Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding.

Upon attachment to susceptible cells, poliovirus and a number of other picornaviruses undergo conformational transitions which result in changes in antigenicity, increased protease sensitivity, the loss of the internal capsid protein VP4, and a loss of the ability to attach to cells. These conformationally altered particles have been characterized by using a number of sequence-specific probes, including two proteases, a panel of antiviral monoclonal antibodies, and a panel of antisera against synthetic peptides which correspond to sequences from the capsid protein VP1. With these probes, cell-altered virus is clearly distinguishable from native and heat-inactivated virions. The probes also demonstrate that the cell-induced conformational change alters the accessibility of several regions of the virus. In particular, the amino terminus of VP1, which is entirely internal in the native virion, becomes externalized. Unlike native and heat-inactivated virus, cell-altered virions are able to attach to liposomes. The exposed amino terminus of VP1 is shown to be responsible for liposome attachment. We propose that during infection the amino terminus of VP1 inserts into endosomal membranes and thus plays a role in the mechanism of cell entry.     

Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades

The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3(JR-CSF) sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.     

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.     

Conformational changes in the VP1-unique region of native human parvovirus B19 lead to exposure of internal sequences that play a role in virus neutralization and infectivity

The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA(2) activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA(2) potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.     

Identification of a new neutralization antigenic site on poliovirus coat protein VP2

Major neutralization antigenic sites have been previously mapped by us on VP1, the largest capsid protein of poliovirus type 1. Here we report the first identification of the primary sequence of a neutralization antigenic site on capsid protein VP2. Inspection of the amino acid sequence of VP2 led to the selection and synthesis of a peptide (n = 12) that, after linking to a carrier protein, induced an antiviral neutralizing antibody response in rabbits. The response was augmented by a single subsequent inoculation of intact virus; thus, the peptide was also capable of priming the production of neutralizing antibodies. These antibodies were directed only against the site specified by the synthetic peptide. Although the VP2-specific neutralization antigenic site appears not to be strongly immunogenic in the intact virion, it can nevertheless contribute to neutralization of poliovirus. This observation may be important for the development of peptide vaccines.     

Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin

The hemagglutinin of influenza virus undergoes a conformational change at low pH, which results in exposure of a hydrophobic segment of the molecule, crucial to expression of viral fusion activity. We have studied the effects of incubation of the virus at low pH either at 37 or 0 degrees C. Treatment of the virus alone at pH 5.0 induces the virus particles to become hydrophobic, as assessed by measuring the binding of zwitterionic liposomes to the virus. At 37 degrees C this hydrophobicity is transient, electron microscopic examination of the virus reveals a highly disorganized spike layer, and fusion activity toward ganglioside-containing zwitterionic liposomes, measured at 37 degrees C with a kinetic fluorescence assay, is irreversibly lost. By contrast, after preincubation of the virus alone at pH 5.0 and 0 degrees C fusion activity remains unaffected. Yet, the preincubation at 0 degrees C does result in exposure of the hydrophobic segment of hemagglutinin, but now hydrophobicity is sustained and viral spike morphology unaltered. Hydrophobicity also remains to a significant extent upon pH neutralization, but fusion activity is negligible under these conditions. It is concluded that for optimal expression of fusion activity the virus must be bound to the target membrane before exposure to low pH. Furthermore, even after exposure of the hydrophobic segment of hemagglutinin, fusion occurs only at low pH. Finally, fusion occurs only at elevated temperature, possibly reflecting the unfolding of hemagglutinin trimers or the cooperative action of several hemagglutinin trimers in the reaction.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.     

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.     

Neutralizing antibodies to an immunodominant envelope sequence do not prevent gp120 binding to CD4.

Animals immunized with the human immunodeficiency virus type 1 gp160 glycoprotein or certain recombinant envelope components develop potent virus-neutralizing activity. This activity is principally due to antibodies directed toward a hypervariable region of gp120 between cysteine residues 302 and 337 and is virus isolate specific. These antisera, as well as two neutralizing monoclonal antibodies directed against the same hypervariable sequence, do not appreciably block gp120 from binding CD4. In contrast, serum samples from infected humans possess high titers of antibodies that block gp120-CD4 binding; these titers approximately correlate with the serum neutralization titers. Our results suggest that there are at least two targets on the envelope glycoprotein for virus neutralization. The target responsible for the broader neutralizing activity of human serum may be a conserved region of gp120 involved in CD4 binding. The antibodies directed at the hypervariable region of the envelope inhibit a different step in virus infection which is subsequent to receptor binding. The extent to which these two different epitopes of gp120 may be involved in protection against human immunodeficiency virus infection is discussed.     

Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis.

The rotavirus neutralizing antigen, VP7, is a 37,000-molecular-weight glycoprotein which is a major component of the outer shell of the virion. The amino acid sequence of VP7 for strain S2 (human serotype 2) and Nebraska calf diarrhea virus (bovine serotype) has been inferred from the nucleic acid sequence of cloned copies of genomic segment nine. Comparison of the amino acid sequences of these two VP7 proteins with those already determined for other rotavirus strains reveals extensive sequence conservation between serotypes with clusters of amino acid differences sited predominantly in hydrophilic domains of the protein. Six peptides have been synthesized that span the hydrophilic regions of the molecule. Antisera to these peptides both recognize the respective homologous peptides in a solid-phase radioimmunoassay and bind to denatured VP7 in a Western blot. However, none of the antisera either recognize virus or exhibit significant neutralizing activity, indicating that these peptide sequences are not available on the surface of the virus.     

Cleavage of VP1 and modification of antigenic site 1 of type 2 polioviruses by intestinal trypsin.

We have exposed 22 independent type 2 poliovirus isolates to human intestinal fluid and purified trypsin. In all cases the virus retained its infectivity, while polyacrylamide gel electrophoresis of viral proteins showed disappearance of the VP1 bands. Concomitantly, the viruses became resistant to antigenic site 1-specific monoclonal antibodies, indicating that the cleavage took place at the antigenic site 1. Sera from persons immunized solely with the inactivated poliovirus vaccine (IPV) neutralized intact type 2 polioviruses more readily than the corresponding trypsin-cleaved virus preparations. The ratio between the neutralization indices for the intact and trypsin-cleaved type 2 polioviruses was not significantly changed by a dose of trivalent oral poliovirus vaccine given to children previously immunized with IPV. These results indicate that while the antigenic site 1 of type 2 poliovirus is immunogenic in humans when IPV is used, the relative role of this antigenic site in human immunity appears to be less critical than that in the case of type 3 polioviruses. Before we obtained these results, only antigenic site 1 had been shown to be immunogenic in type 2 polioviruses.     

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.     

Structure-based identification of a major neutralizing site in an adenovirus hexon

Virus-specific neutralizing antibodies present an obstacle to the effective use of adenovirus vectors for gene therapy and vaccination. The specific sites recognized by neutralizing antibodies have not been identified for any adenovirus, but they have been proposed to reside within the hexon, in small regions of the molecule that are exposed on the capsid surface and possess sequences that vary among serotypes. We have mapped the epitopes recognized by a panel of seven hexon-specific monoclonal antibodies that neutralize the chimpanzee adenovirus 68 (AdC68). Surface plasmon resonance experiments revealed that the antibodies compete for a single hexon binding site, and experiments with synthetic peptides indicated that this site resides within just one small surface loop. Mutations within this loop (but not in other surface loops) permitted virus to escape neutralization by all seven monoclonal antibodies and to resist neutralization by polyclonal antisera obtained from animals immunized against AdC68. These results indicate that a single small surface loop defines a major neutralization site for AdC68 hexon.     

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.     

Relation of VP7 amino acid sequence to monoclonal antibody neutralization of rotavirus and rotavirus monotype.

The neutralization epitopes of the VP7 of human rotavirus RV-4 were studied by using five neutralizing mouse monoclonal antibodies to select virus variants resistant to neutralization by each of the antibodies. Antibody resistance patterns and sequence analysis of the RV-4 variants revealed that at least four sites on VP7, located at amino acids 94 (region A), 147 to 148 (region B), 213 (region C), and 291, are involved in neutralization of the human G1 rotavirus RV-4. The A-region site elicited antibody cross-reactive between G types and showed species-restricted immunodominance not related to carbohydrate attachment. The monotype 1b rotavirus M37 lacked this site. The B region contained strain-specific and cross-reactive sites, absent in monotype 1c rotaviruses. The C-region site was present in all G1 rotaviruses tested. Monotype 1a rotaviruses contained all these sites of neutralization. Virus monotype and sensitivity to monoclonal antibody neutralization usually related to the presence of a particular amino acid(s) at or next to the positions at which the mutations were selected in the virus variants.     

The deletion of 41 proximal nucleotides reverts a poliovirus mutant containing a temperature-sensitive lesion in the 5'' noncoding region of genomic RNA.

We generated a number of small deletions and insertions in the 5' noncoding region of an infectious cDNA copy of the poliovirus RNA genome. Transfection of these mutated cDNAs into COS-1 cells produced the following phenotypic categories: (i) wild-type mutations, (ii) lethal mutations, (iii) mutations exhibiting slow growth or low-titer properties, and (iv) temperature-sensitive (ts) mutations. The deletion of nucleotides 221 to 224 produced a ts virus, 220D1. Mutant 220D1 was found to have a dramatic reduction in growth, virus-specific protein and RNA synthesis, and the shutoff of host cell protein synthesis at 37 or 39 degrees C compared with 33 degrees C. Temperature shift experiments showed that the mutant viral RNA is not an effective template for protein or RNA synthesis at 39 degrees C and suggested a decreased stability of the 220D1 RNA at 39 degrees C. Selection for a non-ts revertant of 220D1 yielded the virus R2, which was no longer ts for growth or viral protein and RNA synthesis. Sequencing the 5' noncoding region of the genomic RNA from R2 revealed the deletion of 41 proximal nucleotides for an overall deletion of nucleotides 184 to 228. These data suggest that the deleted sequences are nonessential to the poliovirus life cycle during growth in HeLa cells. According to computer-predicted RNA secondary structures of the 5' noncoding region of poliovirus RNA, the R2 revertant virus has deleted an entire predicted stem-loop structure.     

Identification of neutralizing antigenic sites on VP1 and VP2 of type A5 foot-and-mouth disease virus, defined by neutralization-resistant variants.

Five neutralizing monoclonal antibodies (nMAbs) obtained against type A5 Spain-86 foot-and-mouth disease virus were used to generate a series of neutralization-resistant variants. In vitro and in vivo assays showed that the variants were fully refractory to neutralization by the selecting nMAb. On the basis of cross-neutralization and binding assays, two neutralizing antigenic sites have been located on the virus surface; one, located near the C-terminus of VP1, displayed a linear epitope, and the second, located on VP2, displayed two conformational epitopes. Nucleotide sequencing of RNA of the parental and variant capsid protein-coding region P1 has placed the amino acid changes at position 198 of VP1 for the first site and at positions 72 and 79 of VP2 for the related epitopes in the second site. The relative importance of these two sites in the biological properties of foot-and-mouth disease virus is discussed.     

Chido and Rogers antigenic determinant on the fourth component of human complement

The blood group substances Chido (Cha) and Rogers (Rga) represent two electrophoretic variants of human C4. Based on the observation that anti-Cha and anti-Rga antisera agglutinated human red blood cells prepared in sucrose-activated autologous serum (LIS cells) at 37 degrees C, it has been assumed that the Cha and Rga antigenic determinants reside in the C4d fragment of C4. Here, we present evidence indicating that C4d is not present on those cells. In order to identify structurally the C4 fragments deposited, LIS cells were prepared at 37 degrees C and 4 degrees C in autologous serum to which 125I-C4 was added. Membranes of LIS cells were solubilized and analyzed by SDS-PAGE in 5 to 15% gradient gels followed by autoradiography. C4d was not deposited on LIS cells prepared at 37 degrees C, whereas C4c (beta, gamma, alpha 3 alpha 4) was. Cells prepared at 4 degrees C carried C4d (alpha 2) and C4c. Anti-Cha and anti-Rga antisera agglutinated both cell types, although C4d was not present on the cells prepared at 37 degrees C. Purified C4, C4c, C4d, and alpha-, beta- and gamma-chains of C4, as well as alpha 3 and alpha 4, were used to neutralize these antisera. C4 and the alpha-chain C4d and alpha 4 fragment of C4c, but neither the alpha 3 fragment nor the beta- or gamma-chains, were capable of neutralizing anti-Cha and anti-Rga antisera. These results strongly suggest that C4d and alpha 4 share an antigenic determinant, both of which are recognized by anti-Cha and anti-Rga antisera.     

Yellow Fever Virus. II. Factors Affecting the Plaque Neutralization Test

In studies on the factors and conditions influencing the yellow fever (YF) virus plaque neutralization test, 60 min was found to be the minimal time necessary for equilibration of the virus-antibody complex at 23 and 37 C. Maximal virus titers in the diluent controls and the pre- and postinoculation serum-containing mixtures occurred by the 60-min adsorption time. Serum neutralization indices also seemed to level by this time. Heating (56 C for 30 min) decreased the neutralizing capacity of serum. However, the slope of the neutralization curve was not affected. The addition of native (unheated) "serum factor" in the form of fresh guinea pig or monkey sera partially restored the neutralizing activity lost by heating in some, but not all, sera. Many sera contained nonspecific inhibitors of YF virus infectivity and neutralization. Preliminary studies with ether extraction suggest that these inhibitors are lipid in nature.