths of these two icosahedra are 598 .r( and respectively. The 513 A. intersphere and circumsphere radii for bottom, division and top are therefore 360 A, 415 A, 484 8, and 423 8. 4X8x: 569 A, respectively. It is clear that the large range in capsid size, depending on the definition used, makes comparison with the results from other methods highly dependent on the correct choice of spherical equivalent. An alternative view is that the range just cited, of 360 to 484 A for the radius and 445 to 598 a for the equivalent edge, illustrates the possible range of values to be expect.ed in the literature, particularly that of electron microscopy. Nermut (1975) measured the length of t.he central four hexons on an edge from freeze-dried and shadowed adenovirus, and the length of t’he edge from freeze-etched adenovirus. The former technique gave 340 A, or 85 ;i for the diameter of a single hexon, and the latter 440 A. Since t’he latter figure was obtained from hexagonal particles. it can be corrected for t,he foreshort,ening of the hexagonal sides due to the view down the threefold axis. to give 508 8. The agreement is excellent if it is assumed that the lengths were measured for icosahedra enclosing hexon bases alonr. The different’ orientations of the hexons on eit,her side of the twofold axis at t,he capsid edge cannot be seen in electron micrographs of the whole virion so the t’ops are apparently invisible in these virus.

R. M. Burnett

136

The dimensions obtained from X-ray and neutron scattering from adenovirus virions (Devaux et al., 1983) agree only approximately with our results. The distance between the capsid hexons was determined as lOO( f 1) ,& from small-angle X-ray scattering. This value was derived from the agreement of the scattering curve, calculated for a series of scattering centres placed on a T = 25 icosahedral lattice, wit’h the observed scattering curve. Since the agreement is so poor, the conclusion must be that this was an unsuitable model. There is better apparent agreement with the neutron-scattering results, which provided an with three icosahedral model of adenovirus concent.ric shells of differing composition. the innermost being comprised of protein and DNA, and the outer two of protein alone. The t)otal thickness determined for the outer two shells (115 A) was in apparent agreement with the height of hexon. However, the circumsphere radius for the icosahedron was 496 A. which agrees best with the radius to just above the hexon base. This suggests tha,t the density of the hexon top plays little role in the neutron scattering, which is not unreasonable given the appearance of the molecule (Fig. 2). Thr outer shell would then correspond to hexon bases and the middle shell to polypeptide VT, which apparently lies internal to the hexon shell (Everit)t ef al., 1973, 1975). The puzzling finding tha,t the outer shell is 90?,;, protein whereas the middle shell is only 6676 protein. with the rest wat,er! would then be explained. It is clear from our results that. t’he spiky adenovirus surface is due to the array of hexon t,ops. Tf these were contributing to t’he scattering, then the outer shell should have a far lower fraction of protein than the middle shell. The X-ray scattering result probably arose from the model used, in which spheres placed on a T = 25 lattice represented capsomeres. The exact dist’ance between these spheres is then a function of the icosahedral radius. Calculated scattering curves are quite insensitive to parameters such as the viral

radius and it is difficult to accept an a.cc:uracy of The best 1”~ for the hexon-hexon distance. agreement with the model of the capsid proposed here is if the neutron scat,tering radius of 500 L\ corresponds to t,hat of an icosahedron covering the hexon bases. However. there is a more fundament,al difficulty as the separation of t,he scattering points at this radius does not on a T = 25 lattice correspond to the hexon-hexon distance. since hexon is a cylinder and not a sphere. Spheres of equivalent scattering would lie on lines normal to% T = 25 lattice of lower radius. This effect could account for the overestimate of the hexon--hexon distance. A better approximation would be a capsid composed of model hexons as used by Kerger rt cl./. (1978) t,o represent the preliminary X-ray results (Burnett, it nl., 1978). The model consisted of 1200 spheres, arranged to fill the space between the inner and outer molecular envelopes.

4. Viral Architecture (a) Largrr

“adenovirus-type”

cap&d,9

The packing shown by hexon in adenorirus suggests that larger capsids could be constructed by using the same principle to form the edge. The edge. by analogy with carpentry, may be described as a half-lapped finger joint, with its axis parallel to the icosahedral edge (Fig. 8). It can be seen that the joint, requires an even number of hexons, disposed symmetrically about, the stric+t t,wofold axis. “Adenovirus-like” capsids correspond to the members of the non-skew series (a = 2n+ 1. h = 0) for the class of polyhedra consisting of 12 pentagons surrounded by a regular dist,ribution of hexagons (Goldberg, 1937). The indices a and b define the vertices on hexagonal location of adjacent) fivefold axes. The number of each hexon type in various members of the series is listed in Table 2. If it is assumed that each hexon must lie on a $21 net? then even a hexameric hexon could not lie on the st’rict twofold axis. The series is t,hus limited t.o members

Table 2 &kmber n R l+ntons Hexons Total HR.~. Hz,. Hr, Hx, Hr,

of structural 0 1 1” 0 12 0 0 0

1 3 12 80 92 * 0 0

units for 2 5 12 240 252 60 60 0

the non-skew series (a = 2n + 1, b = 0) 3 7 12 480 492 60 180 120

4 9 12 800 812 60 300 320

r 1;’ 12 1200 1212 60 420 600

6 13 12 1680 1692 80 540 960

II 2n+ I 12 4On(n+ 1) 4On(n+l)+l:! 60 60(2n - 3) 40n( n - 2)

The number of structural units required to construct hexagonally close-packed capsids of the nonskewed type for which a = 2n+ 1, b = 0. The number of pentons, and of hexons, Hxl, Hr,, Hxg, are invariant at 12 and 60, respectively. The number of each hexon class in the asymmetric unit can be found bv dividing the individual totals by 60. This indicates that Hx,, HI,, and Hs, are always present in the exact number required by the symmetry of the icosahedral point group (60). Hexons Iix, and Hz, for a > 3 are not constrained to lie in identical positions by icosahedral point symmetry. However, Hz, is constrained by l-dimensional translational symmetry along the edge, and Hx, by it.s presence within a two-dimensional p3 net. The class a = 3 is special as one of the two unique hexons lies on the threefold axis and is surrounded by three units combining functions of Hr, and Hz,.

Hexon Packing

wit’h a = 2n + 1, b = 0. The first two members of this series are special cases. That for a = 1 consists only of pentagons, and that for a = 3 has an outermost hexon with characteristics of both the peripentonal hexon, Hx,, and also that of the hexon adjacent to t’he exact twofold, Hx,. The members of the series for which a > 3 have common features. Each is constructed from a a hexon at the exact peripentonal hexon, Hz,, twofold. Hx,, and a hexon, Hz,, that is adjacent t)o the threefold axis in adenovirus (a = 5, Fig. 6). FJ;r, is always at the edge adjacent to Hz, where together they form half the joint at the twofold. These three hexons are invariant, as they occur only once per asymmetric unit. The fourth hexon, IZx,, also occurs only once in adenovirus, where it is at t.he edge between Hz, and Hx,. Larger capsids than adenovirus could be formed I)y insert’ing two additional copies of Hz, at each edge. one between Hz, and Hz,, and one between HZ, and IZx4. Examples for a = 7 and a = 9 are shown in Figure 10. The edge is progressively lengthened in increments of two hexons, ensuring that the twofold axis remains at the interface between hexons lying on adjacent facets. As the edges are lengthened, addit,ional hexons occur in positions within the facet that are surrounded on all sides by other hexons. This type, Hx,, does not occur m adenovirus, but must be present in increasing quantit)y as the planar area of the facet enlarges. Tt is important to note that the number of different types of bonding never exceeds five, no matter how large the capsid. It should also be noted that other arrangements with hexagonal closepacking are c4early possible, a significant example being the skew series with b = a- 1. (b) Quasi-equivalence The packing symmetry of hexon, which enables a molecule to form a large structure with a small number of identical contacts, requires a revaluation of the principle of quasi-equivalence. Crick & W’atson (1956, 1957) first suggested that identical subunits, if arranged with helical or cubic symmetry. would enable the formation of large structures with great genomic economy. The regular form of viruses would thereby be explained. Caspar & Klug (1962) later showed that multiple sets of 60 identical structure units could be used to form icosahedral shells. Each set of 60 units would occupy a topologically distinct location, but modest structural and bonding differences would be sufficient to adapt t’he st,ructure unit to each location. (‘aspar & Klug (1962) showed how successive subtriangulations of an icosahedron could be used to derive a class of icosadeltahedra with 20T equilateral facets. The triangulation number T was defined by T = Pf’. where P = h2 +hk+ k2, for all pairs of integers h and k having no common factor, and f being any integer. A plane p6 net was folded to create fivefold vertices by bringing together two

in Adenovirus

137

b

Figure 10. An illustration of two additional members of the “adenovirus-like” hvpothet,ical capsids. for which a = 2n+ 1. b = 0. as listed in Table 2. Half of the row of hexons at the edge is provided by each of the adjacent facets. For a > 3. the asymmetric unit is formed from one hexon at each of the peripentonal. twofold, and threefold positions. Larger facets are obtained by adding pairs of Hz, as neighbors of Hx, and filling in the central portion of the facet with Hx5. Adrnovirus is the first member of the series. with a = 5. (a) The next member of the series. with a = 7. (b) The situation fnr a = 9 where t.he threefold axis coincides with the position of a hexon. In general. this will occur when the index. II. is a multiple of three.

rows of triangular edges, 60” apart on the plane, to create a convex surface. They realized that the contacts made by three structure units forming each small triangle would be almost t,he same, whether the unit was in a planar or in a convex array. This was described as quasi-equivalence as it resolved the dilemma posed by the existence of icosahedral viral capsids with more than 60 identical subunits. The arrangement maintained the physical principle behind the arguments of Crick & Watson (1956), that the same chemical contacts must be used repetitively, by relaxing a strict requirement for mathematical equivalence. The appearance of icosahedral viruses was explained by

138

R. M. Burnett

clustering of structure units about local fivefold or sixfold symmetry which axes, successfully explained the forms of most known viruses. The analysis of hexon packing in adenovirus suggests that a re-evaluation of Caspar-Klug theory is in order. The structures of three small plant viruses are known to high resolution. Satellite tobacco necrosis virus (Liljas et al., 1982) is a T = 1 virus, for which all contacts made by the 60 subunits are by definition equivalent. Tomato bushy stunt virus (Olson et al., 1983) and southern bean mosaic virus (Rossmann et al., 19836) are both examples of T = 3 capsids. The latter structures reveal that contacts between subunits are essentially identical between the A subunits surrounding the fivefold axis. These contacts are also identical to those of one set between the B and C subunits surrounding the quasi-sixfold axes, but the second set is quite different. The contacts around the threefold axis are again almost exactly conserved. Except at certain locations, such as the hinge region, quasi-equivalence does not hold. The individual subunits do not make approximately ident,ical bonds with their neighbors by small distortions. The bonding is either identical, or quite different. In adenovirus, most contacts are conserved in the four topologically independent hexon positions. The variations from the conserved contacts are discrete and not a gradual distortion of a basic bonding pattern. This is analogous to the two states of the individual subunits in the T = 3 plant viruses, but achieved geometrically rather than by a hinge. The importance of the original Caspar-Klug concept lay in its ability to allow conservation of contacts, and it is this aspect that should be emphasized over any particular geometrical realization. (c) Triangulation

numbers

An important conclusion from hexon packing is that each facet is a small two-dimensional crystal, with the hexameric shape of the structure unit permitting close-packing both within the facet and also at the capsid edge. Since the facet is a twodimensional array, identical contacts are made by each of the structure units, with the exception of contacts at the edges. A “crystalline” facet thus permits the expansion of the capsid to any size without increasing the number of topologically distinct locations beyond a finite small number. Each additional structure unit will have a new posit’ion, but no new environment will be created (Fig. 10). It is this use of translational symmetry that achieves the high degree of conservation, and that distinguishes the current model of adenovirus from that proposed earlier. In the Caspar-Klug model the triangulation number grows as a function of the viral radius, assuming a constant size for the structure unit. As an example, 9360 primitive structure units arranged on a T = 156 lattice would be required to form the 1562 morphological units of the Sericesthis and Tipula iridescent insect viruses

(Wrigley, 1969, 1970). A polypeptide could imaginably bind in the three distinct ways required for a T = 3 capsid, but the 156 required for the insect capsids is an argument of reductio ad absurdum. The difficulty of accurate assembly would be insurmountable. An additional difficulty with the Caspar-Klug approach has been that it fails to explain t,he angularity observed in many viruses, including adenovirus, since the capsid curvature should be distributed equally over all structure units to maximize quasi-equivalence. The demonstration that adenovirus facets are small crystals explains the regular, facetted, appearance of the capsid and suggests a similar construction for other large icosahedral viruses. It is also now clear why the Caspar-Klug theory correctly explained the appearance of the adenovirus capsid, despite the incorrect assumptions regarding the chemical nature of the coat protein. Pentons and hexons are located at the sites of exact fivefold and local sixfold symmetry on the proposed T = 25 lattice. Clusters of primitive be structure units about these axes would indistinguishable in the electron microscope from complex structure units, such as hexons and pentons. at these locations. The link between t,hr two approaches is hexagonal close-packing. Caspar and Klug emphasized the conservation of bonding by quasi-equivalence and imagined that close-packing resulted from clustering about the vertices of a plane p6 net. The current approach emphasizes the importance of close-packing for a virus shell, since it allows the formation of an impenetrable coat. and shows how bonding is conserved for the whole packing unit rather t’han the polypeptide. The relationship between t’he &spar-Klug approach using structure units. and the possible structures that could arise by close-packing pentagons and hexagons on a capsid, has been pointed out by Horne & Wildy (1963). Earlier work (Horne & Wildy, 1961) had sought to define all configurations of capsomeres that can occur when pentagons and hexagons are arranged on a polyhedral capsid. Their approach tended to emphasize these properties, whereas Caspar &, Klug (1962) required a smaller unit’. Goldberg (1937) had already solved the problem of how hexagons and pentagons form close-packed arrays on the surface of dodecahedra. He classified the various possible st)ructures in terms of the positions of neighboring fivefold vertices on a honeycomb arrangement of hexagons. The resultant indices (a, b), relative to axes inclined at 60”> dictate the number of facets bounding the polygon. There are lO(a’ +ab + b’) + 2 such facets, of which 12 are always pentagons. The number of hexamers predicted by Caspar-Klug theory is lO(T- 1) and thus T corresponds to (a*+ab+b”). The Goldberg diagram was put to elegant use by Wrigley (1969, 1970) in assigning the number of morphological units in Srricesthin and Tipula iridescent viruses, which have capsids too large to permit direct counting. The observed ratio

Hexon Packing in Adenovirus of the capsid edge-length to inter-capsomere to the discrete values spacing was compared permitted by the Goldberg diagram. arguments lead inexorably to the These conclusion that the emphasis placed on hexagonal and pentagonal packing units by Horne and Wildy was not misplaced, and that these are probably oft,en multimers such as hexon. The next two of multimeric se&ions contain a discussion structure units and their role in achieving aceurat,e assembly.

(d) (‘omplex

structure units

The identification of hexon as the appropriate structure unit requires the introduction of new terminology. “Complex structure unit” is suggested for units that consist of more than one poiypeptide. knits with one polypeptide, as in the small plant viruses, can be referred to as “primitive”. The original definition of a structure unit as the smallest functionally equivalent building unit of a capsid A (Caspar et al., 1962) can then be maintained. primitive structure unit is defined as one structure polypeptide chain, whereas a complex unit would comprise two or more chains. Complex units may themselves be homopolymers, rest.ricted to one type of chain, or heteropolymers with mixed chains. A major theoretical advantage of choosing a larger unit is that it no longer imposes the requirement that the structure units be limited to hexamers and pentamers: but merely that they can form a hexagonal lattice. The independent existence of complex units need not. be regarded as due to particularly t,enacious clustering of primitive units, even though this may explain their origin. Instead the smallest entit’y is taken as the structure unit that most accurately describes the overall capsid symmetry. Major constituents of a shell that can be identified as independent should be considered for the role of structure unit. Polygonal, or doughnutshaped units. are commonly seen in animal viruses. Their shape, as with hexon, could permit the closepacking that, must be a feature of large shells. This avoids dismissing the independent approach existence of multimers as an irrelevance, and seeks the appropriate unit to achieve a closed symmetric shell. The importance of this approach for adenovirus is that it has revealed the intrinsic capsid symmetr?, and has established that only one class of hexons IS required to construct the capsid. The role of polypeptide 1X in the capsid can also be understood, which in turn explains the progressive dissociation of the capsid into peripentonal hexons, groups-of-nine, and hexons (Rurnett, 1954). (‘omplex structure units also permit large units to be formed from polypeptides of restricted size. (‘aspar & Klug (1962) mentioned the need for large structure units in constructing voluminous shells. This would lessen the need for great quantities of structure unit,s, for which error-free assembly would

139

be difficult. As pointed out by Crane (1950), the use of sub-assemblies can lead to greater accuracy in assembly. This point has been emphasized by Simon (1969) in discussing the importance of hierarchy in building complex systems of any kind. Very few protein sequences are known that are longer than hexon’s 967 residues (Barker et al., 1984). Large structure units therefore must be composed of more than one polypeptide and be complex, as there appears to be an upper limit to the length of a chain. The complex units must have similar properties to those of the primitive units, and be stable entities that are highly resistant to unfolding. To be employable as the viral building blocks, the structure units must be themselves more stable than the structure of which they form a part. This implies internal bonding between the polypeptide chains that is t,ighter than any external bonding that can occur. Hexon is such an example, where three polypeptides are strongly linked to form a symmetric and locally closed complex structure unit. Hexon’s three chains are so tightly interwoven that tryptic digestion leaves a major part apparently untouched although denaturation reveals that t’he chain has been cut in several places (Pereira & Skehel, 1971, Pettersson, 1971). The tight. binding is seen in t,he ext.ensive int.erweaving of the chains in the 2.9 Ah resolution electron density map (Burnett et al., 19&j), where it is very difficult to distinguish one subunit from the next. The resultant stability is achieved at the cost of a complicated assembly process which requires the presence of the adenovirus 100 K protein as a scaffold for correct folding of the very long chains (Cepko & Sharp, 1982). The difficu1t.y of correctly folding a very long chain is probably the major size-limiting factor for a.11 proteins. Evidence for other complex structure units is mainly circumstantial, as our knowledge of the chemical composition of viruses is remarkably poor. However, there is direct evidence that, the capsomeres of Q/? are distinct chemical entities (Takamatsu & Iso, 1982). The Qp coat protein polypeptide forms pentamers and hexamers, each cova.lently linked by disulfide bonds to give two distinct molecular species. The T = 3 Q/I capsid is formed from 12 pentamers and 20 hexamers, so that each species would experience one class of contact,. Several other viruses have been shown to produce particles resembling the morphological units when disrupt’ed, including reovirus (Amano et al., 1971) and polyoma (Friedmann, 1971). The existence of a separate class of pentamer bonds at vertices is demonstrated by the formation of small polyoma (Mattern et al., 1967) and SV40 (Anderer et al., 1967) particles, which have the appropriate size and form to be composed of 12 units. These are analogous to the dodecons of ad3 pentons wit,h their fibres arranged in a star-like array (Norrby, 1966). The pentameric “caps” can thus form selfcomplementary contacts or other unit’s can be interposed to give a larger capsid.

140

R. M. Burnett (e) Accuracy

and spec$city

in assembly

Two of the major requirements for virus architecture are in conflict. Accurate assembly of structure units requires high specificity, but stability of the final structure requires strong binding. For specificity to be high, recognition must be achieved by a large number of weak forces rather than by a few that are strong (Crane, 1950). Specific interactions between two molecules arise from complementary surfaces, in which a large number of weak van der Waals’ forces combine to effect a strong specific bond. The relatively weak interactions ensure that abortive non-specific binding does not occur. This is in opposition to the demand for stability in the final viral structure, which requires strong binding of its components. For hexon, the symmetry of the faces, and their hexagonal distribution, serves to maximize specificity by ensuring that the contact regions are extensive only when the molecules are in their correct orientations. The relative weakness of the binding is seen in the small number of different hexon-hexon interactions that have been observed, reflecting their specificity, and in the need for polypeptide IX as reinforcement of the final structure (Burnett, 1984). The role of complex structure units in accurate assembly is seen if they are regarded as subassemblies in the sense of Crane (1950). Assembly is viewed as a production line, with step-wise incorporation of material into sub-assemblies. Incorrect sub-assemblies are rejected to prevent them from passing t,o the next stage. Crane illustrated his idea with a hypothetical model involving the assembly of 1000 objects from 1000 unit components. He showed that, if each unit had a 99% probability of being incorporated correctly, there was virtually no possibility of a single correct assembly when the final structure was formed directly from single units. If sub-assemblies of 10 and then 100 units were employed. and

incorporated or rejected as before, 740 correct final structures would be formed. The results of applying this idea to adenovirus are shown in Table 3. The probability is again set at 99%, as t’he exact value is not too important for this illustration. The formation of hexon trimers increases the number of correct assemblies by a factor of 100. A further fivefold increase is gained from the introduction of a hypothetical hexon quartet sub-assembly. The actual situation is obviously more complicated as a sixfold excess of individual hexons is found in infected cells (White et al., 1969) rather than the relatively small number shown here. If the analogy of a production line is correct, then rejected components should be seen in the cell. Hexon processing produces the following artifacts: hexon chains that have not been acetylated at the N-terminus (Boulanger et al., 1978); incorrect’ly folded hexons (Cepko & Sharp, 1982); hexons that are not transported from the cytoplasm t,o the nucleus (Kauffman & Ginsburg, 1976); and hexons that fail to become incorporated into virions. The large excess of hexons seen in infected cells thus reflects not only the concentration required to drive assemblv from free hexon, but also the presence of various mcorrectly processed hexons. Mechanisms for the exclusion of incorrect hexons from the assembly process include transportation and “crystallization”. Hexons must be transported from t’he cytoplasm to the nucleus where viral assembly occurs. Correct folding of t’he hexon trimer occurs when it’s polypeptides are complexed with the adenoviral 100 K protein (Cepko & Sharp. 1982). Transport incompetency is seen in virus with mutat’ions in t,he hexon or 100 K polypeptides. Incorrectly folded hexons are thus excluded from t,he assembly site. Transportation sensitivity could compensate for a high error level arising from usurping cell mechanisms optimized for the host. The excess of hexons is sufficiently great to cause crystalline inclusions in the nucleus and t’hese are

Table 3 Various

hypothetical

I’olypeptides size (M,. X 10w3) HeXOIls rejects Quartets rejects Shells rejects

assembly

schemes for the formation

“Caspar-Klug”

Monomer

1,440,000 55

720 000 ‘109

0 1000

1 999

of hexon

shells

HeXOn

Quartet

720,000 109 231.872 71% (30)

720 000 ‘109

87 883

232,872 7128 (30) 55.924 2294 (38) 510 422

The first two columns show direct assembly of shells from individual chains forming hexameric or trimeric capsomeres. The third and last columns show the effects of first forming a trimeric hexon subassembly. and then forming a quartet as a further intermediate. Each line shows the number of units successfully assembled at each stage. The number of rejected incorrect assemblies is shown below, with the equivalent number of whole shells in parentheses. The probability of an incorrect structure after a components are added is (1 -q”), where p is the probability of a successful addition being made. Here. ‘1 is taken to be 0.99.

Hexon Packing in Adenovirus commonly surrounded by viral particles (Morgan et al., 1957). Assembly via the inclusions (Lifchitz et al., 1975), or in proximity to them, would represent a further mechanism for the elimination of defective material. The similarity of assembly and crystallization is emphasized by the crystallinity of the adenovirus facets. The excess pool is a common feature of mammalian cells infected with virus (Fenner et al., 1974), and probably reflects mechanisms similar to t,hose occurring for hexon.

(f) Symm,etrjg

in other

viruses

The choice of a larger unit than the polypeptide was crucial for understanding the construction of adenovirus. It is therefore interesting to consider which detailed structural other viruses, for information exists. These include the structures of polyoma and the plant viruses determined by X-ray cryst,allography and the bacteriophages by electron microscopy. The main thrust of this section will be to present the idea that the correct choice of a complex structure unit, leading to a simpler description of the capsid, may be the key to understanding t!he architecture and assembly of a particular virus. A unit larger than the polypeptide is frequently the motif of a cubic point group. Since closed identical the symmetry is then exact, interactions among the 12, 20 or 60 complex st)ructure units completely define the assembly pathway. If exact symmetry is not present, as in yet larger capsids, then an external limitation of capsid growth is necessary. A recent rontroversy has concerned the structure which of polyoma (Rayment et al., 1982), apparently has an all-pentameric capsid violating the Caspar-Klug assignment’ of 420 subunits to a T = 7d latt.ice. Since capsomeres are seen upon disruption of polyoma (Friedmann, 1971), it is reasonable to consider the pentamers as complex st’ruct’ure units. These building blocks may be divided into two classes, 60 forming a shell and 12 at the vertex locations. Pentamers of the major coat protein. VPl, may form different bonds in the two topologically distinct locat’ions or may be modified by the minor proteins, VP2 and VP3 (Burnett,, 1984; Oostrum & Burnett, 1984). single-polypeptide structure was. A unit emphasized in the analyses of the subunit interactions in tomato bushy stunt virus (Olson et ab., 1983) and sout,hern bean mosaic virus (Rossmann et al., 1983a,h). If these T =3 viruses with 180 polypeptides are considered in terms of hexameric and pentameric clusters, a striking pattern of conserved cont’act,s emerges. These fall into two classes. The five contacts between the pentameric subunits are almost identical to those in three of the six contacts between the hexameric subunits, the other three being quite different. There is only one other set of highly conserved contacts, which relates the three independent subunits at the quasithreefold axis. This axis lies at the centre of a trio,

141

formed from a pentamer and two hexamers. which is stabilized by Ca2’ ions. When calcium is withdrawn from tomato bushy stunt virus, the contacts within pentamers and hexamers remain essentially unchanged but the previously stabilized interfaces move apart and the capsid expands (Robinson & Harrison, 1982). Additionally, fivefold contacts predominate in both southern bean mosaic virus and satellite tobacco necrosis virus (Rossmann et al., 1983a). The structural integrity of the pentamers and hexamers is greater than that of other clusters, which suggests that their possible role in assembly should be considered. The major problem in assembly is to explain how a particular structural subunit “knows” the correct conformation to adopt as it is placed in its location in a growing capsid. This problem does not occur in systems with closed interactions. For example a trimer such as hexon has three extensive contact surfaces between its subunits. When all are in place, the system is closed and no further growth can occur. A system with such interactions is selflimit’ing. Systems without closed interactions must be limited by another mechanism, as in the definition of the length of filamentous bacteriophage by the nucleic acid about which the subunits assemble. An advantage of considering the T = 3 plant viruses as pentamer-hexamer aggregates is that all 20 hexamers form one set of identical contacts, and many of the contacts are identical to those made by the 12 pentamers. Assembly of the small T = 3 plant viruses could occur by the selflimiting aggregation of polypeptides into hexamers and pentamers. utilizing the closed nature of these interact’ions, since the hexamers themselves form a closed set of 20. Assembly has been studied in several plant viruses, and many will form T = 1 particles when the x-terminal arms of the coat proteins are removed. Examples are brome mosaic virus, cowpea chlorotic mottle virus and alfalfa mosaic virus. Assembly from dimers lacking N-terminal arms has been demonstrated in southern bean mosaic virus (Erickson & Rossmann, 1982) and turnip crinkle virus (P. K. Sorger, P. G. Stockley & S. C. Harrison, unpublished results). It is noteworthy that the N-terminal arm stabilizes the hexameric interface that is unlike that found in the pentamer. These experiments suggest that hexamers cannot form without arms, and that pentamer formation alone would lead to the observed T = 1 capsids. Although the purified coat protein is normally assumed to be in dimeric form, there is evidence that dimers are not the only multimers in solution. The polypeptides of brome mosaic virus are present as a dynamic equilibrium of monomers: dimers and higher oligomers (Cuillel et aZ., 1983a). Thus the preponderance of dimers frequently observed most probably reflects the majority species in a pool of components. Assembly of brome mosaic virus is very rapid, taking place in seconds, and t*hrough intermediate species significantly larger than a dimer (Cuillel et aZ., 19836). Assembly is thus

142

R. M. Burnett

autocatalytic, proceeding rapidly to completion once set in motion, which indicates that the assembly path is completely defined as soon as the correct geometry has been established. The dimers in turnip crinkle virus (Golden & Harrison, 1982), which is similar to tomato bushy stunt virus, have subunits joined by their projecting domains. This dimer does not occur within a pentamer or hexamer in the virion, but connects a hexamer to another hexamer or to a pentamer. It is striking that this dimer links two sets of closed interactions, as it then establishes the correct geometry for the vertex. Two linked sets, each initiated by one subunit of the dimer, could link pentamers and give a T = 1 capsid, or link a pentamer to a hexamer and give a T = 3 capsid. The size of RNA, the presence of cations and the pH all influence the assembly of southern bean mosaic virus coat protein into T = 1 or T = 3 virions (Savithri & Erickson, 1983). The data are compatible with pH-dependent capsid geometry, with the role of Ca2+ ions limited to stabilizing the final capsids. This role is consistent with the swelling observed upon removal of calcium. In the small plant viruses, closed interactions about fivefold and sixfold axes linked by dimeric units lead to the correct geometry. In larger systems, such as adenovirus, this mechanism is no longer possible. Since hexons lie on a p3 net, there is no limitation to continuous growth of a facet. However, larger aggregates can spontaneously selfassemble once they have formed. In adenovirus, 20 GONs can form empty shells (Pereira & Wrigley, 1974), and 12 pentons can form dodecons (Norrby, 1966). It is therefore reasonable to conclude that, If more than 60 polypeptides are to self-assemble. then they must be present as oligomers. Even if oligomers form, when there are more than 60 an external limitation will be required. Limitation of assembly could be provided by nucleic acid, as in the filamentous bacteriophage, or by scaffolding proteins. The scaffolding proteins themselves must be capable of self-assembly along the lines described. Two pathways could be imagined for adenovirus. In the first. scaffolding proteins self-assemble into a core. The facets of the core are then coated with hexons, the edges providing the limitation of growth. In the second, hexons are complexed with other proteins to form units that assemble into a very t’hick shell, from which the scaffolding protein is removed. Once a shell has formed, then other proteins can bind to their individual locations without necessarily requiring the same symmetry used in assembly. The basic framework has an intrinsic stability, which is maintained or even improved as other components are added. An analogous situation is seen in the complex isometric bacteriophage heads, such as t’hose of A, #ZbK and T4, which form an icosahedral framework from a large protein. The head expands to its mature form upon insertion of a small protein (Eiserling, 1979). Interactions between assembly subunits must be

in a rather delicate balance and cannot be too strong as otherwise errors in assembly would occur. The conflicting demands of accurate assembly and final stability seem to be resolved in viruses by the addition of a different component once assembly is complete. The calcium ions appear to play a stabilizing role in the plant viruses similar to that of polypeptide IX in adenovirus. I thank Dr ,Janet L. Smith for several helpful discussions and for her critical reading of the manuscript. The investigation was supported by Public Health Service grant AI-17270 from the Xatlonal Institute of Allergy and Infectious Diseases. and by an Irma T. Hirsch1 Career Scientist Award.

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