Dissecting the roles of VP0 cleavage and RNA packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus.

Empty capsids of foot-and-mouth disease virus (FMDV) type A22 Iraq 24/64, whose structure has been solved by X-ray crystallography, are unusual for picornaviruses since they contain VP2 and VP4, the cleavage products of the protein precursor VP0. Both the N terminus of VP1 and the C terminus of VP4, which pack together close to the icosahedral threefold symmetry axis where three pentamers associate, are more disordered in the empty capsid than they are in the RNA-containing virus. The ordering of these termini in the presence of RNA strengthens interactions within a single protomer and between protomers belonging to different pentamers. The disorder in the FMDV empty capsid forms a subset of that seen in the poliovirus empty capsid, which has VP0 intact. Thus, VP0 cleavage confers stability on the picornavirus capsid over and above that attributable to RNA encapsidation. In both FMDV and poliovirus empty capsids, the internal disordering uncovers a conserved histidine which has been proposed to be involved in the cleavage of VP0. A comparison of the putative active sites in FMDV and poliovirus suggests a structural explanation for the sequence specificity of the cleavage reaction.     

Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.     

Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide.

We established a human cell line which was persistently infected (PI) by the normally cytolytic echovirus 6. All of the cultured PI cells contained genome-size viral RNA which was synthesized continuously and incorporated into virus particles. This steady-state infection has been maintained for more than 6 years. In contrast to RNA of wild-type echovirus 6, the viral RNA from PI cells was not lytic when transfected into uninfected, susceptible cells. The capsid polypeptides of the virus particles produced during lytic infections were compared with those of virus particles from PI cells. Wild-type virions contained five polypeptides with molecular masses of 31.5, 27, 25.8, 21.2, and 9.5 kilodaltons. Comparison of polypeptide profiles of virions and empty immature capsids along with peptide analyses by immunoblotting and partial proteolysis of isolated viral proteins identified the cleavage products of the 31.5-kilodalton polypeptide (VP0) as the two smaller polypeptides (VP2 and VP4). The virus particles produced by PI cells as well as cellular extracts of PI cells contained only the three largest proteins (VP0, VP1, and VP3), indicating that VP0 was not processed during persistent infection. The lack of VP2 and VP4 in the defective virus particles coincided with their inability to attach to uninfected, susceptible cells. The maintenance of the steady-state infection of echovirus 6 was not dependent upon the release of virus particles from PI cells.     

Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 A resolution.

The crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 A resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VP0 to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N-terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N-terminal extension of VP1, as well as portions corresponding to VP4 and the N-terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP0 scissile bond is located some 20 A away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoil-shaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA-dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N-terminal network and in generating stable virions.     

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid resistance

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH(4)Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.     

C terminus of infectious bursal disease virus major capsid protein VP2 is involved in definition of the T number for capsid assembly

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.     

The pH stability of foot-and-mouth disease virus

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This review summarized the molecular determinants of the acid stability of FMDV in order to explore the uncoating mechanism of FMDV and improve the acid stability of vaccines.

Background

The foot-and-mouth disease virus (FMDV) capsid is highly acid labile and tends to dissociate into pentameric subunits at acidic condition to release viral RNA for initiating virus replication. However, the acid stability of virus capsid is greatly required for the maintenance of intact virion during the process of virus culture and vaccine production. The conflict between the acid lability in vivo and acid stability in vitro of FMDV capsid promotes the selection of a series of amino acid substitutions which can confer resistance to acid-induced FMDV inactivation. In order to explore the uncoating activity of FMDV and enhance the acid stability of vaccines, we summarized the available works about the pH stability of FMDV.

Main body of the abstract

In this review, we analyzed the intrinsic reasons for the acid instability of FMDV from the structural and functional aspects. We also listed all substitutions obtained by different research methods and showed them in the partial capsid of FMDV. We found that a quadrangle region in the viral capsid was the place where a great many pH-sensitive residues were distributed. As the uncoating event of FMDV is dependent on the pH-sensitive amino acid residues in the capsid, this most pH-sensitive position indicates a potential candidate location for RNA delivery triggered by the acid-induced coat disassociation.

Short conclusion

This review provided an overview of the pH stability of FMDV. The study of pH stability of FMDV not only contributes to the exploration of molecule and mechanism information for FMDV uncoating, but also enlightens the development of FMDV vaccines, including the traditionally inactivated vaccines and the new VLP (virus-like particle) vaccines.

     

Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease viruses from serotypes O, A and C have been crystallized. The particular strains studied include O1K, A10(61), A22 Iraq 24/64, A24 Cruzeiro and C-S8c1. In addition, crystals have been grown of G67, a monoclonal antibody neutralization escape mutant derived from O1K, and of virus R100, recovered after the establishment of a persistent infection in baby hamster kidney cells with C-S8c1. Empty particles, capsids which lack the RNA genome, have also been crystallized for subtypes A22 Iraq 24/64 and A10(61). In almost all cases, crystals suitable for high resolution structure determination were obtained from (NH4)2SO4 or mixtures of polyethylene glycol and NH4Cl.     

A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Lability and Confer Resistance to Acid-Dependent Uncoating Inhibition

The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH₄Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH₄Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH₄Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid.Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus in the family Picornaviridae. FMDV displays epithelial tropism and is responsible for a highly contagious disease of cloven-hoofed animals (23, 60). FMDV populations are quasispecies and exhibit a high potential for variation and adaptation, one consequence of which is the extensive antigenic diversity of this virus, reflected in the existence of seven serotypes and multiple antigenic variants (reviewed in references 17 and 60). Different cellular receptors, including α_vβ integrins and heparan sulfate (HS) glycosaminoglycans, have been described for natural isolates and tissue culture-adapted FMDVs (3, 4, 6, 28-31, 56). However, viruses that are infectious in vivo use integrins as receptors (28). The interaction between FMDV and the integrin molecule is mediated by an Arg-Gly-Asp (RGD) triplet located at the G-H loop of capsid protein VP1 (9, 47). FMDV isolates interacting with integrins gain entry into the cell following clathrin-mediated endocytosis (8, 39, 52). On the other hand, it has been described that a genetically engineered HS-binding mutant uses caveolae to enter into cultured cells (51). After internalization, FMDV must release its genomic RNA molecule of positive polarity into the host cell cytoplasm to establish a productive infection. Early work showed that a variety of lysosomotropic agents, such as weak bases and ionophores that block acidification of endosomes, inhibit FMDV infection (5, 11-13), indicating that genome release is dependent on endosomal acidification. In addition, internalized FMDV particles colocalize with markers from early and recycling endosomes (8, 51, 52) and FMDV infection is reduced by expression of a dominant negative mutant of Rab5 (33), suggesting that FMDV may release its genome from these compartments.The FMDV capsid comprises 60 copies of each of the four structural proteins (VP1 to VP4) arranged in an icosahedral lattice of 12 pentameric subunits. FMDV particles are highly acid labile and disassemble at pH values slightly below neutrality (13). Acid lability is not a feature of the capsids of other picornaviruses, such as Enterovirus. Pentameric subunits are intermediates of FMDV assembly and disassembly (64). A high density of His residues is found close to the interpentameric interface. Protonation of these residues at the acidic pH in the endosomes has been proposed to trigger acid-induced capsid disassembly by electrostatic repulsion between the protonated His side chains (1). His 142 (H142) in VP3 of type A FMDV is involved in a His-α-helix dipole interaction, which is likely to influence the acid lability of FMDV (13). In silico predictions suggested that H142 and H145 in VP3 may have the greatest effect on this process (63). Experimental evidence of the involvement of H142 of VP3 in acid-induced disassembly of FMDV has also been reported (20). Concomitantly with capsid disassembly into pentameric intermediates, internal protein VP4 and viral RNA are released. VP4 is a highly hydrophobic and myristoylated protein (7) whose release has been suggested to mediate membrane permeabilization and ion channel formation, thus facilitating the endosomal exit of viral RNA (15, 16, 34).Besides providing information about the endosomal pH requirements for the release of virus genomes, drugs modifying endosomal acidification can reveal the molecular changes associated with viral resistance to their action. These analyses may also address whether the balance between acid lability and capsid stability required for completion of virus replication allows FMDV, which disassembles at a pH close to neutrality, to escape inhibition by drugs raising the endosomal pH. In this work, we have isolated and characterized FMDV mutants that are able to escape from the inhibition of endosomal acidification exerted by NH₄Cl, a lysosomotropic weak base that raises endolysosomal pH and impairs uncoating and infection of viruses that require transit through acidic endosomal compartments for penetration (5, 26, 53). These mutants showed an increased acid lability, which is likely to allow them to uncoat at more-alkaline pH values. A single amino acid substitution close to the interpentameric interfaces in the capsid of one of these mutants was responsible for a total resistance to the elevation in endosomal pH caused by NH₄Cl treatment and for the acid-labile phenotype.     

Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture.

Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability. Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100). Only 5 of 15 mutations in P1 of R100 were silent. Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date. Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing. The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1. Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies. In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.     

Analysis of the protein-protein interactions in the parvovirus H-1 capsid.

The structure of the icosahedral capsid of the H-1 parvovirus was probed by chemical cross-linking methods. Treatment of empty capsids with high-molecular-weight polyethylene glycols resulted in irreversible aggregation of the minor capsid protein VP1. Multimers of VP1 containing at least five and perhaps six molecules were obtained, but only with empty capsids and not with the full, DNA-containing virus. Cross-linking of the empty capsids with dimethylsuberimidate confirmed the assignments of the products formed after treatment with polyethylene glycol. With dimethylsuberimidate the most abundant product was a heterologous dimer containing VP1 and the major capsid protein VP2'. A small amount of homologous VP2' dimer was also obtained, but the majority of VP2' remained unreacted even at high concentrations of dimethylsuberimidate. The capsid proteins of the full virus, on the other hand, were completely unreactive to dimethylsuberimidate. The data suggest that the minor protein VP1 may be clustered in the capsid and perhaps composes one or two of the morphological units of the icosahedral shell.     

Poliovirus empty capsid morphogenesis: evidence for conformational differences between self- and extract-assembled empty capsids.

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty capsids (isoelectric point, 5.0) were sensitive to both proteinases. Capsid proteins VP0 and VP1 were attacked predominantly, whereas VP3 was resistant to cleavage. Unpolymerized 14S particles possessed a trypsin sensitivity which was qualitatively similar to that of self-assembled empty shells. Surface-specific iodination of virions and procapsids labeled VP1 exclusively. In contrast, radioiodination of self-assembled empty capsids labeled predominantly VP0. After radioiodination the sedimentation coefficient corrected to water at 20 degrees C, the isoelectric point, and the trypsin resistance of the procapsids remained unchanged. Procapsids and extract-assembled empty capsids were N antigenic, whereas self-assembled empty capsids were H antigenic. Self-assembled empty capsids were not converted to pH 6.8 trypsin-resistant structures by incubation with a virus-infected cytoplasmic extract. However, 14S particles assembled in the presence of a mock-infected extract formed empty capsids, 20% of which resembled extract-assembled empty shells as determined by the above-described criteria. These and related findings are discussed in terms of empty capsid structure and morphogenesis.     

A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini

The complex infection process of parvoviruses is not well understood so far. An important role has been attributed to a phospholipase A2 domain which is located within the unique N terminus of the capsid protein VP1. Based on the structural difference between adeno-associated virus type 2 wild-type capsids and capsids lacking VP1 or VP2, we show via electron cryomicroscopy that the N termini of VP1 and VP2 are involved in forming globules inside the capsids of empty and full particles. Upon limited heat shock, VP1 and possibly VP2 become exposed on the outsides of full but not empty capsids, which is correlated with the disappearance of the globules in the inner surfaces of the capsids. Using molecular modeling, we discuss the constraints on the release of the globularly organized VP1-unique N termini through the channels at the fivefold symmetry axes outside of the capsid.     

Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment.

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.     

The molecular basis of the antigenic variation of foot-and-mouth disease virus.

We have cloned and sequenced the viral protein (VP1)-coding regions of two foot-and-mouth disease virus (FMDV) serotypes (C1 and A5). Comparison of the derived amino acid sequences with the known VP1 sequence of FMDV O1K and the two FMDV A subtypes A10 and A12 shows two highly variable regions in the protein, at positions 40-60 and 130-160, as possible antigenic sites. In both variable regions, several sites could be detected where all three sequences of the A subtypes are identical but the three types A, C and O differ from each other. The second variable region overlaps with a major immunogenic determinant of the virus.     

Detecting small changes and additional peptides in the canine parvovirus capsid structure

Parvovirus capsids are assembled from multiple forms of a single protein and are quite stable structurally. However, in order to infect cells, conformational plasticity of the capsid is required and this likely involves the exposure of structures that are buried within the structural models. The presence of functional asymmetry in the otherwise icosahedral capsid has also been proposed. Here we examined the protein composition of canine parvovirus capsids and evaluated their structural variation and permeability by protease sensitivity, spectrofluorometry, and negative staining electron microscopy. Additional protein forms identified included an apparent smaller variant of the virus protein 1 (VP1) and a small proportion of a cleaved form of VP2. Only a small percentage of the proteins in intact capsids were cleaved by any of the proteases tested. The capsid susceptibility to proteolysis varied with temperature but new cleavages were not revealed. No global change in the capsid structure was observed by analysis of Trp fluorescence when capsids were heated between 40 degrees C and 60 degrees C. However, increased polarity of empty capsids was indicated by bis-ANS binding, something not seen for DNA-containing capsids. Removal of calcium with EGTA or exposure to pHs as low as 5.0 had little effect on the structure, but at pH 4.0 changes were revealed by proteinase K digestion. Exposure of viral DNA to the external environment started above 50 degrees C. Some negative stains showed increased permeability of empty capsids at higher temperatures, but no effects were seen after EGTA treatment.     

Lytic replication of Kaposi's sarcoma-associated herpesvirus results in the formation of multiple capsid species: isolation and molecular characterization of A, B, and C capsids from a gammaherpesvirus

Despite the discovery of Epstein-Barr virus more than 35 years ago, a thorough understanding of gammaherpesvirus capsid composition and structure has remained elusive. We approached this problem by purifying capsids from Kaposi's sarcoma-associated herpesvirus (KSHV), the only other known human gammaherpesvirus. The results from our biochemical and imaging analyses demonstrate that KSHV capsids possess a typical herpesvirus icosahedral capsid shell composed of four structural proteins. The hexameric and pentameric capsomers are composed of the major capsid protein (MCP) encoded by open reading frame 25. The heterotrimeric complexes, forming the capsid floor between the hexons and pentons, are each composed of one molecule of ORF62 and two molecules of ORF26. Each of these proteins has significant amino acid sequence homology to capsid proteins in alpha- and betaherpesviruses. In contrast, the fourth protein, ORF65, lacks significant sequence homology to its structural counterparts from the other subfamilies. Nevertheless, this small, basic, and highly antigenic protein decorates the surface of the capsids, as does, for example, the even smaller basic capsid protein VP26 of herpes simplex virus type 1. We have also found that, as with the alpha- and betaherpesviruses, lytic replication of KSHV leads to the formation of at least three capsid species, A, B, and C, with masses of approximately 200, 230, and 300 MDa, respectively. A capsids are empty, B capsids contain an inner array of a fifth structural protein, ORF17.5, and C capsids contain the viral genome.     

Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids

Adeno-associated virus type 2 empty capsids are composed of three proteins, VP1, VP2 and VP3, which have relative molecular masses of 87, 72 and 62 kDa, respectively, and differ in their N-terminal amino acid sequences. They have a likely molar ratio of 1:1:8 and occupy symmetrical equivalent positions in an icosahedrally arranged protein shell. We have investigated empty capsids of adeno-associated virus type 2 by electron cryo-microscopy and icosahedral image reconstruction. The three-dimensional map at 1.05 nm resolution showed sets of three elongated spikes surrounding the three-fold symmetry axes and narrow empty channels at the five-fold axes. The inside of the capsid superimposed with the previously determined structure of the canine parvovirus (Q. Xie and M.S. Chapman, 1996, J. Mol. Biol., 264, 497–520), whereas the outer surface showed clear discrepancies. Globular structures at the inner surface of the capsid at the two-fold symmetry axes were identified as possible positions for the N-terminal extensions of VP1 and VP2.     

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.