Difference imaging of adenovirus: bridging the resolution gap between X-ray crystallography and electron microscopy.

While X-ray crystallography provides atomic resolution structures of proteins and small viruses, electron microscopy provides complementary structural information on the organization of larger assemblies at lower resolution. A novel combination of these two techniques has bridged this resolution gap and revealed the various structural components forming the capsid of human type 2 adenovirus. An image reconstruction of the intact virus, derived from cryo-electron micrographs, was deconvolved with an approximate contrast transfer function to mitigate microscope distortions. A model capsid was calculated from 240 copies of the crystallographic structure of the major capsid protein and filtered to the correct resolution. Subtraction of the calculated capsid from the corrected reconstruction gave a three-dimensional difference map revealing the minor proteins that stabilize the virion. Elongated density penetrating the hexon capsid at the facet edges was ascribed to polypeptide IIIa, a component required for virion assembly. Density on the inner surface of the capsid, connecting the ring of peripentonal hexons, was assigned as polypeptide VI, a component that binds DNA. Identification of the regions of hexon that contact the penton base suggests a structural mechanism for previously proposed events during cell entry.     

Visualization of alpha-helices in a 6-angstrom resolution cryoelectron microscopy structure of adenovirus allows refinement of capsid protein assignments

The structure of adenovirus was determined to a resolution of 6 A by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that alpha-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed alpha-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly alpha-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in approximately 1.5 copies per hexon trimer and is predicted to have two long alpha-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long alpha-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long alpha-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal alpha-helix from protein IX.     

Cryoelectron microscopy map of Atadenovirus reveals cross-genus structural differences from human adenovirus

A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-A resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.     

Image reconstruction reveals the complex molecular organization of adenovirus

The three-dimensional structure of adenovirus has been determined by image reconstruction from cryo-electron micrographs. Comparison with the high resolution X-ray crystal structure of hexon, the major capsid protein, enabled an unusually detailed interpretation of the density map and confirmed the validity of the reconstruction. The hexon packing in the capsid shows more extensive intermolecular interfaces between facets than previously proposed. The reconstruction provides the first three-dimensional visualization of the vertex proteins, including the penton base and its associated protruding fiber. Three minor capsid proteins that stabilize and modulate capsomer interactions are revealed. One of these components stabilizes the group-of-nine hexons in the center of each facet and the other two bridge hexons in adjacent facets. The strategic positions of these proteins highlight the importance of cementing proteins in stabilizing a complex assembly.     

Finding and using local symmetry in identifying lower domain movements in hexon subunits of the herpes simplex virus type 1 B capsid

A characteristic of virus assembly is the use of symmetry to construct a complex capsid from a limited number of different proteins. Many spherical viruses display not only icosahedral symmetry, but also local symmetries, which further increase the redundancy of their structural proteins. We have developed a computational procedure for evaluating the quality of these local symmetries that allows us to probe the extent of local structural variations among subunits. This type of analysis can also provide orientation parameters for carrying out non-icosahedral averaging of quasi-equivalent subunits during three-dimensional structural determination. We have used this procedure to analyze the three types of hexon (P, E and C) in the 8.5 A resolution map of the herpes simplex virus type 1 (HSV-1) B capsid, determined by electron cryomicroscopy. The comparison of the three hexons showed that they have good overall 6-fold symmetry and are almost identical throughout most of their lengths. The largest difference among the three lies near the inner surface in a region of about 34 A in thickness. In this region, the P hexon displays slightly lower 6-fold symmetry than the C and E hexons. More detailed analysis showed that parts of two of the P hexon subunits are displaced counterclockwise with respect to their expected 6-fold positions. The most highly displaced subunit interacts with a subunit from an adjacent P hexon (P'). Using the local 6-fold symmetry axis of the P hexon as a rotation axis, we examined the geometrical relationships among the local symmetry axes of the surrounding capsomeres. Deviations from exact symmetry are also found among these local symmetry axes. The relevance of these findings to the process of capsid assembly is considered.     

A quasi-atomic model of human adenovirus type 5 capsid

Adenoviruses infect a wide range of vertebrates including humans. Their icosahedral capsids are composed of three major proteins: the trimeric hexon forms the facets and the penton, a noncovalent complex of the pentameric penton base and trimeric fibre proteins, is located at the 12 capsid vertices. Several proteins (IIIa, VI, VIII and IX) stabilise the capsid. We have obtained a 10 A resolution map of the human adenovirus 5 by image analysis from cryo-electron micrographs (cryoEMs). This map, in combination with the X-ray structures of the penton base and hexon, was used to build a quasi-atomic model of the arrangement of the two major capsid components and to analyse the hexon-hexon and hexon-penton interactions. The secondary proteins, notably VIII, were located by comparing cryoEM maps of native and pIX deletion mutant virions. Minor proteins IX and IIIa are located on the outside of the capsid, whereas protein VIII is organised with a T=2 lattice on the inner face of the capsid. The capsid organisation is compared with the known X-ray structure of bacteriophage PRD1.     

Capsid-like arrays in crystals of chimpanzee adenovirus hexon

The major coat protein, hexon, from a chimpanzee adenovirus (AdC68) is of interest as a target for vaccine vector modification. AdC68 hexon has been crystallized in the orthorhombic space group C222 with unit cell dimensions of a = 90.8 A, b = 433.0 A, c = 159.3 A, and one trimer (3 x 104,942 Da) in the asymmetric unit. The crystals diffract to 2.1 A resolution. Initial studies reveal that the molecular arrangement is quite unlike that in hexon crystals for human adenovirus. In the AdC68 crystals, hexon trimers are parallel and pack closely in two-dimensional continuous arrays similar to those formed on electron microscope grids. The AdC68 crystals are the first in which adenovirus hexon has molecular interactions that mimic those used in constructing the viral capsid.     

The structure of the human adenovirus 2 penton

The adenovirus penton, a noncovalent complex of the pentameric penton base and trimeric fiber proteins, comprises the vertices of the adenovirus capsid and contains all necessary components for viral attachment and internalization. The 3.3 A resolution crystal structure of human adenovirus 2 (hAd2) penton base shows that the monomer has a basal jellyroll domain and a distal irregular domain formed by two long insertions, a similar topology to the adenovirus hexon. The Arg-Gly-Asp (RGD) motif, required for interactions with cellular integrins, occurs on a flexible surface loop. The complex of penton base with bound N-terminal fiber peptide, determined at 3.5 A resolution, shows that the universal fiber motif FNPVYPY binds at the interface of adjacent penton base monomers and results in a localized structural rearrangement in the insertion domain of the penton base. These results give insight into the structure and assembly of the adenovirus capsid and will be of use for gene-therapy applications.     

CryoEM structure at 9A resolution of an adenovirus vector targeted to hematopoietic cells

We report a sub-nanometer resolution cryo-electron microscopy (cryoEM) structural analysis of an adenoviral vector, Ad35F, comprised of an adenovirus type 5 (Ad5) capsid pseudo-typed with an Ad35 fiber. This vector transduces human hematopoietic cells via association of its fiber protein with CD46, a member of the complement regulatory protein family. Major advances in data acquisition and image processing allowed a significant improvement in resolution compared to earlier structures. Analysis of the cryoEM density was enhanced by docking the crystal structures of both the hexon and penton base capsid proteins. CryoEM density was observed for hexon residues missing from the crystal structure that include hypervariable regions and the epitope of a neutralizing monoclonal antibody. Within the penton base, density was observed for the integrin-binding RGD loop missing from the crystal structure and for the flexible beta ribbon of the variable loop on the side of the penton base. The Ad35 fiber is flexible, consistent with the sequence insert in the third beta-spiral repeat. On the inner capsid surface density is revealed at the base of the hexons and below the penton base. A revised model is presented for protein IX within the virion. Well-defined density was assigned to a conserved domain in the N terminus of protein IX required for incorporation into the virion. For the C-terminal domain of protein IX two alternate conformations are proposed, either binding on the capsid surface or extending away from the capsid. This model is consistent with the tolerance of the C terminus for inserted ligands and its potential use in vector retargeting. This structural study increases our knowledge of Ad capsid assembly, antibody neutralization mechanisms, and may aid further improvements in gene delivery to important human cell types.     

Using cryoEM Reconstruction and Phase Extension to Determine Crystal Structure of Bacteriophage ϕ6 Major Capsid Protein

Bacteriophage ?6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ?6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C222₁. Matthews’s coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ?6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.     

Revised Crystal Structure of Human Adenovirus Reveals the Limits on Protein IX Quasi-Equivalence and on Analyzing Large Macromolecular Complexes

We report the revised crystal structure of a pseudo-typed human adenovirus at 3.8-Å resolution that is consistent with the atomic models of minor proteins determined by cryo-electron microscopy. The diffraction data from multiple crystals were rescaled and merged to increase the data completeness. The densities for the minor proteins were initially identified in the phase-refined omit maps that were further improved by the phases from docked poly-alanine models to build atomic structures. While the trimeric fiber molecules are disordered due to flexibility and imposition of 5-fold symmetry, the remaining major capsid proteins hexon and penton base are clearly ordered, with the exception of hypervariable region 1 of hexons, the RGD containing loop, and the N-termini of the penton base. The exterior minor protein IX together with the interior minor proteins IIIa and VIII stabilizes the adenovirus virion. A segment of N-terminal pro-peptide of VI is found in the interior cavities of peripentonal hexons, and the rest of VI is disordered. While the triskelion substructures formed by the N-termini of IX conform to excellent quasi 3-fold symmetry, the tetrameric coiled-coils formed by the C-termini and organized in parallel and anti-parallel arrangement do not exhibit any quasi-symmetry. This observation also conveys the pitfalls of using the quasi-equivalence as validation criteria for the structural analysis of extended (non-modular) capsid proteins such as IX. Together, these results remedy certain discrepancies in the previous X-ray model in agreement with the cryo-electron microscopy models.     

The structure of the adenovirus capsid. II. The packing symmetry of hexon and its implications for viral architecture

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 polyoma 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.     

Symmetry types, systems and their multiplicity in the structure of adenovirus capsid. I. Symmetry networks and general symmetry motifs

Each of the more than 1500 polypeptide molecules of 7 different types building up the adenovirus capsid--probably even those of their amino-acids--are in symmetrical location. Every kind of polypeptide forms a separately also symmetrical network in the capsid distributed according to their functions in the inner and outer side and the inside of the facets and edges, but always in compliance with the icosahedral symmetry. Therefore, each different polypeptide also means a general symmetry motif in the capsid in its own symmetry network. Hexons can be considered as general symmetry motifs in some special association that is because of their environmental position four kinds of hexon types can be found, which are on every facet, next to one another, like three identical groups of four (GOF) according to the three-fold rotational symmetry. Two polypeptides of a peripentonal hexon of each GOF orient toward the penton and the third toward the other penton located further on the same edge. There are two versions of the arrangement of the GOFs: the hexons surround either a polypeptide IX or a polypeptide IlIa. The two versions of GOFs on 20 facets symmetrically recurring 60 times as general hexon symmetry motifs form the capsid in combination with the network of other polypeptides. Ideally, the surface of the hexon trimer shows three-fold rotational and three-fold reflexional symmetries. In the arrangement of hexons in the facets the translational, rotational, horizontal and vertical reflexional symmetry and the combination of these, as well as the glide reflexion and the antisymmetry can be found. Each hexon has six nearest neighbours and every hexon takes part in the construction of three hexon rows. Every facet and every vertex made up of five facets has an antisymmetrical pair located on the opposite side of the capsid. Every triangular facet participates in forming three vertices and every facet has three nearest neighbouring facets. In the facets, the polypeptide subunits of polypeptide IX centered GOF hexons have identical counter-clockwise orientation but the orientation of the neighbouring facets is always opposite compared to each other. On the five-fold symmetry axis, any facet can be "turned on" to the adjacent facet or "rotated" to all the others and will take the symmetry and orientation of the facet it got turned on or rotated to. Thus, every facet together with the polypeptides attached to it shows a twenty-fold symmetry and multiplicity. An other type of symmetry and multiplicity in the capsid is that perpendicular to the 6 five-fold rotation axes run a geodetic (equatorial) ribbon like motif (superfieces) altogether six made up of 10 x 10 triangular facets and bent ten-times with an angle of 36 degrees. A triangular facet participates in forming three ribbon-like motifs, which intersect with each other on the given facet, but the same three motifs intersect repeatedly only on the antisymmetrically located facet.     

The structure of the adenovirus capsid. III. Hexon packing determined from electron micrographs of capsid fragments

The orientation and relative positions of all 240 hexons in the icosahedral outer capsid of adenovirus have been determined. Two types of capsid fragments, obtained after selective disruption of the virion, were analyzed using electron microscopy and image-processing techniques. Planar inverted groups-of-nine, arising from the central region of the capsid facet, were minimally stained to reveal the morphology of restricted regions of their component hexons. Images shown to be related by correspondence analysis were averaged and features of the individual hexon molecule, known from an X-ray crystallographic investigation, were used in their interpretation. The study confirms earlier observations that the hexons in the group-of-nine are distributed on a p3 net, shows that the hexons form a close-packed array using the pseudo-hexagonal shape of the hexon base, and provides their relative positions. Twenty interlocking groups-of-nine account for 180 of the 240 hexons present in the viral capsid. The orientation of the remaining 60 peripentonal hexons was obtained from a rotationally averaged image of a quarter-capsid, a novel viral fragment comprising five complete facets. Each peripentonal hexon forms planar asymmetric interactions with two neighbors in an adjacent group-of-nine so that it lies on an extension of the p3 net. The complete facet thus consists of 12 hexons arranged on a planar p3 net, with a shape that permits interlocking of hexons at the capsid edge. The relative positions of the hexons have been determined to within 5 A using the molecular model, and indicate that the pseudo-hexagonal basal regions are close-packed in a manner that maximizes the hexon-hexon contacts. The results confirm the model proposed earlier for the arrangement of hexons within the adenovirus capsid (Burnett, 1985), and show the power of the inter-disciplinary approach.     

Symmetry types, systems and their multiplicity in the structure of adenovirus capsid. II. Rotational facet groups of five-, three- and two-fold symmetry axes

The icosahedral adenovirus capsid has three rotational symmetry axes of different types. The six five-fold, ten three-fold and the fifteen two-fold axes have two superficial points each, altogether 62. The axes determine the number and location of the identical rotational facet groups and that during the different rotational phases which other regular facets and with what multiplicity shall be covered by them. The number of rotational facets of the five-, three- and two-fold rotational symmetry axes is 4, 6.66 and 10, respectively. In all the three cases, there are two kinds of possible arrangements of the facets. During the rotation--when the facets of the facet group placed on one by one to the neighbouring identical facet groups--at the five-fold axes, the facets of the rotational facet group get into cover position 12 times with all the 20 regular capsid facets, 20 times at the three-fold axes, and 30 times at the two-fold axes in a way that a different facet combination (facet hit) falls to every facet, and the original symmetry is not disturbed. After all, this means 240, 400 and 600 facet combinations, i.e. multiplicity in case of five-, three- and two-fold symmetry axes respectively, and these numbers correspond with that of the theoretically possible variations. The same results can be calculated by multiplying the number of real rotations of the capsid bringing the body into itself, i.e. the number 60 with the number of facets contributing to the five-, three- and two-fold rotational phases. The other way of the determination of multiplicity takes into account that all the facet groups of the capsid rotate simultaneously during all the rotational phases, and this multiplies the number of multiplicity with the number of the rotational types five-, three- and two-fold which result in one and the same multiplicity number in the case of five-, three- and two-fold symmetry, alike 1200. Perpendicular to the five-fold symmetry axes with the line of intersection drawn horizontally in the middle along the 6 geodetic ribbon like motifs a regular decagonal intersection forms and the capsid can be cut into two equal parts, in which the polypeptides show a 72 degree rotation from each other, but with a proper rotation the polypeptides get into a congruent position, which means 300 or 600 specific facet combinations. The capsid similar to the icosahedron has also 15 virtual mirror planes which divide the capsid into two, identically arranged halves, forming six right angle triangles on each facet, altogether 120 smaller rectangular so-called Mobius-triangles on the surface. In the three-fold symmetry axis of the facets, these triangles in two separate groups of three can be rotated symmetrically with 120 degrees according to the orientation of the polypeptide subunits in a way that the hexon and other polypeptides too nearly cover each other. Consequently, the adenovirus capsid is a symmetrically arranged body in which several various symmetry types and symmetry systems can be found and their structural symmetry elements exist simultaneously and covering each other. The icosahedral symmetry types and systems are valid and functional simultaneously and in parallel with great multiplicity, but the existence of more than 1500 structural elements in several depth levels, their order of location and distribution make the symmetry of the capsid richer and more complex.     

Structure of a Cell Entry Defective Human Adenovirus Provides Insights into Precursor Proteins and Capsid Maturation

Maturation of adenoviruses is distinguished by proteolytic processing of several interior minor capsid proteins and core proteins by the adenoviral protease and subsequent reorganization of adenovirus core. We report the results derived from the icosahedrally averaged cryo-EM structure of a cell entry defective form of adenovirus, designated ts1, at a resolution of 3.7 Å as well as of the localized reconstructions of unique hexons and penton base. The virion structure revealed the structures and organization of precursors of minor capsid proteins, pIIIa, pVI and pVIII, which are closely associated with the hexons on the capsid interior. In addition to a well-ordered helical domain (a.a. 310–397) of pIIIa, highlights of the structure include the precursors of VIII display significantly different structures near the cleavage sites. Moreover, we traced residues 4–96 of the membrane lytic protein (pVI) that includes an amphipathic helix occluded deep in the hexon cavity suggesting the possibility of co-assembly of hexons with the precursors of VI. In addition, we observe a second copy of pVI ordered up to residue L40 in the peripentonal hexons and a few fragments of density corresponding to 2nd and 3rd copies of pVI in other hexons. However, we see no evidence of precursors of VII binding in the hexon cavity. These findings suggest the possibility that differently bound pVI molecules undergo processing at the N-terminal cleavage sites at varying efficiencies, subsequently creating competition between the cleaved and uncleaved forms of VI, followed by reorganization, processing, and release of VI molecules from the hexon cavities.     

Binding of adenovirus and its external proteins to Triton X-114. Dependence on pH

35S-Labeled adenovirus type 2 (Ad2) (10 ng/ml) was incubated with 1% Triton X-114 at various pH values varying from 3.0 to 8.0. The detergent phase was separated from the aqueous phase by centrifugation, and the amounts of Ad2 were determined in the two phases. At pH 7.0-8.0, less than 5% of Ad2 was associated with the detergent phase; at pH 5.0 or below, about 60% of Ad2 was associated with the detergent phase. When a mixture of 35S-labeled capsid proteins was used at pH 7.0, 60-70% of the total proteins were associated with the detergent at pH 5.0, but less than 5% of the proteins interacted with detergent at pH 7.0. Among the three major external proteins (hexon, penton base, and fiber), penton base had the highest association with Triton X-114 at pH 5.0. Both intact virus and the capsid proteins that were associated with Triton X-114 at pH 5.0 were released into the aqueous phase on subsequent incubation at pH 7.0. On the basis of these results, it is suggested that mildly acidic pH induces amphiphilic properties in adenovirus capsid proteins and may help Ad2 escape from acidic endocytic vesicles.     

Model of the trimeric fiber and its interactions with the pentameric penton base of human adenovirus by cryo-electron microscopy

Adenovirus invades host cells by first binding to host receptors through a trimeric fiber, which contains three domains: a receptor-binding knob domain, a long flexible shaft domain, and a penton base-attachment tail domain. Although the structure of the knob domain associated with a portion of the shaft has been solved by X-ray crystallography, the in situ structure of the fiber in the virion is not known; thus, it remains a mystery how the trimeric fiber attaches to its underlying pentameric penton base. By high-resolution cryo-electron microscopy, we have determined the structure of the human adenovirus type 5 (Ad5) to 3.6-Å resolution and have reported the full atomic models for its capsid proteins, but not for the fiber whose density cannot be directly interpreted due to symmetry mismatch with the penton base. Here, we report the determination of the Ad5 fiber structure and its mode of attachment to the pentameric penton base by using an integrative approach of multi-resolution filtering, homology modeling, computational simulation of mismatched symmetries, and fitting of atomic models into cryo-electron microscopy density maps. Our structure reveals that the interactions between the trimeric fiber and the pentameric penton base are mediated by a hydrophobic ring on the top surface of the penton base and three flexible tails inserted into three of the five available grooves formed by neighboring subunits of penton base. These interaction sites provide the molecular basis for the symmetry mismatch and can be targeted for optimizing adenovirus for gene therapy applications.     

Determination of the nucleotide sequence for the penton-base gene of human adenovirus type 5

The major structural proteins of adenovirus (Ad), which form the external capsid, are hexon, penton base and fiber. The primary structure of the Ad5 penton base has been deduced from the nucleotide sequence of the corresponding gene. It has 98.6% homology with the sequence of the analogous protein from Ad2. This result is in contrast with the significantly lower homology found for the two other major structural proteins, the hexon and the fiber.     

Viral evolution revealed by bacteriophage PRD1 and human adenovirus coat protein structures.

The unusual bacteriophage PRD1 features a membrane beneath its icosahedral protein coat. The crystal structure of the major coat protein, P3, at 1.85 A resolution reveals a molecule with three interlocking subunits, each with two eight-stranded viral jelly rolls normal to the viral capsid, and putative membrane-interacting regions. Surprisingly, the P3 molecule closely resembles hexon, the equivalent protein in human adenovirus. Both viruses also have similar overall architecture, with identical capsid lattices and attachment proteins at their vertices. Although these two dsDNA viruses infect hosts from very different kingdoms, their striking similarities, from major coat protein through capsid architecture, strongly suggest their evolutionary relationship.