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PubMed1993

Development of type-specific and cross-reactive serological probes for the minor capsid protein of human papillomavirus type 33.

Human papillomavirus type 33 (HPV33) is associated with malignant tumors of the cervix. In an attempt to develop immunological probes for HPV33 infections, antisera against various bacterial fusion proteins carrying sequences of the minor capsid protein encoded by L2 were raised in animals. Antigenic determinants on the HPV33 L2 protein were identified by using truncated fusion proteins and were classified as type specific or cross-reactive with respect to HPV1, -8, -11, -16, and -18. Cross-reactive epitopes map to amino acids 98 to 107 or to amino acids 102 to 112 and 107 to 117, respectively, depending on the fusion protein used for immunization. Antibodies directed toward these epitopes detect L2 proteins of HPV11, -16, and -18, but not of HPV1 and -8, in Western immunoblots and enzyme-linked immunosorbent assays. HPV33 L2 amino acids 82 to 94 and 117 to 130 induce type-specific antibodies, with the major response directed to amino acids 117 to 130. By using a synthetic peptide corresponding to L2 amino acids 117 to 130, high-titered, type-specific antisera were obtained. These antisera should be useful as immunological probes for HPV33 infection.

Amino Acid Sequence↗
PubMed1993

Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence.

Simian rotavirus (RRV) and murine rotavirus (EDIM-RW) differ dramatically in the oral inoculum required to cause diarrheal disease in neonatal mouse pups and in their ability to spread and cause disease in uninoculated littermates. A genetic approach was used to explore the molecular basis of these differences. Reassortant viruses were produced in vivo by coinfecting infant mice with RRV and EDIM-RW. Reassortant viruses were isolated by plaque purification of progeny virus obtained from mouse pup intestines on MA104 cells. The plaque-purified reassortants were evaluated for 50% diarrhea dose (DD50) and for the ability to spread and cause diarrhea in uninoculated littermates. The parental RRV strain had a DD50 of 10(5) PFU per animal, while the EDIM-RW parental strain had a DD50 of less than 1 PFU per animal. RRV never spreads from inoculated to uninoculated littermates and causes disease. Twenty-three reassortants were tested. Of great interest were the reassortants D1/5 and C3/2, which derived genes 4 and 7 (encoding VP4 and VP7) from RRV. These viruses had a DD50 similar or identical to that of EDIM-RW and spread efficiently from inoculated mouse pups to uninoculated pups. We conclude that the major outer capsid proteins VP4 and VP7 are not primarily responsible for virulence or host range restriction in the mouse model using a homologous murine rotavirus.

Animals↗
PubMed1993

Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins.

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gt11-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-beta-galactosidase. Serum of a rabbit immunized with BFRF3-beta galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-beta-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens.

Amino Acid Sequence↗
PubMed1994

Identification of the threonine phosphorylation sites on the polyomavirus major capsid protein VP1: relationship to the activity of middle T antigen.

Phosphorylation of the polyomavirus major capsid protein VP1 was examined after in vivo 32P labeling of virus-infected cells. Two phosphorylated peptide fragments of VP1 were identified by protease digestion, high-performance liquid chromatography purification, mass spectrometry, and N-terminal sequencing. The peptides from residues 58 to 78 and residues 153 to 173 were phosphorylated on threonine. Site-directed mutations were introduced at these threonine sites, and mutant viruses were reconstructed. A threonine-to-glycine change at residue 63 (mutant G63) and a threonine-to-alanine change at residue 156 (mutant A156) resulted in viruses defective in phosphorylation of the respective peptides after in vivo labeling. Growth of the mutant G63 virus was similar to that of the wild-type virus, but the mutant A156 was inefficient in assembly of 240S viral particles. Polyomavirus nontransforming host range (hr-t) mutants are defective in VP1 threonine phosphorylation when grown in nonpermissive cells (R. L. Garcea, K. Ballmer-Hofer, and T. L. Benjamin, J. Virol. 54:311-316, 1985). Proteolytic mapping of VP1 peptides after in vivo labeling from hr-t mutant virus infections demonstrated that both residues T-63 and T-156 were affected. These results suggest that the block in virion assembly in hr-t mutant viruses is associated with a defect in phosphorylation of threonine 156.

Amino Acid Sequence↗
PubMed1994

Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.

Base Sequence↗
PubMed1994

The herpes simplex virus type 1 major capsid protein (VP5-UL19) promoter contains two cis-acting elements influencing late expression.

The herpes simplex virus type 1 (HSV-1) major capsid protein VP5 gene (UL19) is expressed with beta gamma (gamma 1 [leaky late]) kinetics. We have previously described the construction of recombinant HSV-1 in which the VP5 promoter was engineered to control the expression of the bacterial beta-galactosidase gene as a reporter (C.-J. Huang, S. A. Goodart, M. K. Rice, J. F. Guzowski, and E. K. Wagner, J. Virol. 67:5109-5116, 1993). Here we describe further mutational analysis in recombinant viruses. We have precisely defined the boundaries of the VP5 promoter and identified two regions important for both the level and the kinetics of expression. The 5' boundary was located at -48 relative to the initiation site of transcription by analyzing a series of nested deletions in the upstream sequence, and although a number of cis-acting sites influencing transient expression have been identified upstream of this point, these sites have no role in promoter activity during productive infection. Deletion of an Sp1-binding site located between -48 and the TATA box at -30 greatly reduced VP5 promoter activity late but not early after infection. A cis-acting element whose sequence resembles the human immunodeficiency virus type 1 initiator was located between -2 and +10 in the VP5 sequence by characterizing a series of deletions and site-directed block mutations downstream the TATA box. This element defines the 3' limit of the VP5 promoter, and like the upstream element, disruption of this element also inhibited promoter activity late in the productive cycle.

Base Sequence↗
PubMed1995

Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres.

The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, delta NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or delta NCOVP1 were determined to be 0.37 +/- 0.23 microM for VP2 and 0.18 +/- 0.21 microM for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.

Amino Acid Sequence↗
PubMed1997

The loss of outer capsid protein P2 results in nontransmissibility by the insect vector of rice dwarf phytoreovirus.

A transmission-defective (TD) isolate of rice dwarf phytoreovirus lacked the ability to infect cells when derived from the virus-free insect vector Nephotettix cincticeps. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified virus showed that among six structural proteins, the P2 outer capsid protein (encoded by genome segment S2) was absent from the TD isolate, whereas all six proteins were present in the transmission-competent (TC) isolate. P2 was not detected on immunoblots of rice plants infected with the TD isolate. Genome segment S2 and its transcript were detected in both TD and TC isolates. Sequence analysis of the S2 segment of the TD isolate revealed the presence of a termination codon due to a point mutation in the open reading frame, which might explain the absence of P2 in the TD isolate. These results demonstrate that the P2 protein is one of the factors essential for infection by the virus of vector cells and, thus, influences transmissibility by vector insects.

Amino Acid Sequence↗
PubMed1999

Infectious pancreatic necrosis virus: identification of a VP3-containing ribonucleoprotein core structure and evidence for O-linked glycosylation of the capsid protein VP2.

Virions of infectious pancreatic necrosis virus (IPNV) were completely disintegrated upon dialysis against salt-free buffers. Direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. By using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein VP3. Isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire VP3 content of the virion was recovered together with the radiolabeled genomic material forming the unique threadlike ribonucleoprotein complexes. In a sensitive blotting assay, the outer capsid component of IPNV, i.e., the major structural protein VP2, was shown to specifically bind lectins recognizing sugar moieties of N-acetylgalactosamine, mannose, and fucose. Three established metabolic inhibitors of N-linked glycosylation did not prevent addition of sugar residues to virions, and enzymatic deglycosylation of isolated virions using N-glycosidase failed to remove sugar residues of VP2 recognized by lectins. However, gentle alkaline beta elimination clearly reduced the ability of lectins to recognize VP2. These results suggest that the glycosylation of VP2 is of the O-linked type when IPNV is propagated in RTG-2 cells.

Animals↗
PubMed1999

Common neutralization epitope in minor capsid protein L2 of human papillomavirus types 16 and 6.

Studies of virus neutralization by antibody are a prerequisite for development of a prophylactic vaccine strategy against human papillomaviruses (HPVs). Using HPV16 and -6 pseudovirions capable of inducing beta-galactosidase in infected monkey COS-1 cells, we examined the neutralizing activity of mouse monoclonal antibodies (MAbs) that recognize surface epitopes in HPV16 minor capsid protein L2. Two MAbs binding to a synthetic peptide with the HPV16 L2 sequence of amino acids (aa) 108 to 120 were found to inhibit pseudoinfections with HPV16 as well as HPV6. Antisera raised by immunizing BALB/c mice with the synthetic peptide had a cross-neutralizing activity similar to that of the MAb. The data indicate that HPV16 and -6 have a common cross-neutralization epitope (located within aa 108 to 120 of L2 in HPV16), suggesting that this epitope may be shared by other genital HPVs.

Animals↗
PubMed2000

Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4.

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.

Amino Acid Sequence↗
PubMed2000

Apoptosis in coxsackievirus B3-caused diseases: interaction between the capsid protein VP2 and the proapoptotic protein siva.

Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. Apoptotic events are present in CVB3-induced disease, but it is unclear how CVB3 is involved in apoptosis and which viral proteins may induce the apoptotic pathway. In this report we demonstrate that the human and murine proapoptotic protein Siva specifically interact with the CVB3 capsid protein VP2. Furthermore, the transcription of Siva is strongly induced in tissue of CVB3-infected mice and is present in the same area which is positively stained for apoptosis, CD27, and CD70. It has been proposed that Siva is involved in the CD27/CD70-transduced apoptosis. Therefore, we suggest a molecular mechanism through which apoptotic events contributes to CVB3-caused pathogenesis.

Amino Acid Sequence↗
PubMed2001

Avian reovirus major mu-class outer capsid protein influences efficiency of productive macrophage infection in a virus strain-specific manner.

We determined that the highly pathogenic avian reovirus strain 176 (ARV-176) possesses an enhanced ability to establish productive infections in HD-11 avian macrophages compared to avian fibroblasts. Conversely, the weakly pathogenic strain ARV-138 shows no such macrophagotropic tendency. The macrophage infection capability of the two viruses did not reflect differences in the ability to either induce or inhibit nitric oxide production. Moderate increases in the ARV-138 multiplicity of infection resulted in a concomitant increase in macrophage infection, and under such conditions the kinetics and extent of the ARV-138 replication cycle were equivalent to those of the highly infectious ARV-176 strain. These results indicated that both viruses are apparently equally capable of replicating in an infected macrophage, but they differ in the ability to establish productive infections in these cells. Using a genetic reassortant approach, we determined that the macrophagotropic property of ARV-176 reflects a post-receptor-binding step in the virus replication cycle and that the ARV-176 M2 genome segment is required for efficient infection of HD-11 cells. The M2 genome segment encodes the major mu-class outer capsid protein (muB) of the virus, which is involved in virus entry and transcriptase activation, suggesting that a host-specific influence on ARV entry and/or uncoating may affect the likelihood of the virus establishing a productive infection in a macrophage cell.

Animals↗
PubMed2001

Functional analysis of adenovirus protein IX identifies domains involved in capsid stability, transcriptional activity, and nuclear reorganization.

The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively participate in the stability of the viral icosahedron, acting as a capsid cement. We have previously demonstrated that pIX is also a transcriptional activator of several viral and cellular TATA-containing promoters, likely contributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-terminal domain are crucial for incorporation of the protein into the virion; specific residues of the C-terminal leucine repeat are responsible for pIX interactions with itself and possibly other proteins, a property that is critical for pIX transcriptional activity. We also show that pIX takes part in the virus-induced nuclear reorganization of late infected cells: the protein induces, most likely through self-assembly, the formation of specific nuclear structures which appear as dispersed nuclear globules by immunofluorescence staining and as clear amorphous spherical inclusions by electron microscopy. The integrity of the leucine repeat appears to be essential for the formation and nuclear retention of these inclusions. Together, our results demonstrate the multifunctional nature of pIX and provide new insights into Ad biology.

Adenoviruses, Human↗
PubMed2001

Expression of major capsid protein VP-1 in the absence of viral particles in thymomas induced by murine polyomavirus.

Thymomas induced by polyomavirus strain PTA in mice are known to express the major capsid protein VP-1. Since the expression of a late structural protein such as VP-1 is considered a sign of virus replication, the present work attempted to clarify the implication of the presence of this protein in tumor cells. Electron microscopy of tumors showed a striking absence of viral particles in the vast majority of the cells. However, immunoelectron microscopy of the same samples demonstrated intranuclear VP-1 in most cells despite the absence of viral particles. Very little infectious virus was recovered from tumors. A change in the electrophoretic mobility of VP-1 from thymomas was detected compared with VP-1 from productively infected cells. The data presented in this work prove that the expression of VP-1 in polyomavirus-induced tumors is not synonymous with the presence of infectious virus, suggesting a possible defect in viral encapsidation.

Animals↗
PubMed2002

Antibodies to rotavirus outer capsid glycoprotein VP7 neutralize infectivity by inhibiting virion decapsidation.

The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90 degrees light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound antibody under conditions that generated Fab fragments but not under conditions that generated F(ab')(2) fragments. Electron microscopy showed that RRV virions reacted with an anti-VP7 MAb stayed as triple-layered particles in the presence of excess EDTA. Furthermore, the infectivity of rotavirus neutralized via VP8*, but not that of rotavirus neutralized via VP7, could be recovered by lipofection of neutralized particles into MA-104 cells. These data are consistent with the notion that antibodies directed at VP8* neutralize by inhibiting binding of virus to the cell. They also indicate that antibodies directed at VP7 neutralize by inhibiting virus decapsidation, in a manner that is dependent on the bivalent binding of the antibody.

Animals↗
PubMed2002

Identification of rotavirus VP6 residues located at the interface with VP2 that are essential for capsid assembly and transcriptase activity.

Rotavirus has a complex triple-layered icosahedral capsid. The external layer consists of VP7 and VP4, the intermediate layer consists of VP6 trimers, and the internal layer consists of VP2. Double-layered particles (DLP) derived from the virus by solubilization of VP4 and VP7 are transcriptionally competent and extrude capped mRNA from their vertices. Analysis of the pseudoatomic model of the VP6 layer, obtained by placing the atomic structure of VP6 into electron microscopy reconstructions of the DLP, has identified the regions of the protein involved in interactions with the internal layer. To study the role of VP6 both in the assembly of DLP and in transcription, 13 site-specific substitution mutations of VP6, targeting the contacts between the two inner layers, were constructed and expressed in the baculovirus system. The effects of these mutations on VP6 expression, trimerization, and formation of macromolecular assemblies were investigated. Using either in vitro reconstituted DLP derived from purified viral cores and recombinant VP6 or in vivo self-assembled virus-like particles resulting from the coexpression of VP2 and VP6 in the baculovirus-Sf9 system (VLP2/6), we have identified the amino acids essential for recovery of transcription or assembly. All VP6 mutants formed stable trimers which, like wild-type VP6, assembled into tubular structures. The ability of VP6 to interact with VP2 was examined by several assays, including electron microscopy, coimmunoprecipitation, purification of VLP2/6, and monitoring of the transcriptase activity of reconstituted DLP. Of the 13 VP6 mutants examined, 3 were unable to assemble with VP2 and 3 others partially assembled. These mutants either did not rescue the transcriptase activity of core particles or did so only marginally. Four mutants as well as the wild-type VP6 assembled and transcribed very well. Three mutants assembled well on cores but, surprisingly, did not rescue the transcriptase activity of reconstituted DLP. Our results indicate that hydrophobic interactions between VP6 and VP2 residues are responsible for the stability of the DLP. They also show that subtle electrostatic interactions between VP6 and the underlying transcriptase machinery can be essential for mRNA synthesis.

Amino Acid Sequence↗
PubMed2002

Immune responses to the major capsid protein during parvovirus infection of rats.

Rat virus (RV) is a common parvovirus of laboratory rodents which can disrupt rat-based research. Prenatal or perinatal infection can be pathogenic or lead to persistent infection, whereas infection of adult rats is typically self-limiting. Effects on the host immune system have been documented during RV infection, but little is known about immune responses necessary for viral clearance. Our studies were conducted to identify humoral and cellular responses to the predominant capsid protein, VP2, during experimental infection of adult rats. We observed VP2-specific proliferation, gamma interferon production, and an immunoglobulin G2a humoral response that is maintained for at least 35 days following RV infection. These results strongly suggest the induction of virus-specific Th1-mediated immunity.

Animals↗