Picornavirus Capsid Research Articles

An Increase in Acid Resistance of Foot-and-Mouth Disease Virus Capsid Is Mediated by a Tyrosine Replacement of the VP2 Histidine Previously Associated with VP0 Cleavage

The foot-and-mouth disease virus (FMDV) capsid is highly acid labile, but introduction of amino acid replacements, including an N17D change in VP1, can increase its acid resistance. Using mutant VP1 N17D as a starting point, we isolated a virus with higher acid resistance carrying an additional replacement, VP2 H145Y, in a residue highly conserved among picornaviruses, which has been proposed to be responsible for VP0 cleavage. This mutant provides an example of the multifunctionality of picornavirus capsid residues.

Structures of the Procapsid and Mature Virion of Enterovirus 71 Strain 1095

Enterovirus 71 (EV71) is an important emerging human pathogen with a global distribution and presents a disease pattern resembling poliomyelitis with seasonal epidemics that include cases of severe neurological complications, such as acute flaccid paralysis. EV71 is a member of the Picornaviridae family, which consists of icosahedral, nonenveloped, single-stranded RNA viruses. Here we report structures derived from X-ray crystallography and cryoelectron microscopy (cryo-EM) for the 1095 strain of EV71, including a putative precursor in virus assembly, the procapsid, and the mature virus capsid. The cryo-EM map of the procapsid provides new structural information on portions of the capsid proteins VP0 and VP1 that are disordered in the higher-resolution crystal structures. Our structures solved from virus particles in solution are largely in agreement with those from prior X-ray crystallographic studies; however, we observe small but significant structural differences for the 1095 procapsid compared to a structure solved in a previous study (X. Wang, W. Peng, J. Ren, Z. Hu, J. Xu, Z. Lou, X. Li, W. Yin, X. Shen, C. Porta, T. S. Walter, G. Evans, D. Axford, R. Owen, D. J. Rowlands, J. Wang, D. I. Stuart, E. E. Fry, and Z. Rao, Nat. Struct. Mol. Biol. 19:424-429, 2012) for a different strain of EV71. For both EV71 strains, the procapsid is significantly larger in diameter than the mature capsid, unlike in any other picornavirus. Nonetheless, our results demonstrate that picornavirus capsid expansion is possible without RNA encapsidation and that picornavirus assembly may involve an inward radial collapse of the procapsid to yield the native virion.

Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance

The genome diversity of RNA viruses allows for rapid adaptation to a wide variety of adverse conditions. Accordingly, viruses can escape inhibition by most antiviral compounds targeting either viral or host factors. Here we exploited the capacity of RNA viruses for rapid adaptation to explore the evolutionary constraints of chaperone-mediated protein folding. We hypothesized that inhibiting a host molecular chaperone required for folding of a viral protein would force the virus to evolve an alternate folding strategy. We identified the chaperone Hsp90 as an essential factor for folding and maturation of picornavirus capsid proteins. Pharmacological inhibition of Hsp90 impaired the replication of poliovirus, rhinovirus, and coxsackievirus in cell culture. Strikingly, anti-Hsp90 treatment did not yield drug-resistant viruses, suggesting that the complexity of capsid folding precludes the emergence of alternate folding pathways. These results reveal tight evolutionary constraints on chaperone-mediated protein folding, which may be exploited for viral inhibition in vivo. Indeed, Hsp90 inhibitors drastically reduced poliovirus replication in infected animals without the emergence of drug-resistant escape mutants. We propose that targeting folding of viral proteins may provide a general antiviral strategy that is refractory to development of drug resistance.

Putative autocleavage of outer capsid protein micro1, allowing release of myristoylated peptide micro1N during particle uncoating, is critical for cell entry by reovirus.

Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein micro1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments micro1N (4 kDa; myristoylated) and micro1C (72 kDa). In this study, we found that micro1 cleavage to yield micro1N and micro1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective micro1 (asparagine --> alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including micro1 conformational change and sigma1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin alpha-sarcin). In addition, after these particles were allowed to enter cells, the delta region of micro1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective micro1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of micro1N/micro1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal micro1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

Conformational changes during proteolytic processing of a picornavirus capsid proteins.

We have used synthetic peptide antibodies to probe conformational changes that occur during the cleavage cascade which generates the capsid proteins of a picornavirus. The initial translation product of 97 kDa, the precursor of all four structural proteins, is cleaved to form a 63 kDa fragment which, we show, has significantly different folding characteristics to both its larger parent and its products. We demonstrate that proteolytic cleavages as distant as 520 residues from epitopes confer sufficiently large conformational changes as to render them unrecognisable. To our knowledge, this is the first demonstration of this phenomenon in the picornavirus system.

The Refined Crystal Structure of Cowpea Mosaic Virus at 2.8 Å Resolution

Comoviruses are a group of plant viruses in the picornavirus superfamily. The type member of comoviruses, cowpea mosaic virus (CPMV), was crystallized in the cubic space group I23, a = 317 Å and the hexagonal space group P6122, a = 451 Å, c = 1038 Å. Structures of three closely similar nucleoprotein particles were determined in the cubic form. The roughly 300-Å capsid was similar to the picornavirus capsid displaying a pseudo T = 3 (P = 3) surface lattice. The three β-sandwich domains adopt two orientations, one with the long axis radial and the other two with the long axes tangential in reference to the capsid sphere. T = 3 viruses display one or the other of these two orientations. The CPMV capsid was permeable to cesium ions, leading to a disturbance of the β-annulus inside a channel-like structure, suggesting an ion channel. The hexagonal crystal form diffracted X rays to 3 Å resolution, despite the large unit cell. The large (∼200 Å) solvent channels in the lattice allow exchange of CPMV cognate Fab fragments. As an initial step in the structure determination of the CPMV/Fab complex, the P6122 crystal structure was solved by molecular replacement with the CPMV model determined in the cubic cell.

Trans-encapsidation of a poliovirus replicon by different picornavirus capsid proteins.

A trans-encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successful trans-encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount of trans-encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans, >2 x 10(6) infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans, all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used. trans-encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.

The structure of tobacco ringspot virus: a link in the evolution of icosahedral capsids in the picornavirus superfamily.

Tobacco ringspot virus (TRSV) is a member of the nepovirus genus of icosahedral RNA plant viruses that cause disease in fruit crops. Nepoviruses, comoviruses and picornaviruses are classified in the picornavirus superfamily. Crystal structures of comoviruses and picornaviruses and the molecular mass of the TRSV subunit (sufficient to accommodate three beta-barrel domains) suggested that nepoviruses may represent a link in the evolution of the picornavirus capsids from a T = 3 icosahedral virus. This evolutionary process is thought to involve triplication of the capsid protein gene, to encode a three-domain polyprotein, followed by development of cleavage sites in the interdomain linking regions. Structural studies on TRSV were initiated to determine if the TRSV subunit corresponds to the proposed uncleaved three-domain polyprotein. The 3.5 A resolution structure of TRSV shows that the capsid protein consists of three beta-barrel domains covalently linked by extended polypeptides. The order of connectivity of the domains in TRSV confirms the proposed connectivity for the precleaved comovirus and picornavirus capsid polyprotein. Structural differences between equivalent domains in TRSV and comoviruses are confined to the external surface loops, interdomain connecting polypeptides and N termini. The three different domains within TRSV and comoviruses are more closely related at the structural level than the three individual domains within picornaviruses. The structural results confirm the notion of divergent evolution of the capsid polyproteins of nepoviruses, comoviruses and picornaviruses from a common ancestor. A number of residues were found to be conserved among various nepoviruses, some of which stabilize the quaternary structure of the three domains in the TRSV capsid protein subunit. Two conserved regions were identified on the external surface of TRSV, however, mutational studies will be needed to understand their functional significance. Nepoviruses transmitted by the same nematode species do not share regions with similar amino acid composition on the viral surface.

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.

Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues.

The first full-length hexon protein DNA and deduced amino acid sequences of a subgenus D adenovirus (AV) were determined from candidate AV48 (85-0844). Comprehensive comparison of this sequence with hexon protein sequences from human subgenera A, B, C, D, F, bovine AV3, and mouse AV1 revealed seven discrete hypervariable regions (HVRs) among the 250 variable residues in loops 1 and 2. These regions differed in length between serotypes, from 2 to 38 residues, and contained > 00% of hexon serotype-specific residues among human serotypes. Alignment with the published crystal structure of AV2 established the location and structure of the type-specific regions. Five HVRs were shown to be part of linear loops on the exposed surfaces of the protein, analogous to the serotype-specific loops or "puffs" in picornavirus capsid proteins. The HVRs were supported by a common framework of conserved residues, of which 68 to 75% were hydrophobic. Unique sequences were limited to the seven HVRs, so that one or more of these regions contain the type-specific neutralization epitopes. A neutralizing AV48 hexon-specific antiserum recognized linear peptides that corresponded to six HVRs by enzyme immunoassay. Affinity-purification removal of all peptide-reactive antibodies did not significantly decrease the neutralization titer. Eluted peptide-reactive antibodies did not neutralize. Human antisera that neutralized AV48 did not recognize linear peptides. Purified trimeric native hexon inhibited neutralization, but monomeric heat-denatured hexon did not. We conclude that the AV48 neutralization epitope(s) is complex and conformational.

SCH 38057: a picornavirus capsid-binding molecule with antiviral activity after the initial stage of viral uncoating

The activity of a new water-soluble molecule, SCH 38057, against picornaviruses is described. SCH 38057 inhibited plaque formation of selected entero- and rhinoviruses in a range of 10.2 to 29.1 μM (50% endpoint) and had a therapeutic index of 10 against poliovirus type 2 (polio 2) in HeLa cells. When administered orally or subcutaneously, SCH 38057 protected mice infected with either coxsackievirus B3 (CVB3) or echovirus-9 from mortality. The molecule provided a low level of protection against thermal inactivation of virus, indicating that SCH 38057 interacts with the picornavirus capsid. Binding studies with [ 3H]SCH 38057 revealed that the molecule binds to CVB3 and human rhinovirus 14 (HRV14) in a ratio of 29 and 19 molecules per viral particle, respectively. The affinity constant for SCH 38057 binding to CVB3 was 7.0 × 10 −4 M. When added to cultures of infected cells at 3 h after infection, SCH 38057 markedly inhibited viral RNA synthesis. This finding with lack of inhibition of attachment and loss of infectious virus after attachment were interpreted to indicate that, although SCH 38057 binds to the viral capsid, the molecule exerts its antiviral effect after the initial stage of picornavirus uncoating, i.e., after conversion of the 156S infectious viral particle to smaller subviral species.

Structure Determination of Antiviral Compound SCH 38057 Complexed with Human Rhinovirus 14

SCH 38057 (1-[6-(2-chloro-4-methoxyphenoxy)-hexyl]imidazole hydrochloride) is a new, water-soluble antiviral that has inhibitory activities against a number of picornavirus infections. The structure of the human rhinovirus 14 (HRV14) complex with SCH 38057 was determined at 3·0 Å resolution by single-crystal diffraction techniques using synchrotron X-radiation. SCH 38057 was found to bind at the innermost end of the hydrophobic pocket within the capsid protein VP1, a locus of binding of other antipicornaviral agents; however, the complex differs from previously reported complexes in two important aspects. It leaves a considerable volume near entrance to the binding pocket unoccupied. In addition, the alterations in the conformation of the VP1 polypeptide are similar to, but more extensive than those observed in HRV14 complexes with other antiviral agents. Although only 9 amino acids of VP1 have close contacts with the SCH 38057 molecule (within 3·6 Å), at least 36 amino acids from both VP1 and VP3 have significantly altered conformations (C α movement >0·5 Å versus native). The structures of complexes of HRV14 with SCH 38057 and WIN 51711 are compared. Aromatic ring interactions between picornavirus capsid residues and antiviral inhibitors are proposed to be among the major determinants for positioning of these compounds.

An insect picornavirus may have genome organization similar to that of caliciviruses

Computer-assisted analysis of the amino acid sequence of the product encoded by the sequenced 3′ portion of the cricket paralysis virus (CrPV), an insect picornavirus, genome showed that this protein is homologous not to the RNA-directed RNA polymerases, as originally suggested, but to the capsid proteins of mammalian picornaviruses. Alignment of the CrPV protein sequence with those of picornavirus and calicivirus capsid proteins demonstrated that the sequenced portion of the insect picornavirus genome encodes the C-terminal part of VP3 and the entire VP1. Thus CrPV seems to have a genome organization distinct from that of other picornaviruses but closely resembling that of caliciviruses, with the capsid proteins encoded in the 3′ part of the genome. On the other hand, the tentative phylogenetic trees generated from the VP3 alignment revealed grouping of CrPV with hepatitis A virus, a true picornavirus, not with caliciviruses. Thus CrPV may be a picornavirus with a calicivirus-like genome organization. Different options for CrPV genome expression are discussed.

Functional aspects of the capsid structure of mengo virus

The three-dimensional structure of the Mengo virus capsid has been determined at a resolution of 3.0 Å. This achievement is discussed in an historical context, and the general features of picornavirus capsid design are presented. The dynamic functional aspects of the Mengo virus capsid—namely its ability to interact with specific receptors on host cells, to dissociate and release the viral genomic RNA into the cellular cytoplasm, to assemble with progeny RNA molecules and form new virions, and to alter its external surface in order to evade neutralization by circulating antibodies—are discussed. Comparisons with other picornaviruses whose capsid structures have also been elucidated (poliovirus serotypes 1 and 3, human rhinovirus types 14 and 1A, and foot-and-mouth disease virus type O) illustrate both the similarities and the distinctive features of capsid design found within this family of mammalian viruses.

Conformational variability of a picornavirus capsid: pH-dependent structural changes of mengo virus related to its host receptor attachment site and disassembly

The structure of Mango virus had been determined from crystals grown in the presence of 100 m M phosphate buffer at pH 7.4. It is shown that Mengo virus is poorly infectious at the phosphate concentration similar to that in which it was crystallized. Maximal infectivity is achieved at 10 m M phosphate or less in physiological saline. The phosphate effect is ameliorated when the pH is lowered to 4.6. Although it has not been possible to study the crystal structure of the virus at low phosphate concentrations, it is shown that increasing the CI − concentration at pH 6.2 or decreasing the pH to 4.6 causes substantial conformational changes confined to the “pit,” a deep surface depression. These structural changes involve a movement of the “FMDV loop” (GH loop) in VP1, an ordering of the “VP3 loop” (GH loop in VP3) between 3176 and 3182, the displacement of a bound phosphate near the “FMDV loop” H loop in VP1), and movement of the carboxy terminus of VP2. The changes in conformation are correlated with the dissociation of the virion into pentamers at pH 6.2 and 150 mM CI −. The localization of the conformational changes and the correlated role of the phosphate in controlling infectivity support the hypothesis that the “pit” is the receptor attachment site.

Myristylation of picornavirus capsid protein VP4 and its structural significance

We have obtained evidence that poliovirus and other picornavirus particles are specifically modified by having myristic acid covalently bound to a capsid protein. The electron density map of poliovirus confirms the position of the myristate molecule and defines its location in the virus particle. Analogies with other myristylated proteins suggest that the myristate moiety in picornaviruses may be involved in capsid assembly or in the entry of virus into cells.

Implications of the picornavirus capsid structure for polyprotein processing.

Mature picornaviral proteins are derived by progressive, posttranslational cleavage of a precursor polyprotein. These cleavages play a role in the control of virus functions. Although the processed termini are separated by as much as 75 A in the native virus capsid, the fold and arrangement of polypeptide chains in a protomer before proteolysis are likely to be similar to that found in the mature virus. The three-dimensional structures of rhinovirus and Mengo virus suggest that the cleavage sites within the protomeric precursor are in structurally flexible regions. The final proteolytic processing event, maturation of the virion peptide VP0 (also called peptide 1AB) appears to occur by an unusual autocatalytic serine protease-type mechanism possibly involving viral RNA basic groups that would serve as proton-abstractors during the cleavage reaction.

The structure of picornaviruses: Classification of the bonding networks

An icosahedral shell with 60 each of three nonidentical polypeptide chains must contain at least four intersubunit bonds. A set of four connections defines a minimal bonding network. A classification scheme has been devised to count all of the minimal bonding networks; it has been found that there are 240 distinct possible four-bonded nets. The data of the previous paper by Scraba and co-workers ( Hordern, Leonard, and Scraba, 1979, Virology 97, 131–140) suggest that such an approach to the description of the picornavirus capsid might have validity. In turn, the minimal bonding net theory can be applied to the data obtained for the Mengo virion. The results of such an analysis suggest that, of the 240 possible minimal nets, there are two minimal nets that are completely consistent with the data obtained. Because the analysis is general and complete, there are no other possibilities. Deciding between the two remaining possible minimal nets will require further experiments.

Evidence for the existence of protomers in the assembly of encephalomyocarditis virus

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.