Recovery and altered neutralization specificities of chimeric viruses containing capsid protein domain exchanges from antigenically distinct strains of feline calicivirus

Feline calicivirus (FCV) strains can show significant antigenic variation when tested for cross-reactivity with antisera produced against other FCV strains. Previous work has demonstrated the presence of hypervariable amino acid sequences in the capsid protein of FCV (designated regions C and E) that were postulated to constitute the major antigenic determinants of the virus. To examine the involvement of hypervariable sequences in determining the antigenic phenotype, the nucleotide sequences encoding the E regions from three antigenically distinct parental FCV strains (CFI, KCD, and NADC) were exchanged for the equivalent sequences in an FCV Urbana strain infectious cDNA clone. Two of the three constructs were recovered as viable, chimeric viruses. In six additional constructs, of which three were recovered as viable virus, the E region from the parental viruses was divided into left (N-terminal) and right (C-terminal) halves and engineered into the infectious clone. A final viable construct contained the C, D, and E regions of the NADC parental strain. Recovered chimeric viruses showed considerable antigenic variation from the parental viruses when tested against parental hyperimmune serum. No domain exchange was able to confer complete recognition by parental antiserum with the exception of the KCD E region exchange, which was neutralized at a near-homologous titer with KCD antiserum. These data demonstrate that it is possible to recover engineered chimeric FCV strains that possess altered antigenic characteristics. Furthermore, the E hypervariable region of the capsid protein appears to play a major role in the formation of the antigenic structure of the virion where conformational epitopes may be more important than linear in viral neutralization.     

Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies

Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2. 1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.     

Identification of epitopes of trichosanthin by phage peptide library

The phage displayed random peptide library has recently emerged as a powerful technique for analyzing Ab-Ag interactions. In this study, the method was employed to identify epitopes of trichosanthin. Two monoclonal Abs (4B5, 2E9) which recognized different epitopes of trichosanthin (TCS) were selected and a phage-peptide library with nine amino acids (9 aa) was used to screen the positive phage clones that have high affinity to the mAbs. Two groups of phage clones that carried peptide-specific binding to mAbs were identified by the screen. The identified phage clones carried peptide-specific binding to 4B5 and 2E9 mAbs were immunized in mice. To evaluate mimotope of selected phages, the specific binding activity to TCS was measured in the serum from phage-immunized mice. They all showed positive results. The conserved interaction motifs were deduced from the peptide sequences of each group of selected phage clones. When compared the motif sequence with the sequence of TCS, it was predicted that 4B5-corresponding epitope was located at 27-37 aa of TCS protein and 2E9-corresponding epitope was located at 41-48 aa of TCS. The predicted sequence of 4B5-corresponding epitope was further confirmed by site-directed mutation of TCS protein. The data showed that the expressed TCS protein mutated in 4B5-corresponding epitope was unable to bind 4B5 mAb. The results suggested that the phage display peptide library is useful to identify Ag epitopes and to raise Ab in disease diagnosis and treatment.     

Two-way antigenic cross-reactivity between severe acute respiratory syndrome coronavirus (SARS-CoV) and group 1 animal CoVs is mediated through an antigenic site in the N-terminal region of the SARS-CoV nucleoprotein

In 2002, severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged in humans, causing a global epidemic. By phylogenetic analysis, SARS-CoV is distinct from known CoVs and most closely related to group 2 CoVs. However, no antigenic cross-reactivity between SARS-CoV and known CoVs was conclusively and consistently demonstrated except for group 1 animal CoVs. We analyzed this cross-reactivity by an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using specific antisera to animal CoVs and SARS-CoV and SARS patient convalescent-phase or negative sera. Moderate two-way cross-reactivity between SARS-CoV and porcine CoVs (transmissible gastroenteritis CoV [TGEV] and porcine respiratory CoV [PRCV]) was mediated through the N but not the spike protein, whereas weaker cross-reactivity occurred with feline (feline infectious peritonitis virus) and canine CoVs. Using Escherichia coli-expressed recombinant SARS-CoV N protein and fragments, the cross-reactive region was localized between amino acids (aa) 120 to 208. The N-protein fragments comprising aa 360 to 412 and aa 1 to 213 reacted specifically with SARS convalescent-phase sera but not with negative human sera in ELISA; the fragment comprising aa 1 to 213 cross-reacted with antisera to animal CoVs, whereas the fragment comprising aa 360 to 412 did not cross-react and could be a potential candidate for SARS diagnosis. Particularly noteworthy, a single substitution at aa 120 of PRCV N protein diminished the cross-reactivity. We also demonstrated that the cross-reactivity is not universal for all group 1 CoVs, because HCoV-NL63 did not cross-react with SARS-CoV. One-way cross-reactivity of HCoV-NL63 with group 1 CoVs was localized to aa 1 to 39 and at least one other antigenic site in the N-protein C terminus, differing from the cross-reactive region identified in SARS-CoV N protein. The observed cross-reactivity is not a consequence of a higher level of amino acid identity between SARS-CoV and porcine CoV nucleoproteins, because sequence comparisons indicated that SARS-CoV N protein has amino acid identity similar to that of infectious bronchitis virus N protein and shares a higher level of identity with bovine CoV N protein within the cross-reactive region. The TGEV and SARS-CoV N proteins are RNA chaperons with long disordered regions. We speculate that during natural infection, antibodies target similar short antigenic sites within the N proteins of SARS-CoV and porcine group 1 CoVs that are exposed to an immune response. Identification of the cross-reactive and non-cross-reactive N-protein regions allows development of SARS-CoV-specific antibody assays for screening animal and human sera.     

Junctional adhesion molecule 1 is a functional receptor for feline calicivirus

The life cycle of calicivirus is not fully understood because most of the viruses cannot be propagated in tissue culture cells. We studied the mechanism of calicivirus entry into cells using feline calicivirus (FCV), a cultivable calicivirus. From the cDNA library of Crandell-Rees feline kidney (CRFK) cells, feline junctional adhesion molecule 1 (JAM-1), an immunoglobulin-like protein present in tight junctions, was identified as a cellular-binding molecule of the FCV F4 strain, a prototype strain in Japan. Feline JAM-1 expression in nonpermissive hamster lung cells led to binding and infection by F4 and all other strains tested. An anti-feline JAM-1 antibody reduced the binding of FCV to permissive CRFK cells and strongly suppressed the cytopathic effect (CPE) and FCV progeny production in infected cells. Some strains of FCV, such as F4 and F25, have the ability to replicate in Vero cells. We found that regardless of replication ability, FCV bound to Vero and 293T cells via simian and human JAM-1, respectively. In Vero cells, an anti-human JAM-1 antibody inhibited binding, CPE, and progeny production by F4 and F25. In addition, feline JAM-1 expression permitted FCV infection in 293T cells. Taken together, our results demonstrate that feline JAM-1 is a functional receptor for FCV, simian JAM-1 also functions as a receptor for some strains of FCV, and the interaction between FCV and JAM-1 molecules may be a determinant of viral tropism. This is the first report concerning a functional receptor for the viruses in the family Caliciviridae.     

Mapping of the Feline Calicivirus Proteinase Responsible for Autocatalytic Processing of the Nonstructural Polyprotein and Identification of a Stable Proteinase-Polymerase Precursor Protein

Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.     

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.     

Identification of two epitopes on the dengue 4 virus capsid protein recognized by a serotype-specific and a panel of serotype-cross-reactive human CD4+ cytotoxic T-lymphocyte clones.

We analyzed the CD4+ T-lymphocyte response of a donor who had received an experimental live-attenuated dengue 4 virus (D4V) vaccine. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious dengue virus (DV) antigens showed the highest proliferation to D4V antigen, with lesser, cross-reactive proliferation to D2V antigen. We established CD4+ cytotoxic T-lymphocyte clones (CTL) by stimulation with D4 antigen. Using recombinant baculovirus antigens, we identified seven CTL clones that recognized D4V capsid protein. Six of these CTL clones were cross-reactive between D2 and D4, and one clone was specific for D4. Using synthetic peptides, we found that the D4V-specific CTL clone recognized an epitope between amino acids (aa) 47 and 55 of the capsid protein, while the cross-reactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2V and D4V. This region of the capsid protein induced a variety of CD4+ T-cell responses, as indicated by the fact that six clones which recognized a peptide spanning this region showed heterogeneity in their recognition of truncations of this same peptide. The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84 to 92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84 to 92. These results indicate that this donor exhibits memory CD4+ T-cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level but in bulk culture responses as well. Since previous studies have indicated that the CTL responses to DV infection seem to be directed mainly against the envelope (E) and NS3 proteins, these results are the first to indicate that the DV capsid protein is also a target of the antiviral T-cell response.     

Application of chimeric feline foamy virus-based retroviral vectors for the induction of antiviral immunity in cats

In order to define the potential and applicability of replication-competent foamy virus-based vaccine vectors, recombinant feline foamy virus (FFV) vectors encoding defined segments of the feline calicivirus (FCV) capsid protein E domain were constructed. In cell cultures, these FFV-FCV vectors efficiently transduced and expressed a hybrid fusion protein consisting of the essential FFV Bet protein and the attached FCV E domains. The stability of the vectors in vitro was inversely correlated to the size of the heterologous insert. The deletion of a part of the FFV U3 sequence in these FFV-FCV vectors did not interfere with replication and titer in cell cultures but increased the genetic stability of the hybrid vectors. Selected chimeric vectors were injected into immunocompetent cats and persisted in the transduced host concomitant with a strong and specific humoral immune response against vector components. In a substantial number of cats, antibodies directed against the FCV E domain were induced by the FFV-FCV vectors, but no FCV-neutralizing activities were detectable in vitro. When the vaccinated cats were challenged with a high-titer FCV dose, sterile immunity was not induced by any of the hybrid FFV-FCV vectors. However, the FFV-FCV vector with a truncated U3 region of the long terminal repeat promoter significantly reduced the duration of FCV shedding after challenge and suppressed the appearance of FCV-specific ulcers. Possible mechanisms contributing to the partial protection will be discussed.     

The Feline Calicivirus Leader of the Capsid Protein Is Associated with Cytopathic Effect

Open reading frame 2 (ORF2) of the feline calicivirus (FCV) genome encodes a capsid precursor that is posttranslationally processed to release the mature capsid protein (VP1) and a small protein of 124 amino acids, designated the leader of the capsid (LC). To investigate the role of the LC protein in the virus life cycle, mutations and deletions were introduced into the LC coding region of an infectious FCV cDNA clone. Three cysteine residues that are conserved among all vesivirus LC sequences were found to be critical for the recovery of FCV with a characteristic cytopathic effect in feline kidney cells. A cell-rounding phenotype associated with the transient expression of wild-type and mutagenized forms of the LC correlated with the cytopathic and growth properties of the corresponding engineered viruses. The host cellular protein annexin A2 was identified as a binding partner of the LC protein, consistent with a role for the LC in mediating host cell interactions that alter the integrity of the cell and enable virus spread.     

Some human B and T cell epitopes of bovine serum albumin,the major beef allergen

Bovine serum albumin (BSA) is the major beef allergen. Since IgE and T cell recognitions are central to the specific immune response to allergens, the identification and immunologic characterization of B and T cell epitopes of BSA represent important steps in the development of treatments for beef allergy. Prior to our experiments, we hypothesized that BSA-specific antibodies and T cells react primarily with sequential epitopes in which the amino acid sequences differ greatly between bovine and human albumin. To clarify this hypothesis, 16 peptides corresponding to such regions were synthesized as candidate epitopes. Among them, at least two regions, aa336-345 and aa451-459, were found to be B cell (IgE-binding) epitopes. In inhibition ELISA experiments, EYAV (aa338-341) and LILNR (aa453-457) bound to patient IgE antibodies and were found to be the cores of the IgE-binding epitopes. Three regions, DDSPDLPKLKPDPNTLC (aa107-123), PHACYTSVFDKLKHLVDEP (aa364-382), and LSLILNRLC (aa451-459), were found to induce T cell proliferation in more than half of the patients tested. Of interest was that these three regions were also recognized by B cells. Information concerning human B and T cells epitopes can contribute greatly to the elucidation of the etiology of beef allergy.     

Antigenic and immunogenic epitopes shared by human papillomavirus type 16 and bovine, canine, and avian papillomaviruses.

All types of papillomaviruses (PV) share common, so-called group-specific epitopes. To identify the major group-specific epitopes, we immunized 26 guinea pigs or rabbits with purified bovine PV type 1 (BPV), canine PV, or avian PV from the common chaffinch. The resulting hyperimmune sera, as well as a commercially available rabbit antiserum to BPV and seven monoclonal antibodies to BPV, were tested in an enzyme-linked immunosorbent assay with a set of 66 overlapping 20-amino-acid peptides representing the complete sequence of the major capsid proteins (L1 and L2) of human PV type 16 (HPV 16). Sera from the same animals before immunization were used as controls. The minimal reactive epitopes within each peptide were further characterized by testing of truncated peptides. The cross-reactive epitopes were clustered in two regions of L1, an internal region (at positions 171 to 235), which contained three epitopes, and the more reactive region at the carboxy terminus (at positions 411 to 475), which contained six epitopes. The most reactive of the HPV 16 broadly cross-reactive epitopes was a carboxy-terminal epitope which had the sequence DTYRF and which reacted with nine of the antisera to BPV, canine PV, or avian PV, with the commercially available rabbit antiserum to BPV, and also with a mouse monoclonal antibody to BPV. Antipeptide antisera to all of the HPV 16 L1 peptides and to the most antigenically reactive of their truncated analogs were made in guinea pigs. Antipeptide antisera reactive with BPV were obtained for three of the cross-reactive epitopes, and one of these antisera allowed highly sensitive detection of group-specific PV antigen by immunoperoxidase staining.     

Topological mapping of the P1-adhesin of Mycoplasma pneumoniae with adherence-inhibiting monoclonal antibodies.

Five adherence-inhibiting monoclonal antibodies (mAbs) were used for topological mapping of the binding sites of the 169 kDa membrane-integrated adhesin of Mycoplasma pneumoniae. Antibody binding sites were characterized using overlapping synthetic octapeptides. Three regions of the protein seem to be involved in adherence: the N-terminal region [N-reg, epitopes beginning at amino acid (aa) 1 to aa 14 and aa 231 to aa 238, respectively]; a domain (D1) approximately in the middle of the molecule (beginning at aa 851 to aa 858 and aa 921 to aa 928); and a domain (D2) closer to the C-terminus (beginning at aa 1303 to aa 1310, aa 1391 to aa 1398 and aa 1407 to aa 1414). Each of the mAbs P1.26 and P1.62 reacted with two primary amino acid sequences. Both antibodies bound to the D1 region, but mAb P1.62 showed additional binding to a sequence (aa 231 to aa 238) near the N-terminus, and mAb P1.26 reacted with a second epitope in the D2 domain (aa 1303 to aa 1310). Such dual binding by the two antibodies suggests that in the native protein the epitopes are composed of two sequences which are located on two different sites of the molecule (D1/N-reg and D1/D2, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of a lambda gt11 expression library to localize a neutralizing antibody-binding site in glycoprotein E2 of Sindbis virus.

The Sindbis virus envelope contains two species of integral membrane glycoproteins, E1 and E2. These proteins form heterodimers, and three dimeric units assemble to form spikes incorporated into the viral surface which play an important role in the specific attachment of Sindbis virus to host cells. To map the neutralization epitopes on the surface of the virus, we constructed a lambda gt11 expression library with cDNA inserts 100 to 300 nucleotides long obtained from randomly primed synthesis on Sindbis virus genomic RNA. This library was screened with five different neutralizing monoclonal antibodies (MAbs) specific for E2 (MAbs 50, 51, 49, 18, and 23) and with one neutralizing MAb specific for E1 (MAb 33). When 10(6) lambda gt11 plaques were screened with each antibody, four positive clones that reacted with E2-specific MAb 23 were found. These four clones contained overlapping inserts from glycoprotein E2; the domain from residues 173 to 220 of glycoprotein E2 was present in all inserts, and we concluded that this region contains the neutralization epitope recognized by the antibody. No clones that reacted with the other antibodies examined were found, and we concluded that these antibodies probably recognize conformational epitopes not present in the lambda gt11 library. We suggest that the E2 domain from residues 173 to 220 is a major antigenic determinant of Sindbis virus and that this domain is important for virus attachment to cells.     

Primary structure of bovine lactate dehydrogenase-A isozyme and its synthesis in Escherichia coli

Eleven cDNA clones encoding lactate dehydrogenase(LDH)-A isozyme were isolated from a bovine lymphocyte cDNA library, and the nucleotide sequences of three of the clones (pLDH5, pLDH9 and pLDH12) were determined. With the exception of variation in the 5' portion, two cDNA clones (pLDH9 and pLDH12) appeared to contain the full-length cDNA of 1786 bp, consisting of the protein-coding sequence (996 bp), the 5'- and the 3'-untranslated regions and the poly(dA) tail. The predicted amino acid (aa) sequence of bovine LDH-A (332 aa) showed 96.7% homology with that of pig LDH-A. The protein-coding cDNA region (1650 bp) was inserted into an Escherichia coli expression vector ptac11 and expressed. The protein synthesized in E. coli showed enzyme activity of LDH and was identified by cellogel electrophoresis as LDH-5 isozyme whose subunit M chain is the product of the LDH-A gene.     

Immunochemistry of human Lp[a]: characterization of monoclonal antibodies that cross-react strongly with plasminogen.

Forty different monoclonal antibodies were produced from hybridomas that were raised against human Lp[a]. Of these, 14 strongly cross-reacted with plasminogen on ELISA screening assays while 16 clearly did not and 10 were only marginally cross-reactive. We took advantage of the homology between plasminogen and apo[a] to define the epitopes of 8 strongly cross-reacting monoclonal antibodies. We were able to subdivide these into four general categories based upon site competition assays (using both plasminogen and Lp[a]), and their reactivity with elastolytically derived plasminogen fragments. Group A monoclonal antibodies (F1 1E3, F2 3A3) recognized epitopes within the kringle 5 and protease domains (miniplasminogen) of plasminogen. The group B monoclonal antibody (F6 1A3) reacted solely with plasminogen kringle 4-like domains and appeared to recognize a limited number of sites on Lp[a]. Group C monoclonal antibodies (F6 1B5, F6 1G9) recognized a second, more frequently distributed site within these kringle 4-like domains. The final group, D, monoclonal antibodies (F6 2C3, F6 2G2, F6 3F4) reacted with a cluster of sites found associated with kringle 4-like domains but also reacted with the miniplasminogen domain. Interestingly, only the members of this group were able to interfere with the proteolytic activity of plasmin. Neither periodate treatment of Lp[a] nor incubation of Lp[a] with epsilon-aminocaproic acid affected the binding of any of our monoclonal antibodies.     

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T = 3 capsid protein leads to larger T = 4 capsids

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.     

Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid.

We analyzed a region of the capsid of canine parvovirus (CPV) which determines the ability of the virus to infect canine cells. This region is distinct from those previously shown to determine the canine host range differences between CPV and feline panleukopenia virus. It lies on a ridge of the threefold spike of the capsid and is comprised of five interacting loops from three capsid protein monomers. We analyzed 12 mutants of CPV which contained amino acid changes in two adjacent loops exposed on the surface of this region. Nine mutants infected and grew in feline cells but were restricted in replication in one or the other of two canine cell lines tested. Three other mutants whose genomes contain mutations which affect one probable interchain bond were nonviable and could not be propagated in either canine or feline cells, although the VP1 and VP2 proteins from those mutants produced empty capsids when expressed from a plasmid vector. Although wild-type and mutant capsids bound to canine and feline cells in similar amounts, infection or viral DNA replication was greatly reduced after inoculation of canine cells with most of the mutants. The viral genomes of two host range-restricted mutants and two nonviable mutants replicated to wild-type levels in both feline and canine cells upon transfection with plasmid clones. The capsids of wild-type CPV and two mutants were similar in susceptibility to heat inactivation, but one of those mutants and one other were more stable against urea denaturation. Most mutations in this structural region altered the ability of monoclonal antibodies to recognize epitopes within a major neutralizing antigenic site, and that site could be subdivided into a number of distinct epitopes. These results argue that a specific structure of this region is required for CPV to retain its canine host range.     

Several epitopes of p85 glycoprotein (CDw44) are dependent on intact disulphide bonds. Isolation of cDNA clones requires a polyclonal antibody raised against the reduced protein

Monoclonal antibodies 50B4 and 50E6 recognize two distinct epitopes of human p85 glycoprotein (CDw44). Both epitopes are destroyed by reduction of the purified gycoprotein as demonstrated by inhibition of cellular radioimmunoassay and Western blot analysis. Endoglycosidase F treated p85 glycoprotein, with an apparent molecular weight of 73,000 is still reactive with both monoclonal antibodies. Thus both epitopes are conformational determinats of the polypeptide chain. A rabbit antibody produced against purified native p85 glycoprotein also reacted only with the non-reduced form of p85. Repeated immunizations with SDS-dissociated and reduced p85 yielded a polyclonal antibody reactive by Western blot analysis with reduced and non-reduced forms of p85 glycoprotein. When a HOON leukemia cell line cDNA expression library was screened with this polyclonal antibody, two cDNA clones were isolated which reacted specifically with the antiserum and not with the control non-immune serum. Preliminary characterization of these clones indicates that they are p85-related.