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Hepatitis A virus capsid proteins (VP0, VP3, and VP1) have been synthesized in Escherichia coli for use in antigenic and immunogenic analyses. Rabbits immunized with each of these individual recombinant capsid proteins developed a rapid neutralizing antibody response when subsequently challenged with a subimmunogenic dose of whole virus.
Norwalk virus capsid protein was produced by expression of the second and third open reading frames of the Norwalk virus genome, using a cell-free translation system and baculovirus recombinants. Analysis of the expressed products showed that the second open reading frame encodes a protein with an apparent molecular weight of 58,000 (58K protein) and that this protein self-assembles to form empty viruslike particles similar to native capsids in size and appearance. The antigenicity of these particles was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assays of paired serum samples from volunteers who developed illness following Norwalk virus challenge. These particles also induced high levels of Norwalk virus-specific serum antibody in laboratory animals following parenteral inoculation. A minor 34K protein was also found in infected insect cells. Amino acid sequence analysis of the N terminus of the 34K protein indicated that the 34K protein was a cleavage product of the 58K protein. The availability of large amounts of recombinant Norwalk virus particles will allow the development of rapid, sensitive, and reliable tests for the diagnosis of Norwalk virus infection as well as the implementation of structural studies.
An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.
A nested set of 11 overlapping synthetic peptides covering the entire sequence of rubella virus capsid protein was synthesized, purified, and tested against human rubella virus-specific T-cell lines and rubella virus-seropositive sera. T-cell lines derived from four donors responded strongly to four synthetic peptides containing residues 96 to 123, 119 to 152, 205 to 233, and 255 to 280. Only one peptide (residues 255 to 280) was recognized by all four T-cell lines. Two human immunodominant linear B-cell epitopes were mapped to residues 1 to 30 and 96 to 123 by using peptide-specific enzyme-linked immunosorbent assay. All 11 synthetic peptides were highly immunogenic and induced strong antibody responses in rabbits against the respective immunized peptides. Seven of the 11 rabbit antipeptide antisera (anti-1-30, -74-100, -96-123, -119-152, -205-233, -231-257, and -255-280) specifically recognized the capsid protein on immunoblots. Identification of these T- and B-cell epitopes represents the first step toward rational design of synthetic vaccines against rubella.
Little is known about the assembly of the 28-nm nucleocapsid or core particle of hepatitis B virus. Here we show that this assembly process can be reconstituted in Xenopus oocytes injected with a synthetic mRNA encoding the hepatitis B virus capsid protein (p21.5). Injected oocytes produce both a nonparticulate p21.5 species (free p21.5) and capsid particles. We describe rapid and simple methods for fractionating these species on a small scale either with step gradients of 10 to 60% (wt/vol) sucrose or by centrifugation to pellet the particles, and we characterize the oocyte core particles. Free p21.5 exhibits chemical and physical properties distinctly different from those of particles. Free p21.5 is partially cleaved by proteinase K, whereas core particles are almost completely resistant to cleavage. This suggests that the carboxyl-terminal protamine region, the main target for proteases within p21.5, is exposed in free p21.5 but faces the interior of the p21.5 core particle. Finally, pulse-chase experiments demonstrated that free p21.5 can be chased almost quantitatively into core particles, establishing that free p21.5 is fully competent to form particles and represents an assembly intermediate on the pathway for core particle formation. However, core particle assembly appears very dependent on p21.5 concentration and is rapidly compromised if the p21.5 concentration is lowered. The advantages of oocytes for studying assembly are discussed.
The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time.
The capsid protein (CA) (p24) of human immunodeficiency virus (HIV) type 1 expressed in Escherichia coli and purified to greater than 90% homogeneity was used to examine assembly in vitro and to probe the nature of interactions involved in the formation of capsid structures. The protein was detected in dimeric and oligomeric forms as indicated by molecular size measurements by gel filtration column chromatography, sedimentation through sucrose, and nondenaturing gel electrophoresis. Chemical cross-linking of CA molecules was observed with several homobifunctional reagents. Oligomer size was dependent on cross-linker concentration and exhibited a nonrandom pattern in which dimers and tetramers were more abundant than trimers and pentamers. Oligomers as large as dodecamers were detected in native polyacrylamide gels. These were stable in solutions of high ionic strength or in the presence of nonionic detergent, indicating that strong interactions were involved in oligomer stabilization. Limited tryptic digestion converted the putative dodecamers to octamers, suggesting that a region involved in CA protein multimerization was exposed in the structure. This region was mapped to the central portion of the protein. The recombinant CA proteins assembled in vitro into long rodlike structures and were disassembled into small irregular spheres by alterations in ionic strength and pH. The observation that assembly and disassembly of purified HIV type 1 CA protein can be induced in vitro suggests an approach for identifying possible control mechanisms involved in HIV viral core assembly.
The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.
The role of major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T lymphocytes in immunity to rubella virus (RV) infection is unknown. Lymphocytes of RV-immune individuals were prestimulated on an RV-infected MHC class I-matched (or partially matched) fibroblast monolayer which generated CD8+ lymphoblasts capable of lysing RV-infected fibroblast targets in a class I-restricted manner. Using an infectious Sindbis virus (SV) vector which expressed the RV capsid protein (SV/RubC), lymphocytes from 17 of 22 RV-immune individuals prestimulated on RV-infected fibroblast monolayers lysed SV/RubC-infected fibroblast targets. A sequence within the amino terminus of the capsid protein that was previously shown to contain immunodominant class II-restricted T-cell epitopes was evaluated for class I-restricted epitopes. Fibroblast targets pulsed with synthetic peptides representing subsequences within C1 to C29 (subscripts indicate amino acid positions) were lysed effectively when the targets and effectors matched at multiple class I alleles. By limiting the number of matching class I alleles, an A2-restricted epitope was identified within C9 to C22 and an epitope that could be presented by multiple class I molecules was identified within C11 to C29. A sequence such as C1 to C29 which contains both MHC class I- and MHC class II-restricted epitopes recognized by a heterologous human population may serve as a component of an effective synthetic vaccine.
In pseudorabies virus (PrV), an open reading frame that partially overlaps the gene for the essential glycoprotein gII has been shown to encode a protein homologous to the ICP18.5 polypeptide of herpes simplex virus type 1 (N. Pederson and L. Enquist, Nucleic Acids Res. 17:3597, 1989). To study the function of this protein during the viral replicative cycle, a PrV mutant which carries a beta-galactosidase expression cassette interrupting the ICP18.5(PrV) gene was constructed. This mutant could be propagated only on cell lines that were able to provide ICP18.5(PrV) in trans after transformation with a corresponding genomic PrV DNA fragment. Detailed analysis showed that inactivation of the ICP18.5(PrV) gene did not impair infection of noncomplementing cells, nor did it impair early or late gene expression, as shown by immunoprecipitation of glycoproteins gII, gIII, and gp50. Surface localization of glycoproteins as demonstrated by fluorescence-activated cell sorting analyses was also not affected. Southern blot hybridizations, however, showed that cleavage of replicative concatemeric viral DNA did not occur in noncomplementing cells infected by the ICP18.5 mutant PrV. In addition, electron microscopic analysis revealed an accumulation of empty capsids in the nucleus of mutant-infected noncomplementing cells. We conclude that the ICP18.5(PrV) protein is necessary for viral replication and plays an essential role in the process of mature capsid formation.
The roles of different regions of the duck hepatitis B virus (DHBV) core protein on viral capsid assembly and related functions were examined. Twelve deletion and insertion mutations which covered 80% of the DHBV C open reading frame were constructed and expressed in Escherichia coli. The N-terminal region (amino acids 3 to 66) of DHBV core protein was important for its tertiary structure and function in E. coli. The expressed core mutants without this region apparently inhibited E. coli growth. The results of transmission electron microscopy of E. coli thin sections, capsid agarose gel, and sucrose gradient sedimentation demonstrated that a few DHBV core mutants with insertion in the N terminus and deletion in the C terminus retained the ability to form core-like particles in E. coli. However, other mutations in most of N-terminal and central regions strongly inhibited the self-assembly ability of DHBV core protein in E. coli. In addition, the mutant with a C-terminal region deletion (amino acids 181 to 228) lost most of the nucleic acid-binding activity of the DHBV core protein.
The putative capsid open reading frame (ORF2) of the Leishmania RNA virus LRV1-4 was expressed in a baculovirus expression system. The expressed protein was identified by Western immunoblot analysis with polyclonal antiserum raised to purified LRV1-4 virus. Electron microscopy and sedimentation analysis indicated that the expressed protein self-assembles into empty viruslike particles of similar size and shape to authentic virus particles, thus confirming that ORF2 encodes the viral capsid. The expressed particles are present exclusively in the cytoplasm of infected SF9 cells and are able to assemble in the absence of LRV1-4 RNA, viral polymerase, or any Leishmania host factors.
We have generated four mouse monoclonal antibodies (MAbs) to bovine papillomavirus virions that bound type-specific, adjacent, and conformationally dependent epitopes on the L1 major capsid protein. All four MAbs were neutralizing at ratios of 1 MAb molecule per 5 to 25 L1 molecules, but only three effectively blocked binding of the virus to the cell surface. Therefore, antibodies can prevent papillomavirus infection by at least two mechanisms: inhibition of cell surface receptor binding and a subsequent step in the infectious pathway. The neutralizing epitopes of the bovine papillomavirus L2 minor capsid protein were mapped to the N-terminal half of L2 by blocking the neutralizing activity of full-length L2 antiserum with bacterially expressed peptides of L2. In addition, rabbit antiserum raised against amino acids 45 to 173 of L2 had a neutralizing titer of 1,000, confirming that at least part of the N terminus of L2 is exposed on the virion surface.
Rotaviruses are triple-layered particles that contain four major capsid proteins, VP2, VP4, VP6, and VP7, and two minor proteins, VP1 and VP3. We have cloned each of the rotavirus genes coding for a major capsid protein into the baculovirus expression system and expressed each protein in insect cells. Coexpression of different combinations of the rotavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The coexpression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similar to double-layered rotavirus particles. Coexpression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of nonneutralizing and neutralizing epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs. The production of VP2/4/6 particles indicated that VP4 interacts with VP6. Cell binding assays performed with each of the VLPs indicated that VP4 is the viral attachment protein. Chimeric particles containing VP7 from two different G serotypes also were obtained. The ability to express individual proteins or to coexpress different subsets of proteins provides a system with which to examine the interactions of the rotavirus structural proteins, the role of individual proteins in virus morphogenesis, and the feasibility of a subunit vaccine.
Direct evidence is presented for a host-specific role of the cucumovirus capsid protein in long-distance movement within infected plants. Cucumber (Cucumis sativus L.) is a systemic host for cucumber mosaic cucumovirus (CMV). Tomato aspermy cucumovirus, strain 1 (1-TAV), multiplied to the levels of CMV (i.e., replicated, moved from cell to cell, and formed infectious particles) in the inoculated leaves of cucumbers but was completely unable to spread systemically. The defective long-distance systemic movement of 1-TAV was complemented by CMV in mixed infections. Coinfection of cucumbers with 1-TAV RNA with various combinations of transcripts from full-length cDNA clones of CMV genomic RNA 1, RNA2, and RNA3 showed that CMV RNA3 alone complemented 1-TAV long-distance movement. We obtained mutants containing mutations in the two open reading frames in CMV RNA3 encoding the 3a protein and the capsid protein (CP), both of which are necessary for cell-to-cell movement of CMV. Complementation experiments with mutant CMV RNA3 showed that only 3a protein mutants, i.e., those with an intact CP, complemented the long-distance movement of 1-TAV in cucumbers. Since CMV and TAV have common systemic host plants, the results presented here are strong evidence for an active, host-specific function of the CPs of these two cucumoviruses for long-distance spread in the phloem. The results also suggest that the plasmodesmata in the vascular system and/or at the boundary between the mesophyll and the vascular system, involved in long-distance movement through the phloem, and those in the mesophyll, involved in cell-to-cell movement, differ functionally.
The minute virus of mice, prototype strain MVMp, productively infects cultured murine fibroblasts but not T cells. The immunosuppressive strain, MVMi, shows the converse tropism. These reciprocal tropisms are mediated by the viral capsids, in which their determinants have been mapped to a few specific amino acids in the primary sequence shared by VP1 and VP2. Which of these proteins is relevant in presenting these determinants during infection is not known. We have approached this question using a recombinant parvovirus system in which a LuIII-derived transducing genome, containing the luciferase reporter in place of viral coding sequences, can be packaged by capsid proteins from separate helper sources. We generated transducing virions by using helper constructs expressing either VP1 or VP2, containing the MVMp or MVMi tropic determinant region, in various combinations. The virions were used to infect human NB324K cells and murine A9 fibroblasts. Transduction of the human cells (permissive for both MVMp and MVMi) required both VP1 and VP2 and was successful with all combinations of these proteins. In contrast, significant transducing activity for A9 cells was detected only with recombinant virions containing VP2 of MVMp, while the use of either source of VP1 had little effect. We conclude that VP2 from MVMp is necessary to enable infection of murine A9 fibroblasts.
The viral capsid antigen complex of Epstein-Barr virus (EBV), an important serodiagnostic marker of infection with the virus, consists of at least four components, with molecular masses of 150, 110, 40, and 21 kDa. Here we show that the 21-kDa component of the viral capsid antigen consists of products of two EBV genes, BFRF3 and BLRF2. Both products were expressed from late transcripts, were recognized by human antisera, and were present in virions. The BFRF3 product, but not that of BLRF2, fulfilled the definition of ZEBRA-associated protein p21 (ZAP21). In cells in which EBV was lytically replicating, BFRF3 protein was coimmunoprecipitated together with ZEBRA by a rabbit antiserum directed against amino acids 197 to 245 of BZLF1. In EBV-negative cells cotransfected with BZLF1 and BFRF3 expression vectors, BFRF3 was also coimmunoprecipitated with this antiserum. Although this antiserum could not detect BFRF3 on an immunoblot, it was able to immunoprecipitate BFRF3 in the absence of ZEBRA expression. The rabbit antiserum to amino acids 197 to 245 of BZLF1 was found to detect the same epitope at the carboxy end of BFRF3 as was recognized by rabbit antiserum to BFRF3 itself. Thus, coimmunoprecipitation of BFRF3 p21 with ZEBRA appeared to be due to cross-reactivity of the immunoprecipitating antiserum rather than to direct association of ZEBRA and BFRF3 p21.
Recombinant viruses of echovirus 12 carrying mutations of a rhodanine-resistant or -dependent variant, were investigated, and five single mutations each inducing a rhodanine-resistant or -dependent phenotype were defined. Four mutations are localized in the capsid protein VP1, and the fifth exchange is in VP4. All original and recombinant viruses were shown to be stabilized by the antiviral drug rhodanine against heat inactivation. Hence, resistant and dependent variants still seem able to bind rhodanine, and apparently none of the exchanges affects the putative drug binding site. We hypothesize that drug resistance and dependence are consequences of an increased flexibility of the virus capsid.