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At least 1,621 records · Page 90Linked to original sources
PubMed1998

pH-induced destabilization of lipid bilayers by a peptide from the VP3 protein of the capsid of hepatitis A virus.

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.

Capsid↗
PubMed2000

Gene delivery via polyomavirus major capsid protein VP(1), isolated from recombinant Escherichia coli.

In this study we attempted to investigate the feasibility of transferring exogenous DNA into the recipient host via polyomavirus major capsid protein VP(1) pseudocapsids generated from recombinant Escherichia coli, designated PyVP(1,E), both in vitro and in vivo. NIH-3T3 and FR3T3 cells were transfected with pCMV beta and pPyMT-1 plasmid DNA, respectively. In vitro DNA transfection was carried out via Pseudofect- and PyVP(1,E)-mediated methods, or by co-precipitation with calcium phosphate. Expression of beta-galactosidase and PyMT reporter genes was examined by Western-blot analysis. Parallel experiments were performed in vivo by direct injection of pCMV beta and pPyMT-1 plasmid DNA, complexed with PyVP(1,E), into the livers of Wistar rats, followed by Western-blot analysis and histochemical staining. The results obtained from in vitro transfection experiments showed that expression of the reporter genes can be detected in the recipient cells at 48 h post-transfection. PyVP(1,E) was shown to exhibit similarly efficient in vitro DNA transfection properties to Pseudofect, which was obtained from recombinant baculovirus.

3T3 Cells↗
PubMed1995

Polyomavirus VP1 phosphorylation: coexpression with the VP2 capsid protein modulates VP1 phosphorylation in Sf9 insect cells.

The polyomavirus virion has an outer capsid comprised of 72 pentamers of the VP1 protein associated with the minor virion proteins, VP2 and VP3, and the viral minichromosome. To investigate the interaction between VP1 and VP2/VP3, we mapped VP1 phosphorylation sites and assayed VP1 recognition by anti-peptide antibodies after coexpression of VP1 with VP2 or VP3 by using recombinant baculovirus vectors. VP1, expressed either alone or with VP3, was phosphorylated on serine residues, which are not modified during polyomavirus infection of mouse cells. When VP1 was coexpressed with VP2, the nonphysiologic serine phosphorylation of VP1 was decreased, and a tryptic peptide containing Thr-63, a site modified during virus infection of mouse cells, was phosphorylated. An anti-peptide antibody directed against the VP1 BC loop domain containing Thr-63 recognized VP1 expressed alone but not VP1 coexpressed with VP2 or VP3. The change in phosphorylation resulting from coexpression of two structural proteins identifies the potential of the baculovirus system for studying protein-protein interactions and defines a functional role for the VP1-VP2 interaction.

Amino Acid Sequence↗
PubMed2001

The capsid protein of a plant single-stranded RNA virus is modified by O-linked N-acetylglucosamine.

Plum pox virus (PPV) is a member of the Potyvirus genus of plant viruses. Labeling with UDP-[3H]galactose and galactosyltransferase indicated that the capsid protein (CP) of PPV is a glycoprotein with N-acetylglucosamine terminal residues. Mass spectrometry analysis of different PPV isolates and mutants revealed O-linked N-acetylglucosamination, a modification barely studied in plant proteins, of serine and/or threonine residues near the amino end of PPV CP. CP of PPV virions is also modified by serine and threonine phosphorylation, as shown by Western blot analysis with anti-phosphoserine and anti-phosphothreonine antibodies. Thus, "yin-yang" glycosylation and phosphorylation may play an important role in the regulation of the different functions in which the potyviral CP is involved.

Acetylglucosamine↗
PubMed1989

Alteration of amino acid 101 within capsid protein VP-1 changes the pathogenicity of Theiler's murine encephalomyelitis virus.

Chronic Theiler's murine encephalomyelitis virus infection of susceptible mice is an animal model for human demyelinating diseases. Previously we described an altered and diminished pattern of central nervous system disease in immunocompetent SJL/J mice infected with a variant virus. This variant virus H7A6-2 was selected with a neutralizing mAb recognizing the capsid protein VP-1 of Theiler's virus. Here we characterize the variant virus by ELISA and neutralization assays and by sequencing selected regions of the viral RNA genome and relate the alteration to disease. The variant virus contains one single point mutation within a neutralizing epitope of VP-1. This nucleotide change lead to an amino acid replacement at amino acid 101 of VP-1, a threonine (wild type) to an isoleucine (variant). Model building based on sequence alignments and the known structure of the related Mengo virus indicates that the altered amino acid is located in an exposed loop on the surface of the virus at the periphery of a site that has been proposed to be the receptor binding site. The results of ELISA, neutralization assay, and direct RNA sequencing provide for the first time an opportunity to precisely map an important structural determinant of neurovirulence.

Amino Acid Sequence↗
PubMed2000

Homotypic protection against rotavirus-induced diarrhea in infant mice breast-fed by dams immunized with the recombinant VP8* subunit of the VP4 capsid protein.

The outer capsid proteins VP4 and VP7 induce neutralizing antibody against rotavirus. We have investigated in a mouse model the protection mediated by immunization with VP8*, the amino-terminal tryptic fragment of VP4. BALB/c female mice immunized with simian rotavirus SA11 VP6 and VP8* proteins expressed in Escherichia coli were mated with seronegative males. Litters were orally challenged with the SA11 strain (P5B[2], G3) or with the murine rotavirus strain EDIM (P10[16], G3) to verify the degree of protection against diarrhea induced in the newborns. Only those pups born to dams immunized with VP8* did not develop diarrhea after having been orally challenged with the SA11 strain. Pups born to naive dams but foster nursed by VP8*-immunized dams did not develop diarrhea after having been orally infected with the SA11 strain, but they suffered diarrhea when challenged with the EDIM strain. These results support the concepts that (1) VP8* is a highly immunogenic polypeptide that induces effective homotypic protection against disease in pups born to dams immunized with this antigen and (2) in newborn mice the protection against disease is mediated by neutralizing secretory antibodies present in the milk rather than by serum antibodies transferred through the placenta to the offspring.

Animals↗
PubMed2000

An immunodominant, cross-reactive B-cell epitope region is located at the C-terminal part of the hamster polyomavirus major capsid protein VP1.

The VP1 represents the major capsid protein of the hamster polyomavirus (HaPV). Here we describe the mapping of epitopes along the VP1 using Escherichia coli-expressed VP1-dihydrofolate reductase (DHFR) fusion proteins and PepScan analysis. By use of DHFR fusion proteins an immunodominant region was localized in the C-terminal part of VP1 between amino acids 320-384. Further epitopes are located in the regions amino acids 1-133 and amino acids 133-320, respectively. There were no obvious differences in the reactivity between sera of tumor-bearing and papilloma-free naturally HaPV-infected hamsters. In contrast, PepScan analysis revealed linear epitopes in the regions amino acids 79-97 and amino acids 353-367 for tumor-bearing animals and amino acids 101-113 and amino acids 165-179 for papilloma-free animals. The region between amino acids 320-384 of HaPV-VP1 was found to be involved in cross-reactivity of VP1 from HaPV and other polyomaviruses. Previously we have demonstrated that heterologous expression of HaPV-VP1 allowed the formation of virus-like particles (VLPs). From epitope mapping data and structural predictions it has been suggested that HaPV-VP1-VLPs may tolerate foreign peptides in the region amino acids 81-88 and the C-terminal part of VP1.

Amino Acid Sequence↗
PubMed1998

Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry.

A complex of the polyomavirus internal protein VP2/VP3 with the pentameric major capsid protein VP1 has been prepared by co-expression in Escherichia coli. A C-terminal segment of VP2/VP3 is required for tight association, and a crystal structure of this segment, complexed with a VP1 pentamer, has been determined at 2.2 A resolution. The structure shows specific contacts between a single copy of the internal protein and a pentamer of VP1. These interactions were not detected in the previously described structure of the virion, but the location of VP2 in the recombinant complex is consistent with features in the virion electron-density map. The C-terminus of VP2/VP3 inserts in an unusual, hairpin-like manner into the axial cavity of the VP1 pentamer, where it is anchored strongly by hydrophobic interactions. The remainder of the internal protein appears to have significant flexibility. This structure restricts possible models for exposure of the internal proteins during viral entry.

Amino Acid Sequence↗
PubMed2001

Structure of the reovirus outer capsid and dsRNA-binding protein sigma3 at 1.8 A resolution.

The crystallographically determined structure of the reovirus outer capsid protein sigma3 reveals a two-lobed structure organized around a long central helix. The smaller of the two lobes includes a CCHC zinc-binding site. Residues that vary between strains and serotypes lie mainly on one surface of the protein; residues on the opposite surface are conserved. From a fit of this model to a reconstruction of the whole virion from electron cryomicroscopy, we propose that each sigma3 subunit is positioned with the small lobe anchoring it to the protein mu1 on the surface of the virion, and the large lobe, the site of initial cleavages during entry-related proteolytic disassembly, protruding outwards. The surface containing variable residues faces solvent. The crystallographic asymmetric unit contains two sigma3 subunits, tightly associated as a dimer. One broad surface of the dimer has a positively charged surface patch, which extends across the dyad. In infected cells, sigma3 binds dsRNA and inhibits the interferon response. The location and extent of the positively charged surface patch suggest that the dimer is the RNA-binding form of sigma3.

Amino Acid Sequence↗
PubMed1994

Localization and diagnostic application of immunodominant domains of the BFRF3-encoded Epstein-Barr virus capsid protein.

The Epstein-Barr virus (EBV) open reading frame BFRF3 encodes a viral structural capsid protein or tegument protein, VCA p18, that is highly immunogenic in humans. In this study, a cluster of immunodominant epitopes in the C-terminus of VCA p18 has been identified. These epitopes were combined into a single synthetic peptide that was analyzed for diagnostic value in an ELISA. This VCA combined peptide appeared to be an excellent VCA marker with remarkable applications for IgG- and IgM-related EBV diagnostics. The combined peptide reacted with 95% of 159 sera that were IgG VCA-positive by immunofluorescence assay. In addition, 95% of sera from 67 persons with confirmed infectious mononucleosis were positive for IgM antibodies to this VCA combined peptide. An IgG-specific seroconversion could be demonstrated by subsequent serum samples during the acute phase of primary EBV infections. Although 97% of the tested sera from nasopharyngeal carcinoma patients had IgG antibodies reacting positively with the VCA combined peptide, only 61% contained IgA anti-VCA combined peptide.

Amino Acid Sequence↗
PubMed2001

Binding of external ligands onto an engineered virus capsid.

The development of novel delivery systems for therapeutic substances includes targeting of the carriers to a specific site or tissue within the body of the recipient. This can be accomplished by appropriate receptor-binding domains and requires linking of these domains to the carrier. We have used recombinantly expressed polyomavirus-like particles as a model system and inserted the sequence of a WW domain into different surface loops of the viral capsid protein VP1. In one variant, the WW domain maintained its highly selective binding properties of proline-rich ligands and showed an increased affinity but also an accelerated association/dissociation equilibrium compared to the isolated WW domain, thus allowing a short-term coupling of external ligands onto the surface of the virus-like particles.

Animals↗
PubMed1991

Protection conferred by TrpE fusion proteins containing portions of the C-terminal region of capsid protein VP1 of foot-and-mouth disease virus.

Major immunogenic sites of foot-and-mouth disease virus (FMDV) have been mapped to the C-terminal third of capsid protein VP1; we studied the immunogenicity of a series of TrpE-FMDV fusion proteins containing this region of FMDV strain O1 Campos. Fusion protein TrpE-dCN, which contains a dimer of VP1 amino acid sequences consisting of amino acids 200 to 213 linked by a diproline spacer to amino acids 141 to 158 (200-213 approximately P-P-G approximately 141-158), induced the best response. A single inoculation of guinea-pigs with 100 micrograms TrpE-dCN elicited high levels of neutralizing antibodies and protected all the animals against challenge infection with homologous virus. Although the closely related FMDV strains O1 Campos and O1 Caseros induced high levels of cross-protection, TrpE-dCN-vaccinated guinea-pigs were poorly protected against challenge infection with heterologous FMDV strain O1 Caseros. Nucleotide sequence analysis revealed that amino acid differences at residues 149 and 152 were critical for the induction of cross-protection and that neutralizing epitopes not present in TrpE-dCN are likely to be responsible for conferring a high level of cross-protection between FMDV strains O1 Campos and O1 Caseros.

Amino Acid Sequence↗
PubMed1991

A Gly1 to Ala substitution in poliovirus capsid protein VP0 blocks its myristoylation and prevents viral assembly.

Capsid protein VP4 of poliovirus is acylated with myristic acid via an amide linkage to its N-terminal glycine residue. Our previous studies suggested that myristic acid plays a role in poliovirus assembly and in the early events of infection. In order to understand better its role in the assembly process, we introduced a Gly1 to Ala amino acid substitution in the myristoylation signal sequence of VP4. This substitution prevented VP0 myristoylation in vivo and abolished the infectivity of genomic transcripts harbouring the mutation. These mutated RNAs were still able to replicate in the transfected cells but the assembly processes were inefficient and no mature virions could be detected.

Alanine↗
PubMed1993

Mapping of linear B cell epitopes on capsid proteins of bovine papillomavirus: identification of three external type-restricted epitopes.

Immunodominant, conserved and type-restricted external epitopes of bovine papillomavirus (BPV) major (L1) capsid protein have been identified using BPV particles and synthetic peptides. Antisera to disrupted BPV-1 recognized BPV-1 and BPV-2 particles in immune electron microscopy (IEM) studies and inhibited BPV-2-induced focus formation of NIH/3T3 cells. Thus BPV-1/BPV-2 cross-reactive epitopes occur on the surface of virions. The L1 protein appeared to be immunodominant as the antisera reacted with three dominant BPV-1/BPV-2 conserved B cell epitopes (amino acids 111 to 125, 131 to 145 and 191 to 205) in Pepscan assays of BPV-1 L1, whereas no common epitopes and less frequent antibody binding to peptides were detected in Pepscans of the L2 protein of BPV-1. Four discrete variable regions were identified in the sequences of L1 proteins of BPV-1 and BPV-2. Antisera against synthetic peptides corresponding to three of the four variable regions (amino acids 42 to 56, 435 to 449 and 485 to 499) of BPV-2 L1 caused clumping of BPV-2, but not of BPV-1, particles as examined by IEM, and antisera to one peptide (amino acids 485 to 499) inhibited BPV-2-induced focus formation of NIH/3T3 cells. These data suggest that these regions are type-specific BPV-2 L1 epitopes and that they occur on the virion surface. Although conformation-dependent epitopes remain to be identified on papillomaviruses, the linear epitopes identified in this study may be worthy of further study as constituents of experimental prophylactic vaccines.

3T3 Cells↗
PubMed1995

Proteolytic cleavage of VP2, an outer capsid protein of African horse sickness virus, by species-specific serum proteases enhances infectivity in Culicoides.

Purified African horse sickness virus (AHSV) was fed, as part of a blood meal, to adult females from a susceptible colony of Culicoides variipennis, established in the insectories at the Institute for Animal Health, Pirbright Laboratory, UK. The meal consisted of heparinized blood obtained from ovine, bovine, equine (horse and donkey) or canine sources spiked with AHSV serotype 9 (AHSV9). The infectivity levels observed for C. variipennis varied significantly, according to the source of the blood sample. Comparison of the protein profiles obtained from AHSV9 incubated with the individual serum of plasma samples indicated that some species-specific serum proteases were able to cleave the outer capsid protein, VP2. The blood samples containing serum proteases that were able to cleave VP2 also showed an increase in infectivity for the insect vector when spiked with purified AHSV.

African Horse Sickness Virus↗
PubMed1996

Recombinant baculovirus-synthesized African horsesickness virus (AHSV) outer-capsid protein VP2 provides protection against virulent AHSV challenge.

African horsesickness virus serotype 4 (AHSV-4) outer-capsid proteins VP2 or VP2 and VP5, prepared from single or dual recombinant baculovirus expression vectors grown in Sf9 insect cells, were administered in different amounts to horses and the neutralizing antibody responses were measured. Control and vaccinated horses were challenged with virulent AHSV-4 6 months later and monitored post challenge. The results indicated that two inoculations of extracts containing VP2 and VP5, or VP2 alone, in doses of 5 micrograms VP2 or more per horse, were sufficient to elicit protection against African horsesickness (AHS) disease. The recombinant VP2 protein is a potential candidate vaccine for AHS in horses.

African Horse Sickness↗
PubMed1996

Expression of muscovy duck parvovirus capsid proteins (VP2 and VP3) in a baculovirus expression system and demonstration of immunity induced by the recombinant proteins.

The gene encoding the muscovy duck parvovirus (DPV)-strain 89384 capsid proteins VP2 and VP3 was cloned in a baculovirus expression system and expressed in insect cells. The recombinant proteins were found to react with specific anti-DPV serum by Western blotting and to be located in the nucleus of insect cells (Sf9) as shown by immunofluorescence. Empty virus-like particles (VLPs) identical in size and appearance to DPV virions were observed by electron microscopy. The antigenicity and immunogenicity of the recombinant proteins were evaluated by ELISA and seroneutralization. Immunization of 3-week-old muscovy ducklings induced anti-DPV antibodies; neutralizing antibody titres were consistent with those observed in ducklings inoculated with a commercial inactivated vaccine. The way to develop these promising results is discussed.

Animals↗
PubMed1997

The gene encoding the capsid protein P82 of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus: sequencing, transcription and characterization by immunoblot analysis.

A gene encoding a capsid-associated viral structural protein has been identified and sequenced in the genome of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV). The gene has a 1872 nucleotide open reading frame (ORF) encoding 624 amino acids with a predicted molecular mass of 71.4 kDa. Transcription, which appeared to be initiated from a conserved GTAAG motif of baculovirus late genes, was detected at 12 h, reached a maximum at 48 h and declined at 72 h post-infection (p.i.). Part of the ORF was cloned in frame into a prokaryotic expression vector, pMAL-c2, and the fusion protein was used to generate antibodies in rabbits. It was shown, with the aid of the polyclonal antiserum, that this viral protein was detectable at 24 h p.i. in infected cells. The protein appeared as an 82 kDa band in occlusion-derived virus and as an 82 kDa band and a 72 kDa band in budded virus. Amino acid sequence comparisons revealed that this ORF had high homology with the ORF p87 (77% similarity) of Orgyia pseudotsugata (Op) MNPV and the ORF p80 (60% similarity) of Autographa californica (Ac) MNPV. Immunoblots confirmed that the CfMNPV protein had antigenic similarities to the P87 protein of OpMNPV, but not to the P80 of AcMNPV.

Amino Acid Sequence↗