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PubMed1996

Selective cleavage by trypsin of the capsid protein VP1 of type 3 poliovirus results in improved sorting of cell bound virions.

A large proportion of host cell-bound virions of poliovirus type 1 strain Mahoney (PV1/M) is known to elute to the culture medium during incubation at 37 degrees C, and only a fraction of the virions remaining cell-associated will successfully uncoat and contribute to the new replication cycle. We found that while the proportion of inoculum type 3 poliovirus strain Saukett (PV3/S) bound to GMK cells was of the same order as that of PV1/M, the bound PV3/S virions uncoated much less efficiently, as judged by velocity sedimentation analysis of virion disintegration. Rather, the majority of the cell-associated PV3/S viruses remained apparently unaffected for several hours within an unidentified intracellular compartment. Incubation of PV3/S with intestinal trypsin is known to result in selective cleavage of the capsid protein VP1 and striking antigenic changes. Trypsin treatment of stock PV3/S preparations did not affect the infectivity titre or modify single-cycle progeny virus yields significantly. However, the fate of the cell-bound inoculum virus was profoundly altered. Trypsin-treated PV3/S virions (PV3/S-Try) attached to GMK cells less tightly than the untreated PV3/S virus or PV1/M, and a relatively larger proportion of the cell-bound virus eluted to the medium during subsequent incubation at 36 degrees C. However, the fraction of virions remaining cell-associated rapidly disintegrated suggesting efficient uncoating. In accordance with these observations, one step growth curves of PV3/S-Try in all cell lines tested showed lowered eclipse phase titres compared to those obtained with the untreated PV3/S inocula. Similar effects were also demonstrated for type 3 poliovirus strain Sabin while trypsin-sensitive strains of the other two serotypes of poliovirus remained unaffected in this sense. The putative biological significance of the altered sorting of cell-bound PV3/S-Try virions is not known. It might be related to the observations that sensitivity of type 3 poliovirus strains to trypsin is conserved in spite of the fact that the target site of trypsin action is flanked by highly variable motives in an immunodominant antigenic site.

Animals↗
PubMed1997

Comparative amino acid sequence analysis of the outer capsid protein VP4 from four lapine rotavirus strains reveals identity with genotype P[14] human rotaviruses.

The genes encoding the outer capsid VP4 proteins of four lapine rotavirus strains, three isolated in the US (ALA, C-11 and BAP-2) and one isolated in Japan (R-2) were sequenced, and the predicted amino acid (aa) sequence was compared to all known rotavirus genotypes. A high degree of aa identity (96.8-98.9%) was found among the American lapine strains, while the Japanese rotavirus strain R-2 shared less aa identity (89.5-90.0%) with the American strains. The four lapine rotaviruses shared the closest aa identity (90.6-94.9%) with the P[14] genotype, consisting of viruses isolated from humans in Italy, Finland and Thailand. These results indicate that the VP4 protein of the four lapine strains are genotype P14, and that among lapine strains there are possibly two subtypes, one represented by the American lapine strains and the other by the Japanese R-2 strain.

Amino Acid Sequence↗
PubMed1997

Sequence analysis of genome segment 10 encoding the major outer capsid protein (VP7) of genogroup B aquareovirus and its relationship with the VP7 protein of genogroup A aquareovirus.

Genome segment 10 of the coho salmon (CSR) strain of genogroup B aquareovirus has been cloned and sequenced. The gene is at least 936 nucleotides in length and has a major open reading frame encoding a protein of 293 amino acids with a calculated molecular mass of 31.7 kDa. Comparison of nucleotide and deduced amino acid sequences of genome segment 10 of genogroup A and genogroup B strains indicated 55.7 and 36.5% identity at the nucleotide and amino acid level, respectively. Baculovirus expression and serological identification of the genome segment 10 product of the CSR strain indicated that this gene encodes the major outer capsid protein of genogroup B aquareovirus.

Animals↗
PubMed1998

Serotypic characterization of outer capsid spike protein VP4 of vervet monkey rotavirus SA11 strain.

The vervet monkey rotavirus SA11, a prototype strain of group A rotaviruses, has been shown to possess VP7 serotype 3 specificity but its neutralization specificity with regard to the other outer capsid protein VP4 has not been elucidated. We thus determined its VP4 specificity by two-way cross-neutralization with guinea pig antiserum prepared with a single gene substitution reassortant that had only the VP4-encoding gene from the simian rotavirus SA11 strain and remaining ten genes from human rotavirus DS-1 strain (G serotype 2). The SA11 VP4 was related antigenically in a one-way fashion to rhesus monkey rotavirus MMU18006 VP4 (a P5B strain) and marginally to human and canine rotavirus VP4s with P serotype 5A specificity. In addition, the SA11 VP4 was shown to be distinct antigenically from those of other known P serotypes (1-4, and 6-11) as well as those of uncharacterized equine, lapine, and avian rotavirus strains. The SA11 VP4 is thus proposed for classification as a P5B serotype.

Animals↗
PubMed1999

Expression of goose parvovirus VP1 capsid protein by a baculovirus expression system and establishment of fluorescent antibody test to diagnose goose parvovirus infection.

The gene encoding VP1 capsid protein of a goose parvovirus was cloned into a baculovirus transfer vector and a recombinant baculovirus was produced. The recombinant virus expressed a protein of 88 kDa corresponding to the molecular weight of VP1 protein and the protein was detected by immunoblotting. By indirect fluorescent antibody (IFA) test, the expressed protein was detected in the nucleus of the insect cells as big granules and electron microscopy also showed several big granules in the nucleus infected with the recombinant virus. The IFA test was developed for screening antibody in Muscovy ducks.

Animals↗
PubMed2006

Enhanced immunogenicity to food-and-mouth disease virus in mice vaccination with alphaviral replicon-based DNA vaccine expressing the capsid precursor polypeptide (P1).

Recently, alphavirus replicon-based DNA vaccines, also known as suicidal DNA vaccines, have emerged as an important strategy to enhance the potency of DNA vaccines. In this study, two different types of DNA vaccines encoding the capsid precursor polypeptide (P1) of foot-and-mouth disease virus (FMDV) were constructed and the immunogenicity were investigated and compared in mouse model. The first DNA vaccine, pcDP1, is a conventional plasmid DNA vaccine in which P1 was driven directly by a cytomegalovirus promoter. The second DNA vaccine, pSCAP1, is a Semliki Forest virus (SFV) replicon-based DNA vaccine encoding the same antigen. In vitro expression and characterization indicated that two vaccine vectors could correctly produce the P1 antigen. However, pSCAP1 could induce obvious apoptosis of the transfected cells. After immunization in BALB/c mice, the P1-specific ELISA antibodies, neutralizing antibodies, as well as lymphocyte proliferative responses induced by pSCAP1 were significantly higher than those obtained in mice immunized with pcDP1. Notably, mice immunized with the pSCAP1 had the determined ability of clearing virus in their sera after FMDV challenge. These results indicate that the SFV replicon-based DNA vaccine pSCAP1 are more effective than conventional DNA vaccine and it can be considered a promising approach for the development of a safety and efficacious vaccine against FMDV.

Alphavirus↗
PubMed1995

Use of a gene-targeted phage display random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein VP5.

We describe the use of a gene-targeted random epitope library for the mapping of antigenic determinants. A DNA clone encoding the target antigen was digested randomly with DNase I to generate a population of DNA fragments of different sizes and sequences. After size fractionation, small DNA fragments (100-200 bp) were isolated and cloned into the phage expression vector fUSE2 to form an expression library displaying random polypeptide sequences as fusion proteins at the N terminus of the phage gene III protein. This library, termed a gene-targeted random epitope library to distinguish it from totally random synthetic epitope libraries, was then screened by affinity selection for recombinant phages which were specifically bound by the antibody of interest. Using this approach, we have mapped a monoclonal antibody (mAb)-defined epitope on the bluetongue virus outer capsid protein VP5. This epitope is not accessible on the intact virus surface, but is recognised by the immune system of sheep and cattle during virus infection. Although the example given here utilised a DNA fragment of known sequence and the library was screened for a mAb-defined epitope, the strategy described should be equally applicable to genes of unknown sequence and for screening of epitopes using polyclonal antibodies. The approach can also be extended to identify immunodominant epitope from much more complex genome-targeted random epitope library for virus, bacteria and eukaryotic organisms. Other applications of recombinant phages expressing defined immunodominant epitopes include serodiagnosis and vaccine development.

Amino Acid Sequence↗
PubMed1989

Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype.

Analyses of reassortant and parental strains of BTV serotypes 3 and 10, in serum neutralization tests, confirmed the major role of outer capsid protein VP2 in determination of virus serotype and its involvement in serum neutralization. However, a reassortant BTV strain (R70), containing protein VP5 derived from BTV 3 and VP2 derived from BTV 10, cross-neutralized with both parental virus strains (BTV 3 and BTV 10). It is concluded that VP5 also plays some part in serotype determination of these virus isolates, as analyzed by serum-neutralization, but its role may be less significant than that of VP2.

Animals↗
PubMed1990

Sequence analysis of the gene (6) encoding the major capsid protein (VP6) of group C rotavirus: higher than expected homology to the corresponding protein from group A virus.

Two overlapping c-DNA clones, which hybridized in Northern blots to RNA segment 6 of the prototype strain (Cowden) of group C rotavirus, were selected from a c-DNA library in pBR322 and sequenced. The gene 6 sequence obtained was 1349 nucleotides and contained a single long open reading frame encoding a protein of 394 amino acids (total MW, 44,479) which is in line with the size of the major capsid protein VP6. Comparison of the group C sequence with that of the corresponding group A rotavirus gene revealed homology levels of 55 and 42% for nucleotides and amino acids, respectively. These values were surprisingly high in view of previous immunological and nucleic acid hybridization data which failed to show any cross-reaction between group A and non-group A rotaviruses. The epidemiological implications of these observations are discussed.

Amino Acid Sequence↗
PubMed1991

Expression of the outer capsid protein VP5 of two bluetongue viruses, and synthesis of chimeric double-shelled virus-like particles using combinations of recombinant baculoviruses.

We have previously reported the assembly of virus-like particles (VLPs), consisting of the four major structural proteins of bluetongue virus (BTV), in Spodoptera frugiperda cells coinfected with recombinant baculoviruses (French et al. (1990). J. Virol. 64, 5695-5700). In this paper we report further studies using this system to assemble heterologous VLPs containing the outer capsid proteins (VP2 and VP5) of a range of different BTV serotypes. S. frugiperda cells were coinfected with three recombinant baculoviruses; a dual recombinant expressing VP3 and VP7 (of BTV-17 and -10, respectively) in combination with a single recombinant expressing VP2 of BTV-1, -2, -10, -11, 13, or -17 and an additional single recombinant expressing VP5 of BTV-2, BTV-10, or BTV-13. The resultant VLPs were purified and analyzed by electronmicroscopy, Western immunoblotting, and hemagglutination assays to determine whether double-shelled VLPs had been assembled. In the course of these experiments the VP2 proteins of all six available serotypes were successfully incorporated into VLPs. Particles from two different combinations of chimeric VLPs (having VP2 derived from BTV-1 or that of BTV-17) were used to raise antisera in guinea pigs. Both of these sera showed high neutralizing antibody titers against live BTV, indicating that heterologous VLPs may have potential for use in anti-BTV vaccines.

Animals↗
PubMed1991

Nucleotide sequence and transcriptional mapping of the major capsid protein gene of pseudorabies virus.

The gene encoding the 142-kDa major capsid protein (MCP142) of pseudorabies virus (PrV) was isolated and sequenced. Nucleotide sequence analysis revealed that the MCP142 gene has a single open reading frame of 3993 nucleotides (nt) encoding 1330 amino acids. The 4400-nt major RNA from the MCP142 gene was detected in PrV-infected cells. The 5' end of the transcript was located 60 nt upstream of the initiation codon. The 3' end of the transcript was located 18 nt downstream of a putative poly(A) signal sequence TATAAA and 133 nt downstream of the termination codon. In comparing amino acid sequence homology between MCP142 of PrV and other available herpesviruses MCP was shown to have 58% homology with herpes simplex virus type 1 and varicella-zoster virus, 27% with Epstein-Barr virus, and 24% with human herpesvirus 6 and human cytomegalovirus. It has greater homology with those of the alpha-herpesviruses than with those of the beta-herpesviruses and the gamma-herpesviruses.

Amino Acid Sequence↗
PubMed1991

Characterization of a nuclear localization sequence in the polyomavirus capsid protein VP1.

Expression of VP1-beta-galactosidase fusion proteins in the yeast Saccharomyces cerevisiae was used to identify a domain of the polyomavirus VP1 capsid protein which targets this protein to the nucleus. Fusion of the first 17 amino acids of VP1 to beta-galactosidase was sufficient for nuclear localization, whereas fusion of the first 12 amino acids gave a "mixed" cytoplasmic-nuclear phenotype. Mutation of a putative targeting sequence MAPKR(5)K from R to S changed the localization of a 21 amino acid fusion protein from the nucleus to cytoplasm. These results define a nuclear location signal in the amino terminus of polyomavirus VP1 and separate this function from the high-affinity DNA binding function previously defined for this region.

Animals↗
PubMed1992

The trypsin-sensitive RVER domain in the capsid proteins of minute virus of mice is required for efficient cell binding and viral infection but not for proteolytic processing in vivo.

Analysis of a series of mutations in the trypsin-sensitive RVER region of the amino terminal domain in the capsid proteins (VP1 and VP2) of the autonomous parvovirus, minute virus of mice (MVM), demonstrates that this sequence is not essential for proteolytic processing of VP2 into VP3 in vivo, but specific amino acids within this domain are important for viral infection. Analysis of the most deficient of these mutants, VP(delta 2842-2863), a 7-aa deletion of aa 159-165 in VP1 and 17-23 in VP2, has identified at least two steps in MVM infection in which this domain is important. VP(delta 2842-2863) was 3-fold defective in binding to murine A9(2L) cells and, when an equivalent amount of virus was bound to cells, additionally 10-fold deficient compared to wild-type in initiating a productive infection. However, in those cells effectively infected, VP(delta 2843-2863) replicated similar to wild-type. These results suggest that these seven amino acids constitute a region important for both binding and a subsequent step prior to the start of DNA replication such as viral uptake or transport to the nucleus.

Amino Acid Sequence↗
PubMed1992

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2.

A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of gene 5 encoding the inner capsid protein (VP6) of bovine group C rotavirus: comparison with corresponding genes of group C, A, and B rotaviruses.

To further study the molecular characteristics of group (gp) C rotaviruses, we produced, cloned, and sequenced cDNA to gene 5 of the Shintoku strain of bovine gp C rotavirus. The resulting clone was specific for gene 5 and was genetically related to the human and porcine gp C rotaviruses, as demonstrated by Northern blot hybridization analysis. The Shintoku gene 5 is 1352 nucleotides in length and has one open reading frame encoding a polypeptide of 395 amino acids with a predicted molecular mass of 44.5 kDa. Comparative sequence analysis indicated that: (i) the Shintoku gene 5 protein shared 88.4 to 90.6% homology with the VP6 of the human (Bristol and 88-220) and porcine (Cowden) strains of gp C rotaviruses, but only low homology with the VP6 of bovine gp A (RF) and human gp B (ADRV) rotaviruses (41.3 and 16.3%, respectively); (ii) the predicted secondary structure was highly conserved among the gene 5 proteins of the bovine, porcine, and human gp C rotaviruses; and (iii) seven highly conserved regions were identified for the first time in the deduced primary amino acid sequences of gene 5 of gp C and gene 6 of gp A rotaviruses. However, only three of these highly conserved areas were present in the regions of VP6, where the secondary structure was predicted to be similar for the rotavirus strains examined. These three regions may contribute to common epitopes between the two groups of rotaviruses. Our results, in comparison with data for other rotaviruses, indicate that gene 5 of the bovine gp C rotavirus codes for the major inner capsid protein (VP6).

Amino Acid Sequence↗
PubMed1982

Assembly of adenovirus major capsid protein is mediated by a nonvirion protein.

The assembly of hexon, the major capsid protein of adenovirus, was investigated with the use of conformation-specific monoclonal antibodies. The hexon capsomere is a trimer of three identical monomers folded into a highly conserved and stable structure. The unique nature of this structure is indicated by the lack of common antigenic determinants between the capsomere and either monomeric or denatured hexon. The assembly of the trimer requires the action of a nonvirion protein, the 100K protein. The virus-encoded 100K protein forms a tight complex with hexon polypeptides. This 100K-hexon complex can form on the polyribosomes while hexon is a nascent chain. Exclusion chromatography revealed that the complex has a molecular weight of 800,000. The complex contains only pretrimer hexon; no mature hexon capsomeres can be found bound to 100K. Kinetic analysis of hexon trimerization and hexon-100K binding indicated that trimerization and the release of hexon from the complex occur concomitantly.

Adenoviruses, Human↗
PubMed1988

Epitope-mapping on the Epstein-Barr virus major capsid protein using systematic synthesis of overlapping oligopeptides.

Systematic solid-phase synthesis of all possible overlapping nonapeptides of the 1381 amino acid sequence of the Epstein-Barr virus major capsid protein (EBV-MCP) was used to identify the position of linear antigen epitopes on this protein as recognised by human polyclonal antisera. Antisera were selected for reactivity with EBV-MCP on immunoblots. The results show that antibodies from different individual donors may recognise EBV-MCP through binding to a variety of different epitopes. These epitopes are localized at random over the protein backbone though some non-binding areas are also present. In addition, ten 'hot-spots' were identified containing closely-spaced reactive peptides (epitope-clusters) recognised by most (greater than or equal to 70%) individuals. No significant correlation was found between the actual location of these epitope-clusters and computer predictions using either hydrophilicity plots, secondary structure plots or a combination of (additional) parameters. Epitope-clusters generally were located in regions of indifferent or hydrophilic nature and mostly contained predicted beta-turn configurations. Only one epitope-cluster was located within a region of sequence homology with the MCPs of herpes simplex virus type 1 and varicella-zoster virus. The present study demonstrates the potential of using systematic peptide synthesis to define serologically relevant linear epitopes on large and relatively unexplored viral polypeptides.

Amino Acid Sequence↗
PubMed1995

Expression of the major capsid protein of human papillomavirus type 16 in Escherichia coli.

Major capsid proteins (MCPs) of various papillomaviruses have recently been expressed in heterologous cells as soluble and functional polypeptides. The host cells for producing these proteins have so far been of eukaryotic origin; however, E. coli has potential utility a host, with advantages over eukaryotic cells such as relatively simple culture requirements and greater ease of mutation of expressed sequences. We studied the expression by E. coli of the MCP of human papillomavirus type 16 (HPV16) using the gene derived from the 'prototype' HPV16 genome. Using expression vector pTrc99A, the protein was produced in full-length unfused form at levels of 3-4% of cell protein. Soluble polypeptide was detected, albeit at low levels. The level of solubility was not increased by growing cells at low temperature and slowing the rate of protein synthesis. The soluble protein was degraded at its carboxy terminus by an outer membrane protease of E. coli, OmpT, giving rise to two slightly shortened protein species of 52K and 56K in addition to the full-length 57K polypeptide. Since the MCP of prototype HPV16 is known to be prone to excessive aggregation compared with other papillomaviral MCPs, the recovery of soluble polypeptide indicates that E. coli is worth consideration as an alternative host to eukaryotes for producing these proteins.

Amino Acid Sequence↗