Myristylation of poliovirus capsid precursor P1 is required for assembly of subviral particles.

The poliovirus capsid precursor polyprotein, P1, is cotranslationally modified by the addition of myristic acid. We have examined the importance of myristylation of the P1 capsid precursor during the poliovirus assembly process by using a recently described recombinant vaccinia virus expression system which allows the independent production of the poliovirus P1 protein and the poliovirus 3CD proteinase (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). We constructed a site-directed mutation in the poliovirus cDNA encoding an alanine at the second amino acid position of P1 in place of the glycine residue required for the myristic acid addition and isolated a recombinant vaccinia virus (VVP1myr-) that expressed a nonmyristylated form of the P1 capsid precursor. The 3CD proteinase expressed by a coinfecting vaccinia virus, VVP3, proteolytically processed the nonmyristylated precursor P1 expressed by VVP1myr-. However, the processed capsid proteins, VP0, VP3, and VP1, did not assemble into 14S or 75S subviral particles, in contrast to the VP0, VP3, and VP1 proteins derived from the myristylated P1 precursor. When cells were coinfected with VVP1myr- and poliovirus type 1, the nonmyristylated P1 precursor expressed by VVP1myr- was processed by 3CD expressed by poliovirus, and the nonmyristylated VP0-VP3-VP1 (VP0-3-1) protomers were incorporated into capsid particles and virions which sedimented through a 30% sucrose cushion. Thus, the nonmyristylated P1 precursor and VP0-3-1 protomers were not excluded from sites of virion assembly, and the assembly defects observed for the nonmyristylated protomers were overcome in the presence of myristylated capsid protomers expressed by poliovirus. We conclude that myristylation of the poliovirus P1 capsid precursor plays an important role during poliovirus assembly by facilitating the appropriate interactions required between 5S protomer subunits to form stable 14S pentamers. The results of these studies demonstrate that the independent expression of the poliovirus P1 and 3CD proteins by using recombinant vaccinia viruses provides a unique experimental tool for analyzing the dynamics of the poliovirus assembly process.     

Identification of the threonine phosphorylation sites on the polyomavirus major capsid protein VP1: relationship to the activity of middle T antigen.

Phosphorylation of the polyomavirus major capsid protein VP1 was examined after in vivo 32P labeling of virus-infected cells. Two phosphorylated peptide fragments of VP1 were identified by protease digestion, high-performance liquid chromatography purification, mass spectrometry, and N-terminal sequencing. The peptides from residues 58 to 78 and residues 153 to 173 were phosphorylated on threonine. Site-directed mutations were introduced at these threonine sites, and mutant viruses were reconstructed. A threonine-to-glycine change at residue 63 (mutant G63) and a threonine-to-alanine change at residue 156 (mutant A156) resulted in viruses defective in phosphorylation of the respective peptides after in vivo labeling. Growth of the mutant G63 virus was similar to that of the wild-type virus, but the mutant A156 was inefficient in assembly of 240S viral particles. Polyomavirus nontransforming host range (hr-t) mutants are defective in VP1 threonine phosphorylation when grown in nonpermissive cells (R. L. Garcea, K. Ballmer-Hofer, and T. L. Benjamin, J. Virol. 54:311-316, 1985). Proteolytic mapping of VP1 peptides after in vivo labeling from hr-t mutant virus infections demonstrated that both residues T-63 and T-156 were affected. These results suggest that the block in virion assembly in hr-t mutant viruses is associated with a defect in phosphorylation of threonine 156.     

Poliovirus Mutants at Histidine 195 of VP2 Do Not Cleave VP0 into VP2 and VP4

The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.     

Myristoylation is important at multiple stages in poliovirus assembly.

The N-terminal glycine of the VP4 capsid subunit of poliovirus is covalently modified with myristic acid (C14 saturated fatty acid). To investigate the function of VP4 myristoylation in poliovirus replication, amino acid substitutions were placed within the myristoylation consensus sequence at the alanine residue (4003A) adjacent to the N-terminal glycine by using site-directed mutagenesis methods. Mutants which replace the alanine residue with a small hydrophobic residue such as leucine, valine, or glycine displayed normal levels of myristoylation and normal growth kinetics. Replacement with the polar amino acid histidine (4003A.H) also resulted in a level of myristoylation comparable to that of the wild type. However, replacement of the alanine residue with aspartic acid (4003A.D) caused a dramatic reduction (about 40 to 60%) in myristoylation levels of the VP4 precursors (P1 and VP0). In contrast, no differences in modification levels were found in either VP0 and VP4 proteins isolated from mature mutant virions, indicating that myristoylation is required for assembly of the infectious virion. The myristoylation levels of the VP0 proteins found in capsid assembly intermediates indicate that there is a strong but not absolute preference for myristoyl-modified subunits during pentamer formation. Complete myristoylation was observed in mature virions but not in assembly intermediates, indicating that there is a selection for myristoyl-modified subunits during stable RNA encapsidation to form the mature virus particle. In addition, even though mutant infectious virions are fully modified, the severe reduction in specific infectivity of both 4003A.D and 4003A.H purified viruses indicates that the amino acid residue adjacent to the N-terminal glycine apparently has an additional role early during viral infection and that mutations at this position induce pleiotropic effects.     

Functional relevance of amino acid residues involved in interactions with ordered nucleic acid in a spherical virus

In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.     

A mutation in VP4 defines a new step in the late stages of cell entry by poliovirus.

During the entry of poliovirus into cells, a conformational transition occurs within the virion that is dependent upon its binding to the cell surface receptor. This conformational rearrangement generates an altered particle of 135S, results in the extrusion of capsid protein VP4 and the amino terminus of VP1 from the virion interior, and leads to the acquisition of membrane-binding properties by the 135S particle. Although the subsequent fate of VP4 is unknown, its apparent absence from purified 135S particles has long suggested that VP4 is not directly involved during virus entry. We report here the construction by site-specific mutagenesis of a nonviable VP4 mutant that upon transfection of the cDNA appears to form mature virus particles. These particles, upon interaction with the cellular receptor, undergo the 135S conformational transition but are defective at a subsequent stage in virus entry. The results demonstrate that the participation of VP4 is required during cell entry of poliovirus. In addition, these data indicate the existence of additional stages in the cell entry process beyond receptor binding and the transition to 135S particles. These post-135S stages must include the poorly understood processes by which nonenveloped viruses cross the cell membrane, uncoat, and deliver their genomes into the cytoplasm.     

Amino acid substitutions in the poliovirus maturation cleavage site affect assembly and result in accumulation of provirions.

The assembly of infectious poliovirus virions requires a proteolytic cleavage between an asparagine-serine amino acid pair (the maturation cleavage site) in VP0 after encapsidation of the genomic RNA. In this study, we have investigated the effects that mutations in the maturation cleavage site have on P1 polyprotein processing, assembly of subviral intermediates, and encapsidation of the viral genomic RNA. We have made mutations in the maturation cleavage site which change the asparagine-serine amino acid pair to either glutamine-glycine or threonine-serine. The mutations were created by site-directed mutagenesis of P1 cDNAs which were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses. The P1 polyproteins expressed from the recombinant vaccinia viruses were analyzed for proteolytic processing and assembly defects in cells coinfected with a recombinant vaccinia virus (VV-P3) that expresses the poliovirus 3CD protease. A trans complementation system using a defective poliovirus genome was utilized to assess the capacity of the mutant P1 proteins to encapsidate genomic RNA (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The mutant P1 proteins containing the glutamine-glycine amino acid pair (VP4-QG) and the threonine-serine pair (VP4-TS) were processed by 3CD provided in trans from VV-P3. The processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor VP4-QG were unstable and failed to assemble into subviral structures in cells coinfected with VV-P3. However, the capsid proteins derived from VP4-QG did assemble into empty-capsid-like structures in the presence of the defective poliovirus genome. In contrast, the capsid proteins derived from processing of the VP4-TS mutant assembled into subviral intermediates both in the presence and in the absence of the defective genome RNA. By a sedimentation analysis, we determined that the capsid proteins derived from the VP4-TS precursor encapsidated the defective genome RNA. However, the cleavage of VP0 to VP4 and VP2 was delayed, resulting in the accumulation of provirions. The maturation cleavage of the VP0 protein containing the VP4-TS mutation was accelerated by incubation of the provirions at 37 degrees C. The results of these studies demonstrate that mutations in the maturation cleavage site have profound effects on the subsequent capability of the capsid proteins to assemble and provide evidence for the existence of the provirion as an assembly intermediate.     

Poliovirus capsid proteins derived from P1 precursors with glutamine-valine cleavage sites have defects in assembly and RNA encapsidation.

Assembly of poliovirus virions requires proteolytic cleavage of the P1 capsid precursor polyprotein between two separate glutamine-glycine (QG) amino acid pairs by the viral protease 3CD. In this study, we have investigated the effects on P1 polyprotein processing and subsequent assembly of processed capsid proteins caused by substitution of the glycine residue at the individual QG cleavage sites with valine (QG-->QV). P1 cDNAs encoding the valine substitutions were created by site-directed mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 precursors. The recombinant vaccinia virus-expressed mutant P1 polyproteins were analyzed for proteolytic processing defects in cells coinfected with a recombinant vaccinia virus (VVP3) that expresses the poliovirus 3CD protease and for processing and assembly defects by using a trans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome that does not express functional capsid proteins (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substituted precursors were proteolytically processed at the altered sites both in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites were slower than those of cleavage at the wild-type QG site in the precursor. Completely processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral structures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substitution at the amino terminus of VP1 (VP1-G001V) assembled empty capsid particles but were deficient in assembling RNA-containing virions. The assembly characteristics of the VP1-G001V mutant were compared with those of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaard and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletion of the first four amino-terminal residues of VP1 (VP1-delta 1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-delta 1-4 precursor also occurred more slowly than complete cleavage of the wild-type precursor, and formation of virions was delayed; however, capsid proteins derived from the VP1-G001V mutant assembled RNA-containing virions less efficiently than those derived from the VP1-delta 1-4 precursor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Catalysis of poliovirus VP0 maturation cleavage is not mediated by serine 10 of VP2.

The maturation of the poliovirus capsid occurs as the result of a single unexplained proteolytic event during which 58 to 59 copies of the 60 VP0 capsid protein precursors are cleaved. An autocatalytic mechanism for cleavage of VP0 to VP4 and VP2 was proposed by Arnold et al. (E. Arnold, M. Luo, G. Vriend, M. G. Rossman, A. C. Palmenberg, G. D. Parks, M. J. Nicklin, and E. Wimmer, Proc. Natl. Acad. Sci. USA 84:21-25, 1987) in which serine 10 of VP2 is activated by virion RNA to catalyze VP4-VP2 processing. The hypothesis rests on the observation that a hydrogen bond was observed between serine 10 of VP2 (S2010) and the carboxyl terminus of VP4 in three mature picornaviral atomic structures: rhinovirus 14, mengovirus, and poliovirus type 1 (Mahoney). We constructed mutant viruses with cysteine (S2010C) or alanine (S2010A) replacing serine 10 of VP2; these exhibited normal proteolytic processing of VP0. While our results do not exclude an autocatalytic mechanism for the maturation cleavage, they do eliminate the conserved S2010 residue as the catalytic amino acid.     

N-terminal amino acid sequences in the major capsid proteins of foot-and-mouth disease virus types A, O, and C.

Sequences of amino acids at the N-termini of virus proteins VP1, VP2, and VP3 were determined for foot-and-mouth disease virus types A12 strain 119, O1Brugge and C3Resende. In the polyacrylamide gel electrophoresis system used to purify the proteins, VP3 migrated faster than VP1 or VP2; and in the virion, VP3 could be cleaved by trypsin into VP3a and VP3b. The N-terminal amino acids for each of the virus types were glycine in VP1, aspartic acid in VP2, and threonine in VP3. No divergences in sequence across the virus types were indicated until at least the fourth position in VP1, and the third in VP3. For virus types A12, O1 and C3, the sequences were, respectively: for VP1 (Gly-ile-phe,pro,val---), (Gly,ile,phe---) and Gly-ile-phe,ala---); for VP2 (Asp,X,met---), (Asp---) and Asp-leu---); and for VP3 (Thr-thr-ala-thr---), (Thr-thr-ser---) and (Thr-thr---). Unresolved mixtures of VP3a and VP3b, from either A12 or O1 viruses, appeared to have the N-terminal amino acids threonine, which is presumed to be the same threonine as in uncleaved VP3 and serine, which is generated by the tryptic cleavage.     

Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1.

Epidemiologic and genetic evidence suggests that influenza A viruses evolve more rapidly than other viruses in humans. Although the high mutation rate of the virus is often cited as the cause of the extensive variation, direct measurement of this parameter has not been obtained in vivo. In this study, the rate of mutation in tissue culture for the nonstructural (NS) gene of influenza A virus and for the VP1 gene in poliovirus type 1 was assayed by direct sequence analysis. Each gene was repeatedly sequenced in over 100 viral clones which were descended from a single virion in one plaque generation. A total of 108 NS genes of influenza virus were sequenced, and in the 91,708 nucleotides analyzed, seven point changes were observed. A total of 105 VP1 genes of poliovirus were sequenced, and in the 95,688 nucleotides analyzed, no mutations were observed. We then calculated mutation rates of 1.5 X 10(-5) and less than 2.1 X 10(-6) mutations per nucleotide per infectious cycle for influenza virus and poliovirus, respectively. We suggest that the higher mutation rate of influenza A virus may promote the rapid evolution of this virus in nature.     

Trypsin sensitivity of the Sabin strain of type 1 poliovirus: cleavage sites in virions and related particles.

Treatment of the Sabin strain of type 1 poliovirus with trypsin produced two stable fragments of capsid protein VP1 which remained associated with the virions. Trypsinized virus was fully infectious and was neutralized by type-specific antisera. The susceptible site in the Sabin 1 strain was between the lysine at position 99 and the asparagine at position 100. A similar tryptic cleavage occurred in the Leon and Sabin strains of type 3 poliovirus, probably at the arginine at position 100, but not in the type 1 Mahoney strain, which lacks a basic residue at either position 99 or position 100. Tryptic treatment of heat-treated virus and 14S assembly intermediates produced unique stable fragments which were different from those produced in virions. The implications of our results for future characterization of the surface structures of these particles and structural rearrangements in the poliovirus capsid are discussed.     

Folding and processing of the capsid protein precursor P1 is kinetically retarded in neutralization site 3B mutants of poliovirus.

Poliovirus mutants in neutralizing antigenic site 3B were constructed by replacing the glutamic acid residue at amino acid 74 of capsid protein VP2 (VP2074E), using site-specific mutagenesis methods. All viable mutants display small-plaque phenotypes. Characterization of these mutants indicates that capsid assembly is perturbed. Although the defect in capsid assembly reduces the yield of mutant virus particles per cell, the resultant assembled particle is wild-type-like in structure and infectivity. Analyses of capsid assembly intermediates show a transient accumulation of the unprocessed capsid protein precursor, P1, indicating that cleavage of the mutant P1 by the 3CD protease is retarded. The mutant VP0-VP3-VP1 complex generated upon P1 cleavage appears assembly competent, forming pentamer and empty capsid assembly intermediates and infectious virion particles. Although the structure of the infectious mutant virus is virtually identical with that of the wild-type virus, the thermal stability of the mutant virus is dramatically increased over that of the wild-type virus. Thus, mutations at this residue are pleiotropic, altering the kinetics of capsid assembly and generating a virus that is more thermostable and more resistant to neutralization by the site 3B monoclonal antibodies.     

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.     

A Network of Protein Interactions around the Herpes Simplex Virus Tegument Protein VP22

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22''s major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.     

Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus.

The three-dimensional structure of the Sabin strain of type 3 poliovirus has been determined at 2.4 A resolution. Significant structural differences with the Mahoney strain of type 1 poliovirus are confined to loops and terminal extensions of the capsid proteins, occur in all of the major antigenic sites of the virion and typically involve insertions, deletions or the replacement of prolines. Several newly identified components of the structure participate in assembly-dependent interactions which are relevant to the biologically important processes of viral assembly and uncoating. These include two sites of lipid substitution, two putative nucleotides and a beta sheet formed by the N-termini of capsid proteins VP4 and VP1. The structure provides an explanation for the temperature sensitive phenotype of the P3/Sabin strain. Amino acids that regulate temperature sensitivity in type 3 poliovirus are located in the interfaces between promoters, in the binding site for a lipid substituent and in an assembly-dependent extended beta sheet that stabilizes the association of pentamers. Several lines of evidence indicate that these structural components also control conformational transitions at various stages of the viral life cycle.     

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.     

Analysis of the structure of a common cold virus, human rhinovirus 14, refined at a resolution of 3.0 A

Human rhinovirus 14 has a pseudo T = 3 icosahedral structure in which 60 copies of the three larger capsid proteins VP1, VP2 and VP3 are arranged in an icosahedral surface lattice, reminiscent of T = 3 viruses such as tomato bushy stunt virus and southern bean mosaic virus. The overall secondary and tertiary structures of VP1, VP2 and VP3 are very similar. The structure of human rhinovirus 14, which was refined at a resolution of 3.0 A [R = 0.16 for reflections with F greater than 3 sigma(F)], is here analyzed in detail. Quantitative analysis of the surface areas of contact (proportional to hydrophobic free energy of association) supports the previously assigned arrangement within the promoter, in which interactions between VP1 and VP3 predominate. Major contacts among VP1, VP2 and VP3 are between the beta-barrel moieties. VP4 is associated with the capsid interior by a distributed network of contacts with VP1, VP2 and VP3 within a promoter. As the virion assembly proceeds, the solvent-accessible surface area becomes increasingly hydrophilic in character. A mixed parallel and antiparallel seven-stranded sheet is composed of the beta C, beta H, beta E and beta F strands of VP3 in one pentamer and beta A1 and beta A2 of VP2 and the VP1 amino terminus in another pentamer. This association plays an essential role in holding pentamers together in the mature virion as this contact region includes more than half of the total short non-bonded contacts between pentamers. Contacts between protomers within pentamers are more extensive than the contacts between pentamers, accounting in part for the stability of pentamers. The previously identified immunogenic regions are correlated with high solvent accessibility, accessibility to large probes and also high thermal parameters. Surface residues in the canyon, the putative cellular receptor recognition site, have lower thermal parameters than other portions of the human rhinovirus 14 surface. Many of the water molecules in the ordered solvent model are located at subunit interfaces. A number of unusual crevices exist in the protein shell of human rhinovirus 14, including the hydrophobic pocket in VP1 which is the locus of binding for the WIN antiviral agents. These may be required for conformational flexibility during assembly and disassembly. The structures of the beta-barrels of human rhinovirus 14 VP1, VP2 and VP3 are compared with each other and with the southern bean mosaic virus coat protein.     

A cellular cofactor facilitates efficient 3CD cleavage of the poliovirus P1 precursor.

The production of poliovirus capsid proteins from a capsid protein precursor (P1) is mediated by virus-encoded proteinase 3CD and involves a complicated set of proteinase-substrate interactions. In addition to substrate and enzymatic determinants required for this interaction, we describe a cellular cofactor, which facilitates 3CD recognition of the P1 precursor. Cellular cofactor activity is 3CD dependent and salt dependent. Our analysis shows that proteolytic cleavage of the P1 precursor at the VP0/VP3 cleavage site exhibits a greater dependency on the cellular cofactor than cleavage at the VP3/VP1 site. Such a greater dependency on cellular cofactor activity can be relieved (in part) by the substitution of an Ala residue for the Pro residue at the -4 position of the VP0/VP3 cleavage site. However, mutant viruses containing Pro-to-Ala substitutions at the -4 position of the VP0/VP3 site exhibit defects in viral growth.