A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1.

Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119. The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies). They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104. Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103. A peptide representing the sequence of this region was chemically synthesized. Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies. The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies. We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1.     

Molecular characterization of a subgroup specificity associated with the rotavirus inner capsid protein VP2

Group A rotaviruses are classified into serotypes, based on the reactivity pattern of neutralizing antibodies to VP4 and VP7, as well as into subgroups (SGs), based on non-neutralizing antibodies directed against VP6. The inner capsid protein (VP2) has also been described as a SG antigen; however, little is known regarding the molecular determinants of VP2 SG specificity. In this study, we characterize VP2 SGs by correlating genetic markers with the immunoreactivity of the SG-specific monoclonal antibody (YO-60). Our results show that VP2 proteins similar in sequence to that of the prototypic human strain Wa are recognized by YO-60, classifying them as VP2 SG-II. In contrast, proteins not bound by YO-60 are similar to those of human strains DS-1 or AU-1 and represent VP2 SG-I. Using a mutagenesis approach, we identified residues that determine recognition by either YO-60 or the group A-specific VP2 monoclonal antibody (6E8). We found that YO-60 binds to a conformationally dependent epitope that includes Wa VP2 residue M328. The epitope for 6E8 is also contingent upon VP2 conformation and resides within a single region of the protein (Wa VP2 residues A440 to T530). Using a high-resolution structure of bovine rotavirus double-layered particles, we predicted these epitopes to be spatially distinct from each other and located on opposite surfaces of VP2. This study reveals the extent of genetic variation among group A rotavirus VP2 proteins and illuminates the molecular basis for a previously described SG specificity associated with the rotavirus inner capsid protein.     

Identification of T-helper epitopes in the VP1 capsid protein of poliovirus.

Poliovirus-specific T lymphocytes were isolated from virus-immunized mice of different H-2 haplotypes. Immunological characterization of this population indicates that the effector population involved in the observed poliovirus-specific proliferative response was that of CD4-positive T-helper cells. Proliferative responses also were induced within these T-lymphocyte populations upon stimulation with either purified VP1 capsid protein or VP1 synthetic peptides. By using these synthetic peptides, several T-helper epitopes were identified. Generally, proliferative responses were observed in three regions of VP1. Two regions spanning VP1 residues 86 to 120 and 201 to 241 were recognized by T lymphocytes from BALB/c (H-2d), C57BL/6 (H-2b), and C3H/HeJ (H-2k) backgrounds. Analyses using synthetic peptides of nonoverlapping sequences indicated that the region spanning residues 201 to 241 may contain several T epitopes and may account for the strong proliferative response observed. In addition, for two of the three haplotypes examined, T epitopes were observed within residues 7 to 24 of VP1. Additional epitopes which appeared to be restricted to specific H-2 backgrounds were identified. T epitopes within VP1 that are common between different strains of mice appeared to lie within previously identified neutralizing antigenic sites in poliovirus.     

Mouse neurovirulence determinants of poliovirus type 1 strain LS-a map to the coding regions of capsid protein VP1 and proteinase 2Apro.

Poliovirus type 1 strain LS-a [PV1(LS-a)] is a OV variant adapted to mice by multiple passages through mouse and monkey tissues. To investigate the molecular basis underlying mouse neurovirulence of PV1(LS-a), a cDNA of the viral genome containing nucleotides 112 to 7441 was cloned, and the nucleotide sequence was determined. Compared with that of the mouse avirulent progenitor PV1(Mahoney), 54 nucleotide changes were found in the genome of the PV1(LS-a) virus, resulting in 20 amino acid substitutions in the virus polyprotein. Whereas the nucleotide changes were scattered throughout the genome, the amino acid substitutions were largely clustered in the capsid proteins and, to a certain extent, in the virus proteinase 2Apro. By in vitro mutagenesis, PV1(LS-a)-specific capsid mutations were introduced into a cDNA clone of PV1(Mahoney). We show that neither the individual amino acid mutations nor combinations of mutations in the region encoding VP1 conferred to PV1(Mahoney) the mouse-adapted phenotype of PV1(LS-a). Chimeric cDNA studies demonstrated that a recombinant type 1 virus containing the PV1(LS-a) sequence from nucleotide 2470 to nucleotide 3625 displayed a neurovirulent phenotype in mice. Further dissection of this region revealed that mouse neurovirulence of PV1(LS-a) was determined by multiple mutations in regions encoding both viral proteinase 2Apro and capsid protein VP1. The mouse neurovirulent viruses, PV1(LS-a), W1-M/LS-Pf [nucleotides 496 to 3625 from PV1(LS-a)], and W1-M/LS-NP [nucleotides 2470 to 3625 from PV1(LS-a)], showed increased sensitivity to heat treatment at 45 degrees C for 1 h. Surprisingly, the thermolabile phenotype was also displayed by a recombinant of PV1(Mahoney) carrying a PV1(LS-a) DNA fragment encoding the N-terminal portion of 2Apro. This suggests that base substitutions in the region encoding 2Apro affected capsid stability, thereby contributing to the neurovirulence of the virus in mice.     

Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor.

Poliovirus initiates infection by binding to its cell receptor and undergoing a receptor-mediated conformational alteration. To identify capsid residues that control these interactions, we have isolated and characterized poliovirus mutants that are resistant to neutralization by a soluble form of the poliovirus receptor. Twenty one soluble receptor-resistant (srr) mutants were identified which still use the poliovirus receptor to infect cells. All but one srr mutant contain a single amino acid change at one of 13 different positions, either on the surface or in the interior of the virion. The results of binding and alteration assays demonstrate that both surface and internal capsid residues regulate attachment to the receptor and conformational change of the virus. Mutations that reduce alteration also affect receptor binding, suggesting a common structural basis for early events in poliovirus infection.     

Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.     

Isolation and preliminary characterization of temperature-sensitive mutants of poliovirus type 1.

We isolated six temperature-sensitive mutants of poliovirus type 1 (Mahoney) by hydroxylamine mutagenesis and replica plating at 31, 33 (permissive), and 39 degrees C (restrictive). One of these mutants, designated tsB9, was chosen for more detailed examination. tsB9 accumulated 25% of the wild-type amount of virus-specific RNA at the restrictive temperature. We found that tsB9 was not able to synthesize mature, 35S single-stranded RNA at the restrictive temperature. In spite of the absence of significant RNA synthesis, tsB9 retained the ability to inhibit host protein synthesis during infection at 39 degrees C at about the same rate as wild-type virus.     

Host range determinants located on the interior of the poliovirus capsid.

The inability of certain poliovirus strains to infect mice can be overcome by the expression of human poliovirus receptors in mice or by the presence of a particular amino acid sequence of the B-C loop of the viral capsid protein VP1. We have identified changes in an additional capsid structure that permit host-restricted poliovirus strains to infect mice. Variants of the mouse-virulent P2/Lansing strain were constructed containing amino acid changes, deletions and insertions in the B-C loop of VP1. These variants were attenuated in mice, demonstrating the importance of the B-C loop sequence in host range. Passage of two of the B-C loop variants in mice led to the selection of viruses that were substantially more virulent. The increased neurovirulence of these strains was mapped to two different suppressor mutations in the N-terminus of VP1. Whereas the B-C loop of VP1 is highly exposed on the surface of the capsid, near the five-fold axis of symmetry, the suppressor mutations are in the interior of the virion, near the three-fold axis. Introduction of the suppressor mutations into the genome of the mouse-avirulent P1/Mahoney strain resulted in neurovirulent viruses, demonstrating that the P2/Lansing B-C loop sequence is not required to infect mice. Because the internal host range determinants are in a structure known to be important in conformational transitions of the virion, the host range of poliovirus may be determined by the ability of virions to undergo transitions catalyzed by cell receptors.     

Production and characterization of monoclonal antibodies to polyomavirus major capsid protein VP1.

Four hybridoma cell lines producing monoclonal antibodies against intact polyoma virions were produced and characterized. These antibodies were selected for their ability to react with polyoma virions in an enzyme-linked immunosorbent assay. The antibodies immunoprecipitated polyoma virions and specifically recognized the major capsid protein VP1 on an immunoblot. Distinct VP1 isoelectric species were immunoprecipitated from dissociated virion capsomere preparations. Two-dimensional gel electrophoresis demonstrated antibody reactivity with specific VP1 species. Monoclonal antibodies E7 and G9 recognized capsomeres containing VP1 species D, E, and F, while monoclonal antibodies C10 and D3 recognized capsomeres containing species B and C. Two of the monoclonal antibodies, E7 and G9, were capable of neutralizing viral infection and inhibiting hemagglutination. The biological activity of the monoclonal antibodies correlated well with the biological function of the species with which they reacted.     

Localization of calcium on the polyomavirus VP1 capsid protein.

Our laboratory has previously shown that the divalent cation Ca2+ is an integral part of the polyomavirus and plays a major role in stabilizing the intact virion structure. In this report, we show that calcium is sequestered on the major capsid protein VP1 of polyomavirus. The virion structural proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being transferred to nitrocellulose and probed with 45CaCl2. Autoradiography revealed 45Ca binding exclusively to VP1. Increasing the amount of VP1 transferred to the nitrocellulose resulted in a concomitant increase in 45Ca binding. 45Ca binding to VP1 could be reduced by competition with an excess of unlabeled CaCl2. Separation of the species of VP1 by two-dimensional gel electrophoresis before electroblotting and probing with 45CaCl2 revealed that all six species (A to F) bind the radiolabeled calcium. Formic acid cleavage of the 43-kilodalton (kDa) VP1 protein into 29-, 18-, and 16-kDa fragments before 45Ca-binding analysis revealed that only the 18- and 16-kDa carboxyl-terminal fragments of this protein bind 45Ca.     

Natural type 3/type 2 intertypic vaccine-related poliovirus recombinants with the first crossover sites within the VP1 capsid coding region

Background

Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008.

Principal Findings

Complete genomic sequences revealed their vaccine-related genomic features and showed that their first crossover sites were randomly distributed in the 3′ end of the VP1 coding region. The length of donor Sabin 2 sequences ranged from 55 to 136 nucleotides, which is the longest donor sequence reported in the literature for this type of poliovirus recombination. The recombination resulted in the introduction of Sabin 2 neutralizing antigenic site 3a (NAg3a) into a Sabin 3 genomic background in the VP1 coding region, which may have been altered by some of the type 3-specific antigenic properties, but had not acquired any type 2-specific characterizations. NAg3a of the Sabin 3 strain seems atypical; other wild-type poliovirus isolates that have circulated in recent years have sequences of NAg3a more like the Sabin 2 strain.

Conclusions

10 natural type 3/type 2 intertypic VP1 capsid-recombinant polioviruses, in which the first crossover sites were found to be in the VP1 coding region, were isolated and characterized. In spite of the complete replacement of NAg3a by type 2-specific amino acids, the serotypes of the recombinants were not altered, and they were totally neutralized by polyclonal type 3 antisera but not at all by type 2 antisera. It is possible that recent type 3 wild poliovirus isolates may be a recombinant having NAg3a sequences derived from another strain during between 1967 and 1980, and the type 3/type 2 recombination events in the 3′ end of the VP1 coding region may result in a higher fitness.     

Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry.

A complex of the polyomavirus internal protein VP2/VP3 with the pentameric major capsid protein VP1 has been prepared by co-expression in Escherichia coli. A C-terminal segment of VP2/VP3 is required for tight association, and a crystal structure of this segment, complexed with a VP1 pentamer, has been determined at 2.2 A resolution. The structure shows specific contacts between a single copy of the internal protein and a pentamer of VP1. These interactions were not detected in the previously described structure of the virion, but the location of VP2 in the recombinant complex is consistent with features in the virion electron-density map. The C-terminus of VP2/VP3 inserts in an unusual, hairpin-like manner into the axial cavity of the VP1 pentamer, where it is anchored strongly by hydrophobic interactions. The remainder of the internal protein appears to have significant flexibility. This structure restricts possible models for exposure of the internal proteins during viral entry.     

Molecular basis for linkage of a continuous and discontinuous neutralization epitope on the structural polypeptide VP2 of poliovirus type 1.

We obtained neutralizing monoclonal antibodies against a continuous neutralization epitope on VP2 of poliovirus type 1 strain Mahoney by using a combined in vivo-in vitro immunization procedure. The antibody-binding site was mapped to amino acid residues within the peptide segment (residues 164 through 170) of VP2 by competition with synthetic peptide and sequencing of resistant mutants. Cross-neutralization of these mutants with another neutralizing monoclonal antibody revealed a linkage of the continuous epitope and a discontinuous neutralization epitope involving both loops of the double-loop structure of VP2 at the twofold axis on the surface of the virion.     

Substitutions in the capsids of poliovirus mutants selected in human neuroblastoma cells confer on the Mahoney type 1 strain a phenotype neurovirulent in mice.

Poliovirus (PV) type 1 mutants selected in human neuroblastoma cells persistently infected (PVpi) with the wild-type Mahoney strain exhibited a mouse-neurovirulent phenotype. Four of the five substitutions present in the capsid proteins of a PVpi were demonstrated to extend the host range of the Mahoney strain to mice. These new mouse-neurovirulent determinants were located in the three-dimensional structure of the viral capsid; two of them (residues 142 of VP2 and 60 of VP3) were located in loops exposed at the surface of the protein shell, whereas the other two (residues 43 of VP1 and 62 of VP4) were located on the inside of the capsid. VP1 residue 43 and VP2 residue 142 substitutions were also selected in a PVpi derived from the attenuated Sabin strain. We suggest that the selective pressure of human neuroblastoma cell factor(s) involved in early steps of PV multiplication could be responsible for the arising of amino acid substitutions which confer adaptation to the mouse central nervous system to PV.     

Hydroxyproline in the major capsid protein VP1 of polyomavirus.

Amino acid analysis of [3H]proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of 3H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from [35S]methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.     

Polymorphism in the assembly of polyomavirus capsid protein VP1.

Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid.     

Effect of hypertonic medium on protein synthesis of Mahoney and LSc poliovirus.

The initiation of synthesis of Mahoney virus protein is more sensitive to high osmolarity than initiation of synthesis of LSc virus protein. Production of Mahoney virus protein appears to be only slightly more resistant to high osmolarity than synthesis of HeLa-Rhino cell protein.     

Type-specific and cross-reactive antigenicity of capsid proteins VP1 and VP2 of echovirus type 7

After disruption of echovirus type 7 virions with urea and heat, VP1 and VP2 were separated by isoelectric focusing in urea-containing sucrose gradients. Antisera to these two polypeptides were produced in guinea pigs. In complement fixation, antiserum to VP1 reacted with native and heated virions (N and H antigens, respectively) of homologous virus, and also cross-reacted with heated virions of some other enteroviruses used. Antiserum to VP2 was reactive only with heated virions of homologous and heterologous viruses. Interestingly, the anti-VP2 serum reacted neither with native nor even with heated procapsids (naturally-occurring empty capsids). Antiserum to VP1, but not VP2, showed neutralizing and hemagglutination-inhibiting activities. These results suggest that 1) both VP1 and VP2 possess cross-reactive antigenic determinants which are exposed on the surface of heated virions, and 2) type-specific determinants of VP1 are located on the surface of native virions.     

Identification and molecular characterization of the RNA polymerase-binding motif of infectious bursal disease virus inner capsid protein VP3

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most important infectious poultry diseases. Major aspects of the molecular biology of IBDV, such as assembly and replication, are as yet poorly understood. We have previously shown that encapsidation of the putative virus-encoded RNA-dependent RNA polymerase VP1 is mediated by its interaction with the inner capsid protein VP3. Here, we report the characterization of the VP1-VP3 interaction. RNase A treatment of VP1- and VP3-containing extracts does not affect the formation of VP1-VP3 complexes, indicating that formation of the complex requires the establishment of protein-protein interactions. The use of a set of VP3 deletion mutants allowed the mapping of the VP1 binding motif of VP3 within a highly charged 16-amino-acid stretch on the C terminus of VP3. This region of VP3 is sufficient to confer VP1 binding activity when fused to an unrelated protein. Furthermore, a peptide corresponding to the VP1 binding region of VP3 specifically inhibits the formation of VP1-VP3 complexes. The presence of Trojan peptides containing the VP1 binding motif in IBDV-infected cells specifically reduces infective virus production, thus showing that formation of VP1-VP3 complexes plays a critical role in IBDV replication.