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PubMed1997

Cloning, sequence analysis and expression of the major outer capsid protein gene of an aquareovirus.

The nucleotide and deduced amino acid sequences of genome segment 10 of aquareovirus strain SBR, encoding the major outer capsid protein (VP7), have been determined. Genome segment 10 of SBR virus is 986 nucleotides long and encodes a polypeptide of 298 amino acids with a predicted molecular mass of 32,430 Da. There are 26 non-translated nucleotides at the 5' end and 66 non-translated nucleotides at the 3' end. Using a recombinant baculovirus system, the VP7 protein of SBR virus was expressed to a high level. The baculovirus-produced VP7 protein was similar both in its size and antigenic properties to the authentic aquareovirus VP7 protein. Antiserum from a rabbit immunized with the baculovirus-produced VP7 protein failed to neutralize the homologous aquareovirus strain. As determined by Western blotting, this antiserum reacted with aquareovirus strains belonging to the same genogroup as SBR virus, but did not react with aquareovirus strains belonging to the other genogroups.

Animals↗
PubMed1997

Deletion mapping of functional domains in the rotavirus capsid protein VP6.

VP6, the major capsid protein of rotavirus, oligomerizes into trimers that constitute the intermediate shell of the virions. In order to map functional domains in this protein, we introduced seven internal in-frame deletions within the coding region of gene 6 of human rotavirus strain Wa. Regions of homology among the VP6 proteins of group A and group C rotaviruses were targeted for deletion mutagenesis. The mutant VP6 proteins were expressed in mammalian cells using the recombinant vaccinia virus system and were examined for their ability to oligomerize into trimers as well as to assemble into double-layered virus-like particles upon coexpression with the rotavirus core protein VP2. Deletions that abolished trimerization defined a domain (residues 246 to 314) that maps within a larger region previously found to be critical for oligomerization (amino acids 105 to 328). When the capacity of each mutant to assemble into double-layered virus-like particles was analysed, three different assembly phenotypes were observed. Phenotype I was represented by two deletion mutants lacking residues 246 to 250 and 308 to 314 that produced particles with efficiencies similar to that of wild-type VP6. Phenotype II, characterized by a moderate decrease in the efficiency of particle assembly with respect to that of wild-type VP6, included two mutants with deletions at the C terminus of the protein. Phenotype III was exhibited by three mutants whose abilities to assemble into double-layered virus-like particles were drastically impaired. Two of these mutants define a previously unidentified assembly domain (amino acids 122 to 147) at the N terminus of rotavirus VP6.

Amino Acid Sequence↗
PubMed1999

Genetic and antigenic variation of capsid protein VP7 of serotype G1 human rotavirus isolates.

The deduced amino acid sequences of the outer capsid protein, VP7, of serotype G1 rotavirus clinical isolates collected over a 6 year period (1990-1995) in Melbourne, Australia, were examined. Phylogenetic analysis characterized the sequences into two discrete clusters representing two of the four global lineages of human G1 VP7 proteins. Antigenic characterization using a panel of serotype G1-specific neutralizing monoclonal antibodies classified lineage II isolates (1990-1993) as monotype G1a while lineage I isolates were classified as monotype G1b (1993-1995). Examination of the sequences of the neutralization epitope regions of VP7 revealed a particular amino acid substitution at residue 94 in region A (Asp --> Ser/Thr) that correlated with lineage and monotype designation. Our results indicated that temporal genetic variation of the VP7 of serotype G1 rotaviruses was associated with changes in the antigenicity of these isolates.

Amino Acid Sequence↗
PubMed1999

Site-saturation mutagenesis of the PALTAVETG motif in coxsackievirus A9 capsid protein VP1 reveals evidence of conservation of a periodic hydrophobicity profile.

Enteroviruses possess a highly conserved 9 amino acid stretch of mainly hydrophobic character in the capsid protein VP1. A novel strategy, combining site-saturation mutagenesis and a single-tube cloning and transfection procedure, has been developed for the analysis of this motif in coxsackievirus A9 (CAV-9). Four individual amino acids were separately mutated. Mutagenesis of three of the four positions in CAV-9 resulted in a number of viable but impaired mutant strains, each containing a single amino acid substitution. In contrast, no mutants with amino acid substitutions at leucine 31 were isolated, although three different leucine codons were found among the viruses recovered. Small plaque size was regularly associated with reduced yields of infectious virus and an amino acid substitution at the target site in the viruses isolated from the site-saturated virus pools. From the range of amino acids observed in viable mutants, it was possible to estimate the characteristics that are required at individual amino acid positions. It seems that in the motif studied here, a periodic hydrophobicity profile needs to be conserved. The constraints observed on the ranges of acceptable amino acids presumably reflect the structural-functional requirements that have resulted in the conservation of the motif.

Amino Acid Sequence↗
PubMed2002

Evidence of recombination in the capsid-coding region of type A foot-and-mouth disease virus.

Recombination is one of the factors that contribute to genetic diversity in foot-and-mouth disease virus (FMDV). Similarity and bootscan analyses have provided evidence of recombination in the capsid-coding (P1) region of the virus. In the present study, of the 14 subtype A22 field isolates that were distributed in three previously described genotypes (IV, VI and VII) based on the 1D (VP1-encoding) gene sequence (Tosh et al., 2002 ), one isolate (IND 170/88) was found to be a hybrid of genotypes VI and VII in the P1 region. VP1, VP4, the 5' region of VP2 and the 3' region of VP3 of this virus were characteristic of genotype VI, whereas the remaining 3' region of VP2 and the 5' region of VP3 were characteristic of genotype VII. No insertion or deletion was observed in the recombinant virus. Recombination in the P1 region may provide an escape mechanism for the virus.

Animals↗
PubMed2006

Porcine circovirus type 2 replicase binds the capsid protein and an intermediate filament-like protein.

Porcine circovirus type 2 (PCV2) is an important porcine pathogen that establishes persistent subclinical infections but may, on activation, contribute to the development of post-weaning multisystemic wasting syndrome (PMWS). This disease is characterized by weight loss, respiratory or digestive disorders and enlarged lymph nodes with lymphocyte depletion. The molecular mechanisms behind the development of the disease are completely unknown. In order to clarify functions of the different viral proteins and, if possible, to connect these new findings to molecular mechanisms behind the pathogenesis or the viral life cycle, a bacterial two-hybrid screening of a porcine expression library from PK-15A cells was conducted. Using viral proteins corresponding to ORFs 1, 2, 3 and 4 as bait, a number of interactions were identified and two of them were chosen for further characterization. GST pull-down assays confirmed that viral replicase (Rep) interacted with an intermediate filament protein, similar to human syncoilin, and with the transcriptional regulator c-myc. Furthermore, interactions of the viral proteins to each other revealed an interaction between PCV2 Rep and the capsid (Cap) protein and Cap to itself.

Amino Acid Sequence↗
PubMed1999

Conformational changes of polyomavirus major capsid protein VP1 in sodium dodecyl sulfate solution.

Conformations of polyomavirus (Py) major capsid protein VP1 were analyzed by circular dichroism (CD) and fluorescence spectroscopy in the presence of sodium dodecyl sulfate (SDS). Binding of PyVP1 to SDS induced marked conformational changes of PyVP1, which were reflected on the CD and fluorescence spectra. Abrupt changes in both optical properties occurred within the narrow ranges of SDS concentrations with the transition midpoints closely related to SDS micelle formation. Analysis of circular dichroism spectra showed that the contents of alpha-helices, beta-sheets, beta-turns and random coils in PyVP1 varied upon addition of SDS, demonstrating the exquisite sensitivity of the conformations of the protein to the environment. The interactions of PyVP1 with SDS were shown to be dependent on the ionic strength of the protein solution, suggesting that both hydrophobic and electrostatic forces contribute to the PyVP1-SDS complex formation. The SDS-induced conformational changes of PyVP1 appeared to be a two-stage process.

Capsid↗
PubMed1991

Decreased antibody reactivity to Epstein-Barr virus capsid antigen in type 1 (insulin-dependent) diabetes mellitus.

In this study, antibody levels to Epstein-Barr virus (EBV) capsid antigen (VCA) and EBV early antigens (EA) were analysed by enzyme immunoassay in 54 newly diagnosed type 1 diabetic children and in matched controls. The patients had significantly lower EBV VCA IgG-class antibody levels (p less than 0.02). This was true particularly in young patients and in boys (p less than 0.005). VCA IgA-class antibody levels were also decreased in young patients (p less than 0.02). VCA IgM-class antibodies were observed in two of the patients only. IgG- and IgA-class antibodies to EBV EA or rubella virus antigen showed no differences between patients and controls. The results suggest that EBV infections coincide with the onset of clinical diabetes relatively rarely. However, the abnormally low antibody response to EBV VCA in diabetic children suggests abnormalities in the EBV-specific immune response.

Adolescent↗
PubMed2004

A -1 ribosomal frameshift in the transcript that encodes the major head protein of bacteriophage A2 mediates biosynthesis of a second essential component of the capsid.

The two major capsid proteins of Lactobacillus bacteriophage A2 share their amino termini. The smaller of these (gp5A) results from translation of orf5 and proteolytic processing after residue 123. The larger form (gp5B) originates through a -1 ribosomal frameshift at the penultimate codon of orf5 mRNA, resulting in a product that is 85 amino acids longer than gp5A. Frameshifting needs two cis-acting elements: a slippery region with the sequence C CCA AAA (0 frame), and a stem-loop that begins 9 nucleotides after the end of the slippery sequence. Mutations introduced in the slippery sequence suppress the frameshift. Similarly, deletion of the second half of the stem-loop results in drastic reduction of frameshifting. Both gp5A and gp5B appear to be essential for phage viability, since lysogens harboring prophages that produce only one or the other protein become lysed upon induction with mitomycin C, though no viable phage progeny are observed.

Bacteriophages↗
PubMed1993

Peptide antisera targeted to a conserved sequence in poliovirus capsid VP1 cross-react widely with members of the genus Enterovirus.

Rabbits were immunized with synthetic peptides derived from an immunodominant region of the VP1 capsid protein of enteroviruses. This region shows a high degree of homology among all sequenced members of the genus. Peptide-induced antisera were used for immunoperoxidase staining of cell cultures infected with 41 different serotypes of enterovirus. Specific cytoplasmic staining was readily seen in all but two cases. Echovirus type 22 was previously known to differ genetically from the rest of the enteroviruses, and hence, a negative result was expected. Surprisingly, one of the tested serum samples reacted with echovirus 22-infected cells. Coxsackievirus A7-infected cells could be reliably stained with only one of the tested serum samples. For the remaining 39 serotypes, scattered infected cells resulting from 1 to 2 days of incubation with diluted inocula were easily scored as positive before the cytopathic effect became visible. The same antibodies were also used in a sandwich-type enzyme immunoassay to demonstrate poliovirus antigens in cell extracts as early as 3 h after a high-multiplicity infection. These antibodies are candidates for enterovirus group reagents, being potentially useful in both the laboratory diagnosis of enterovirus infections and research on enterovirus-host interactions.

Amino Acid Sequence↗
PubMed1994

Serum antibody response to recombinant major inner capsid protein following human infection with group B rotavirus.

Recombinant major inner capsid protein (VP6) of the IDIR strain of group B rotavirus (GBR) was incorporated in a solid-phase immunoassay to access antibody response to infection in humans. Expression of VP6 in insect cells permitted design of a highly sensitive assay that avoided the contaminants present in GBR antigens obtained from fecal specimens. Among patients infected with the ADRV strain of GBR in China, increased reactivity with recombinant VP6 was observed in convalescent-phase sera in comparison with sera obtained shortly after infection (P = 0.0084). Anti-VP6 antibodies were detectable as soon as 7 days after onset of gastrointestinal symptoms, and serum reactivity persisted in specimens drawn more than 1 year after infection. Solid-phase immunoassay with recombinant VP6 was next employed in order to assess anti-GBR antibody in 513 serum specimens obtained from 423 Maryland residents (ages, 7 months to 96 years; median age, 42 years). Four individuals (< 1%) exhibited serum antibodies directed against the recombinant VP6 (ages, 54 to 95 years; mean age, 77 years). Examination of 129 additional serum specimens including some from other geographic regions of the United States failed to reveal the presence of anti-GBR antibody. Anti-GBR antibody was also not detected in any of 131 serum specimens from 60 staff and residents of a nursing home in Switzerland. While infection of humans with GBR has been uncommon in these locations outside of China, the detection of serum antibodies in older individuals in the United States either indicated an unknown, age-related risk factor or may have indicated infection in the more distant past. The availability of these reagents should allow surveys for GBR infection among additional populations that have not previously been investigated.

Adolescent↗
PubMed1998

The L1 major capsid protein of human papillomavirus type 16 variants affects yield of virus-like particles produced in an insect cell expression system.

The L1 major capsid proteins of six human papillomavirus type 16 (HPV-16) strains were expressed in insect cells by using recombinant baculoviruses. Virus-like particles (VLPs) which appeared similar to empty virions were identified by electron microscopy for all HPV strains investigated. However, the yield of VLPs produced varied in a range from 1 to 79 depending on the HPV-16 strain. The L1 proteins of these strains differed by up to 15 amino acids from the L1 protein of the prototype HPV-16 strain. Mutations in the amino acid region from residues 83 to 97 seemed to affect the level of expression of the L1 protein. These results are important when considering the development of HPV vaccines and serological tests. They indicate that strains inducing high levels of VLP production must be selected for the development of vaccines. Moreover, the L1 proteins of all strains investigated were able to bind with DNA. We also investigated the seroreactivities of VLPs derived from three different HPV-16 strains from Algeria, Senegal, and the Philippines by testing sera from women from 11 countries in immunoglobulin G-specific enzyme-linked immunosorbent assays. We observed a strong correlation between the reactivities of the three different VLP variants, independent of the geographical origin of the sera investigated. These results indicate that the three strains investigated are serologically cross-reactive despite the fact that their L1 proteins differ in 14 amino acids and suggest that VLPs derived from only one HPV-16 strain could be sufficient for the development of an HPV-16 vaccine and anti-HPV-16 tests.

Amino Acid Sequence↗
PubMed2000

Cloning of the rhesus lymphocryptovirus viral capsid antigen and Epstein-Barr virus-encoded small RNA homologues and use in diagnosis of acute and persistent infections.

Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis and is associated with the development of several human malignancies. A closely related herpesvirus in the same lymphocryptovirus (LCV) genera as EBV naturally infects rhesus monkeys and provides an important animal model for studying EBV pathogenesis. We cloned the small viral capsid antigen (sVCA) homologue from the rhesus LCV and developed a peptide enzyme-linked immunosorbent assay (ELISA) to determine whether epitopes in the rhesus LCV sVCA are a reliable indicator of rhesus LCV infection. In order to define a "gold standard" for rhesus LCV infection, we also cloned the EBV-encoded small RNA 1 (EBER1) and EBER2 homologues from rhesus LCV and developed a reverse transcription (RT)-PCR assay to detect persistent LCV infection in rhesus monkey peripheral blood lymphocytes. Animals from a conventional and a hand-reared colony were studied to compare the prevalence of rhesus LCV infection in the two groups. There was a 100% correlation between the peptide ELISA and EBER RT-PCR results for rhesus LCV infection. In addition, specificity for LCV infection and exclusion of potential cross-reactivity to the rhesus rhadinovirus sVCA homologue could be demonstrated using sera from experimentally infected animals. These studies establish two novel assays for reliable diagnosis of acute and persistent rhesus LCV infections. The rhesus LCV sVCA peptide ELISA provides a sensitive and reliable assay for routine screening, and these studies of the hand-reared colony confirm the feasibility of raising rhesus LCV-naive animals.

Amino Acid Sequence↗
PubMed1989

Synthesis, posttranslational modifications, and nuclear transport of polyomavirus major capsid protein VP1.

Polyomavirus major capsid protein VP1 synthesis was studied in infected primary baby mouse kidney cells. A standard curve of VP1 protein was used to quantitate VP1 in the cytoplasm and nucleus of infected cells during the time course of infection. Polyomavirus VP1 continued to be accumulated in the cytoplasm of the cells until 27 h postinfection, at which time the synthesis of VP1 leveled off. VP1 continued to accumulate in the nucleus of the infected cells throughout the course of infection. The presence of the six isospecies, A to F, of polyomavirus VP1 was also studied to determine the relative quantity of each species during the time course of infection. All six species were found in the cytoplasm and nucleus of infected cells at various times postinfection. However, the relative quantity of each species was different at early as compared with later times of infection. In addition, phosphorylated VP1 was found in isolated polyribosomes of infected cells, suggesting that phosphorylation of VP1 is a cotranslational modification. Examination of the effect of macromolecular synthesis on the transport of VP1 into the nucleus of infected baby mouse kidney cells as well as the rate of its nuclear accumulation during and after protein synthesis inhibition revealed that the continual transport and accumulation of VP1 in the nucleus required protein synthesis.

Animals↗
PubMed1991

Protease digestion of hepatitis A virus: disparate effects on capsid proteins, antigenicity, and infectivity.

High concentrations of either trypsin or chymotrypsin caused nearly complete cleavage of capsid protein VP2 of hepatitis A virus but did not significantly reduce the infectivity, thermostability, or antigenicity of the virus. Chymotrypsin also had a lesser effect on VP1. These findings indicate the presence of a protease-accessible VP2 surface site which neither contributes significantly to the dominant antigenic site nor plays a role in the attachment of the virus to putative cell receptors.

Animals↗
PubMed1992

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

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

Amino Acid Sequence↗
PubMed1992

Transactivation of the major capsid protein gene of herpes simplex virus type 1 requires a cellular transcription factor.

The purpose of this investigation was to identify and characterize the regulatory elements involved in the transcriptional activation of the beta gamma (leaky-late or gamma 1) genes of herpes simplex virus type 1 (HSV-1) by using the major capsid protein (VP5 or ICP5) gene as model. Gel mobility shift assays with nuclear extracts from uninfected and infected HeLa cells enabled us to identify two major protein-DNA complexes involving the VP5 promoter. The mobilities of these two complexes remained unaltered, and no unique complexes were observed when infected cell nuclear extracts were used. DNase I and orthophenanthroline-Cu+ footprint analyses revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, we tested the effect of mutations in this region by using transient expression of a cis-linked chloramphenicol acetyltransferase gene. Deletion of the above sequence resulted in a seven- to eightfold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV-1 immediate-early genes. From these results, we conclude that the LBS sequence and a cellular factor(s) are involved in the transactivation of the VP5 gene. A search of published gene sequences revealed that sequences related to the LBS exist in a number of other HSV-1, cytomegalovirus, retrovirus, and cellular promoters. Sequence homologies of binding sites and results of unpublished competition binding studies suggest that this leaky-late binding factor may be related to, or the same as, a ubiquitous cellular transcriptional factor called YY1 or common factor-1 (also known as NF-E1, delta, and UCRBP).

Base Sequence↗
PubMed1993

Characterization of the DNA binding properties of polyomavirus capsid protein.

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

Amino Acid Sequence↗