Peripentonal Hexons Research Articles

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.

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.

Structure and function of cement proteins in human adenovirus

Human adenoviruses (HAdVs) are large (~150nm in diameter, 150MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause respiratory, ocular, and enteric diseases. The capsid shell of adenovirus (Ad) comprises multiple copies of three major capsid proteins (MCP: hexon, penton base and fiber) and four minor/cement proteins (IIIa, VI, VIII and IX) that are organized with pseudo T=25 icosahedral symmetry. In addition, six other proteins (V, VII, μ, IVa2, terminal protein and protease) are encapsidated along with the 36Kb dsDNA genome inside the capsid. The crystal structures of all three MCPs are known and so is their organization in the capsid from prior X-ray crystallography and cryoEM analyses. However structures and locations of various cement proteins are of considerable debate. We have determined and refined the structure of an entire human adenovirus employing X-ray crystallpgraphic methods at 3.8Å resolution. Adenovirus cement proteins play crucial roles in virion assembly, disassembly, cell entry and infection. Based on the refined crystal structure of adenovirus, we have determined the structure of the cement protein VI, a key membrane-lytic molecule and its associations with proteins V and VIII, which together glue peripentonal hexons beneath vertex region and connect them to rest of the capsid. Following virion maturation, the cleaved N-terminal pro-peptide of VI is observed deep in the peripentonal hexon cavity, detached from the membrane-lytic domain. Furthermore, we have significantly revised the recent cryoEM models for proteins IIIa and IX and both are located on the capsid exterior. Together, the cement proteins exclusively stabilize the hexon shell, thus rendering penton vertices the weakest links of the adenovirus capsid. Adenovirus cement protein structures reveal the molecular basis of the maturation cleavage of VI that is needed for endosome rupture and delivery of the virion into cytoplasm.

Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection.

Adenovirus cement proteins play crucial roles in virion assembly, disassembly, cell entry, and infection. Based on a refined crystal structure of the adenovirus virion at 3.8-Å resolution, we have determined the structures of all of the cement proteins (IIIa, VI, VIII, and IX) and their organization in two distinct layers. We have significantly revised the recent cryoelectron microscopy models for proteins IIIa and IX and show that both are located on the capsid exterior. Together, the cement proteins exclusively stabilize the hexon shell, thus rendering penton vertices the weakest links of the adenovirus capsid. We describe, for the first time to our knowledge, the structure of protein VI, a key membrane-lytic molecule, and unveil its associations with VIII and core protein V, which together glue peripentonal hexons beneath the vertex region and connect them to the rest of the capsid on the interior. Following virion maturation, the cleaved N-terminal propeptide of VI is observed, reaching deep into the peripentonal hexon cavity, detached from the membrane-lytic domain, so that the latter can be released. Our results thus provide the molecular basis for the requirement of maturation cleavage of protein VI. This process is essential for untethering and release of the membrane-lytic region, which is known to mediate endosome rupture and delivery of partially disassembled virions into the host cell cytoplasm.

The Cleaved N-Terminus of pVI Binds Peripentonal Hexons in Mature Adenovirus

Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen–deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule.

Localization of the N-Terminus of Minor Coat Protein IIIa in the Adenovirus Capsid

Minor coat protein IIIa is conserved in all adenoviruses (Ads) and is required for correct viral assembly, but its precise function in capsid organization is unknown. The latest Ad capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and to further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant Ad variants with IIIa encoding six-histidine (6His) tag, 6His, and FLAG peptides, or with 6His linked to FLAG with a (Gly 4Ser) 3 linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the Ad encoding the 6His–linker–FLAG–IIIa gene, the 6His tag was present in light particles, but not in mature virions. Immunoelectron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between the penton base and peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in Ad capsid assembly and disassembly.

Adenovirus Protein VI Mediates Membrane Disruption following Capsid Disassembly

In contrast to enveloped viruses, the mechanisms involved in membrane penetration by nonenveloped viruses are not as well understood. In these studies, we determined the relationship between adenovirus (Ad) capsid disassembly and the development of membrane lytic activity. Exposure to low pH or heating induced conformational changes in wild-type Ad but not in temperature-sensitive Ad (ts1) particles that fail to escape the early endosome. Wild-type Ad but not ts1 particles permeabilized model membranes (liposomes) and facilitated the cytosolic delivery of a ribotoxin. Alterations in wild-type Ad capsids were associated with the exposure of a pH-independent membrane lytic factor. Unexpectedly, this factor was identified as protein VI, a 22-kDa cement protein located beneath the peripentonal hexons in the viral capsid. Recombinant protein VI and preprotein VI, but not a deletion mutant lacking an N-terminal amphipathic alpha-helix, possessed membrane lytic activity similar to partially disassembled virions. A new model of Ad entry is proposed based on our present observations of capsid disassembly and membrane penetration.

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.

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 Å 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.

Correlation averaging of stem images of negatively stained adenovirus groups-of-nine herons

Adenovirus has an icosahedral capsid composed of 252 morphological units of two chemical types. There are 240 copies of the trimeric maior coat protein hexon (Mr 3 x 109,077) and 12 copies of the pentameric penton base and trimeric fibre vertex proteins. The X-ray crystal structure of hexon provides an accurate molecular model for the interpretation of electron micrographs of the virion and its fragments. Dissociation of the virions first releases the 60 peripentonal hexons surrounding the pentons, and then the remaining hexons as planar groups-of-nine hexons (GONs). The disruption pattern of the virion, and the non-random dissociation of the GONs, are at variance with the overall organization of the capsid as small p3 arrays of 12 hexons on each facet.

Interpretation of electron micrographs of adenovirus hexon arrays using a crystallographic molecular model

Two types of two-dimensional arrays of purified adenovirus type 2 hexon have been obtained and analyzed by Fourier filtration of their electron micrographs. One array contained continuously close-packed hexons, distributed on a hexagonal p3 lattice, with a unit cell dimension of 94 ± 2 Å The other array contained close-packed hexons with a regular absence, so that rings of six hexons related by sixfold symmetry formed a p6 unit cell. The cell dimension of the hexagonal array was 153 ± 3 Å with neighboring hexons separated by 88 ± 2 Å. Smaller p6 arrays were also formed by hexons freed from complete virions on the microscope grid by treatment with distilled water. A molecular model of hexon, known from the X-ray crystallographic structure, was used to interpret Fourier-filtered images of the arrays, and to determine the relative orientations of the hexon molecules. The hexon-hexon interaction in the p3 array is that found in the virion facet, whereas that in the p6 array is a planar form of the interaction between peripentonal hexons around the vertex.

Mutual orientation of peripentonal hexons and polypeptide subunits in the adenovirus capsid.

The mutual rotational orientation of the three hexon polypeptide subunits of the peripentonal hexons has been determined in relation to each other, to the penton and to the neighbouring hexons. The possible mutual orientation of all the polypeptide subunits of the adenovirus-building hexon capsomers are presented on a tentative virus model.

Electron microscopic studies on the connections between capsomers in the adenovirus capsid

Electron microscopic observation of negatively stained virions suggests that the interhexonal spaces within the adenovirus capsid are bridged by interhexonal connective elements. Similar connections are also detected between pentons and the five peripentonal hexons. Examinations of electron micrographs by Markham's rotational integration technique reveals the presence of a regular network of the connective elements between the capsomers in the whole capsid. The length of these connections corresponds to about one-third of the diameter of the hexons. The connections consist of two approximately parallel elements, thus every hexon is joined to its six nearest neighbours by 12 connections. A possible mutual orientation pattern of the connective elements in the whole capsid is presented on a tentative virus model.

Structure and assembly of adenoviruses.

Adenoviruses, this year celebrating the 30th anniversary of their discovery by man, are still in the foreground of scientific interest. With their multiple genes they are in fact one of the outstanding model systems for studying gene expression and its control in mammalian cells, as evidenced in numerous reviews (PHILIPSON et al. 1975; DOERFLER 1977; GINSBERG 1979; PHILIPSON 1979; NEVINS and CHEN-KIANG 1981; FLINT and BROKER 1981). The advances in the biochemistry and molecular biology of these viruses have not yet been balanced by equal progress in ultrastructural research of the virion itself and its assembly in the cell, although the architectural problems have recently received attention (PEREIRA and WRIGLEY 1974; EVERITT et al. 1975; BROWN et al. 1975; CORDEN et al. 1976; BURNETT et al. 1978; NERMUT 1980). Although adenoviruses are recovered from several hosts, including among others humans, monkeys, mice, and birds, with distinct but small differences in single polypeptides, the virion itself is of remarkably uniform morphology (Fig. 1). They all have an icosahedral shell and the capsid is built up from 252 capsomers, comprising 240 hexons, each surrounded by six neighboring capsomers, and 12 pentons at the vertices of the icosahedron each surrounded by five peripentonal hexons (VALENTINE and PEREIRA 1965; GINSBERG etal. 1966). Inside the capsid is a nuclear core containing the viral DNA and at least three viral proteins.KeywordsAdenovirus TypeHuman AdenovirusMature VirionEmpty CapsidPenton BaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Antigenic Determinants of Adenovirus Capsids

Abstract We have tested the two principal theories which explain the previous finding that small amounts of type-specific antibody to the adenovirus hexon can neutralize infectivity, whereas even large amounts of cross-reactive antibody do not. It has been suggested that the type-specific determinants are especially prominent in the virion. We have therefore measured the capacity of whole virus to bind appropriate antibodies, using a sensitive radioimmunoprecipitation (RIP) system. In fact, virions bound typespecific and cross-reactive antibodies impartially. Moreover, they bound both much less effectively than did free hexon or disrupted virus, suggesting that many of each kind of determinant are inaccessible in virions. It has been suggested that the type-specific determinants are confined to those hexons located next to the pentons, and that they are the targets for neutralizing antibody. We have therefore studied the antigenicity of peripentonal and nonamer hexons isolated from virions, and found that each possessed both kinds of determinants. Furthermore, these were present in the same proportions as in hexons purified from the soluble antigens in infected cells (“free hexons”). We conclude that the mechanism of neutralization by antibody is complicated, and that the type-specific determinants exposed on the virion must play a crucial role.

Structural proteins of adenoviruses. X. Isolation and topography of low molecular weight antigens from the virion of adenovirus type 2.

Abstract With high resolution SDS-polyacrylamide gel electrophoresis ( Maizel, 1971 ) and a new method to extract the basic proteins, it was ascertained that adenovirus type 2 contains at least 10 distinct polypeptides (II, III, IIIa, IV–X) and possibly more. Five proteins (V, VI, VII, VIII, and X) were purified by selective extraction in urea at high ionic strength, and low pH followed by preparative polyacrylamide electrophoresis toward the cathode at pH 3.4. Antisera, produced against proteins V, VI, and VII were used to reveal that these proteins were antigenically distinct and unrelated to hexons, pentons, and fibers. The location of the polypeptides was investigated by two methods of virion degradation ( Prage et al. , 1970 ). Polypeptides V and VII were associated with the DNA-containing core. Polypeptide VI appeared to be associated with all hexons of the virion. Hexons from the triangular facets of the capsid contained in addition to polypeptides II and VI, polypeptide IX, and possibly also polypeptide VIII. Hexons purified from infected cells contained only polypeptide II. Polypeptide IIIa was preferentially released together with the peripentonal hexons.

Structural proteins of adenoviruses: IV. Sequential degradation of the adenovirus type 2 virion

The composition of highly purified type 2 adenovirion has been studied by different degradation procedures. Dialysis against Tris-maleate buffers in the pH range 6.0–6.6 was used as an initial step, resulting in a quantitative release of pentons from the virions. At room temperature, these pentonless virions rapidly also release 5 or 6 hexons per original penton unit. The remainder of the capsid is further disintegrated by repeated freezing and thawing, which releases hexons in a nonaggregated form and also a precipitated DNA-protein complex. Two major protein components may be extracted from the latter with mineral acids. Amino acid analysis of these internal DNA complexed proteins reveals that they are highly basic, with an arginine content of 19%. Three or more low-molecular weight antigens distinct from hexon, penton base, fiber, and basic core proteins are also released during this procedure. Pyridine at a concentration of 10% degrades virions by a different pattern; hexons originating from the faces of the adenovirus icosahedron are preserved in groups of nine. Peripentonal hexons, fibers, degraded vertex capsomers, and low molecular weight antigens are also released. The internal core is retained in a suspended state and contains 15% of the total virion protein. The virion appears to be composed of 8 or more antigenically distinct proteins.