Heterogeneity in the structural glycoprotein (VP7) of simian rotavirus SA11

The polypeptides of cells infected with a series of plaque isolates of the simian rotavirus SA11 were analyzed by SDS-PAGE. Altered electrophoretic migration of the major outer capsid glycoprotein (VP7) was found with independent virus stocks exhibiting gene products of VP7 ranging in apparent molecular weight from 35.5K to 38K. Similar differences in electrophoretic migration in the polypeptide precursor of the glycoprotein (pVP7) suggested that the heterogeneity resulted from mutations in the gene encoding the glycoprotein. The glycoprotein phenotype was stable on passage; the phenotypes were unchanged for 10 passages at high and low multiplicity. The biologic consequences of heterogeneity in the polypeptide are discussed.     

Genome and polypeptides characterization of Tellina virus 1 reveals a fifth genetic cluster in the Birnaviridae family

We characterized tellina virus 1 (TV-1), a birnavirus isolated from the marine bivalve mollusk Tellina tenuis. Genome sequence analysis established that TV-1 is representative of a viral cluster distant from other birnaviruses. The maturation process of the polyprotein encoded by the genomic segment A was delineated with the identification of the N-termini of the viral protease VP4 and the ribonucleoprotein VP3, and the characterization of peptides deriving from the processing of pVP2, the VP2 capsid protein precursor. One of these peptides was shown to possess a membrane-disrupting domain. Like the blotched snakehead virus, the polyprotein exhibits a non-structural polypeptide (named [X]) located between pVP2 and VP4. Mutagenesis analysis allowed the identification in VP4 of a catalytic Ser-Lys dyad that does not possess the common Gly-X-Ser signature of the serine hydrolases. The genomic segment B encodes the viral RNA-dependent RNA-polymerase VP1 with the unique sequence motif arrangement identified in other birnavirus VP1s.     

蓝舌病毒两外壳蛋白VP2和VP5在昆虫细胞中的表达 …

目的 研究蓝舌病毒（ＢＴＶ）ＶＰ２与ＶＰ５的免疫学特性,为ＢＴＶ基因工程疫苗研究和病毒样颗粒装配打下基础。方法 将ＢＴＶ１０ ＶＰ２和ＶＰ５基因分别插入杆状病毒表达载体ｐＦａｓｔＢａｃ１,转染昆虫细胞获得重组杆状病毒。用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ检测重组杆状病毒对ＶＰ２和ＶＰ５的表达,运用组织培养中和试验和直接血凝试验检测表达产物的生物学活性。     

Absence of subviral particles and assembly activity in HeLa cells infected with defective-interfering (DI) particles of poliovirus

HeLa cells infected with defective-interfering (DI) particles of poliovirus were examined for their capacity to synthesize viral proteins, viral-related particles, and the viral factor(s) that promotes the assembly of 14 S particles into empty capsidsin vitro. As reported by C. N. Cole and D. Baltimore (1973,J. Mol. Biol.76, 325–343) cells infected with purified DI particles failed to synthesize the capsid precursor NCVP 1a or any of the capsid polypeptides VP 0, VP 1, VP 2, or VP 3. Consequently, no 14 S particles, empty capsids, or virions were formed at low to moderate multiplicities. Cytoplasmic extracts prepared from cells infected with purified DI particles do not promote the assembly of viral 14 S particles into empty capsids. We conclude that the presence of assembly activity is dependent on the formation of the capsid precursor protein (NCVP-1a) or its cleavage products.     

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Triatoma virus (TrV) is a non-enveloped + ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.     

Identification of precursors of structural proteins VP1 and VP2 of hepatitis A virus.

The morphogenetic pathway of hepatitis A virus (HAV), classified as a member of the enteroviruses within the Picornaviridae, still remains obscure and seems to differ considerably from that of poliovirus, the most studied representative of this genus. In order to elucidate the precursor/product relationship of HAV structural proteins, subviral particles, which represent more than 50% of the viral antigen produced in infected cells, were separated from mature virions and their polypeptide pattern was analyzed by polyacrylamide gel electrophoresis and immunoblotting using monospecific antisera. Whereas mature virions are composed of viral proteins VP1, VP2, and VP3, subviral particles contained VP0 and smaller polypeptides instead of VP2. Comparison of proteins of different strains of HAV showed that VP0 of strain HAS-15 migrated slower than that of strains MBB or GBM. During the course of the infectious cycle, VP0 accumulated and only small portions were converted to VP2 supporting earlier observations that encapsidation of RNA with concomitant cleavage of VP0 is rate-limiting, leaving a large amount of viral antigen in premature particles. Similar to VP0, accumulation of VP1 was observed and two immunologically related precursor proteins, p38 and p36, were found during the course of infection. Immunological characterization of p38 using antisera directed to the N-terminus of VP1 and to synthetic peptides located at the presumptive C- and N-termini of 2A suggests that p38 is VP1 delta 2A carrying 45 N-terminal amino acids of the P2-region.     

The apoptotic capability of coxsackievirus B3 is influenced by the efficient interaction between the capsid protein VP2 and the proapoptotic host protein Siva

Infections with coxsackievirus B3 (CVB3) are common causes of myocarditis in humans. One detail of CVB3-induced pathogenesis is apoptosis. The interaction between the capsid protein VP2 of the myocardial virus variant CVB3H3 and the proapoptotic host cell protein Siva has recently been observed. In order to characterize the interaction between both proteins more precisely, the binding activity of the CVB3H3 VP2 to Siva was compared to that of the mutant virus CVB3H310A1 VP2. We found that the asparagine at position 165 in VP2 is essential for a stable interaction with Siva influencing also the induction of apoptosis, viral spread, and inflammatory responses in vivo. Furthermore, the specific binding site of Siva to VP2 is located at amino acid positions 118-136. Together, these results show that the interaction between VP2 of CVB3H3 and Siva is a highly specific process involving distinct amino acids on both proteins that most likely influence the outcome of CVB3-caused disease.     

Characterization of cleavage sites and protease activity in the polyprotein precursor of Japanese marine aquabirnavirus and expression analysis of generated proteins by a VP4 protease activity in four distinct cell lines

Summary A polyprotein precursor NH₂-pVP2–VP4–VP3-COOH is encoded in genomic segment A of members of the family Birnaviridae. By N-terminal sequencing analysis, primary cleavage sites of a marine birnavirus (MABV) polyprotein were identified as Ala⁵⁰⁸ ↓ Ser⁵⁰⁹ and Ala⁷³⁴ ↓ Ser⁷³⁵, where the cleavage motif was the same as that of infectious pancreatic necrosis virus (IPNV). However, further VP4 and VP3 cleavages occurred at novel sites. Ser⁶³³ and Lys⁶⁷⁴ mutations affected the cleavage activity by site-directed mutagenesis. Additional catalytic residues including Ile⁵⁴³ and Val⁶⁸⁶ were MABV-specific. As shown by electron microscopy, pVP2 and further cleaved VP3s (fcVP3s) could not form virus-like particles (VLPs). This suggests that VP3 is necessary for VLP formation. By Western blot analysis of the VP3 expression, fcVP3s were found in RSBK-2 cells and FHM cells, while VP3 was cleaved less in EPC cells, suggesting that fcVP3s might merely be a degraded form. Alternatively, if fcVP3s play functional roles other than in viral assembly, the further VP3 cleavage is, at least, not restricted in FHM cells. Strangely, VP3 was not completely further cleaved in CHSE-214 cells despite the fact that this cell line has a potential proteolytic factor, implying that complicated factors are associated with the further VP3 cleavage.     

Biochemical mapping of the foot-and-mouth disease virus genome.

Four primary cleavage products, mol. wt. 10(3) X 100, 88, 56 and 52 (P100, P85, P56 and P52 respectively) are present in BHK 2I cells infected with foot-and-mouth disease virus (FMDV). However, no precursor polyprotein equal to the sum of their mol. wt. was detected, even when amino acid analogues and proteolytic enzyme inhibitors were used. Three of the primary products were shown to cleave to smaller polypeptides, including the capsid polypeptides of the virus. Polypeptide P88, which was shown to be the precursor of the capsid polypeptides, is translated from the gene located at the 5'-end of the genome. The order of the structural polypeptides, determined by the use of emetine, is VP4, VP2, VP3, VP1. The order of the remaining primary cleavage products is P52, P56 and P100. P56 is a stable product, identical with the virus infection associated (VIA) antigen found in virus harvests. The function of the other two products P52 and P100 is not known. EMDV thus differs from other picornaviruses in that there is an extra primary cleavage product, apparently resulting from translation of more of the virus genome.     

Expression of the Foot-and-Mouth Disease Virus VP1 protein using a replication-competent recombinant canine adenovirus type 2 elicits a humoral antibody response in a porcine model

To develop a new type vaccine for Foot-and-Mouth Disease (FMD) prevention by using canine adenovirus as vector, the VP1 cDNA of Foot-and-Mouth Disease Virus (FMDV) type O strain China 99 was amplified by RT-PCR and cloned into pEGFP-C1 by replacing the GFP gene with the VP1 cDNA, resulting in an expression plasmid pVP1-C1. The expression cassette of VP1 composed of the CMV promoter, the VP1 gene and the SV40 early mRNA polyadenylation signal was recovered by Nsi I / Mlu I digestion of pVP1-C1 and cloned into the Canine adenovirus type-2 (CAV-2) genome in which E3 region was partly deleted by removing the Ssp I- Ssp I fragment. The recombinant virus (CAV-2-VP1) was obtained by transfecting the recombinant CAV-2-VP1 genome into MDCK cells with Lipofectamine 2000. Immunization trial in pigs with the recombinant virus, CAV-2-VP1, showed that CAV-2-VP1 could stimulate a specific immune response to both FMDV and the vector virus. Immune response to the VP1 and FMDV after VP1 expression was confirmed by ELISA, western blotting analysis and neutralization test. It was indicated that CAV-2 may serve as a vector for FMD vaccine development in pigs.     

Uncatalyzed assembly of spherical particles from SV40 VP1 pentamers and linear dsDNA incorporates both low and high cooperativity elements

The capsid of SV40 virion is comprised of 72 pentamers of the major capsid protein, VP1. We examined the synergism between pentamer-pentamer interaction and pentamer-DNA interaction using a minimal system of purified VP1 and a linear dsDNA 600-mer, comparing electrophoresis with electron microscopy and size exclusion chromatography. At low VP1/DNA ratios, large tubes were observed that apparently did not survive native agarose gel electrophoresis. As the VP1 concentration increased, electrophoretic migration was slower and tubes were replaced by 200 Å diameter particles and excess free pentamer. At high VP1/DNA ratios, a progressively larger fraction of particles was similar to 450 Å diameter virions. VP1 association with DNA is very strong compared to the concentrations in these experiments yet, paradoxically, stable complexes appear only at high ratios of VP1 to DNA. These data suggest a DNA saturation-dependent nucleation event based on non-specific pentamer-DNA interaction that controls assembly and the ultimate capsid geometry.     

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.     

Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat-inactivated virions

Hybridoma cell lines were established against poliovirus type 1 (Mahoney) heat-denatured virions (C particles). Each anti-C monoclonal antibody (McAb) immunoprecipitated specifically one of the individualized poliovirus capsid polypeptides VP1, VP2, or VP3. One of the anti-C McAb (C-3), reacting with VP1, neutralized homologous virus and immunoprecipitated infectious D particles. Its properties have been compared to those of a neutralizing anti-D McAb (D-Ic). In contrast with the C-3 antigenic site, the D-Ic epitope was not present on C particles nor on individualized structural polypeptide. This demonstrates that C-3 and D-Ic epitopes represent two independent antigenic determinants, both critical for poliovirus neutralization.     

Recombinant VP7-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Colorado tick fever virus

VP6, VP7, VP9, VP10, VP11, and VP12 of Colorado tick fever virus (CTF virus), a virus member of the genus Coltivirus, family Reoviridae, were expressed in bacteria with the pGEX-4T-2 vector. A partial sequence of VP7 (designated pVP7) was chosen to elaborate an enzyme-linked immunosorbent assay (ELISA) for detecting anti-CTF virus immunoglobulin G (IgG) antibodies in humans. This was based on two observations: (i) among all expressed proteins, pVP7 showed the highest immunoreactivity to an anti-CTF virus hyperimmune ascitic fluid; (ii) to provide the highest selectivity of antibody detection, the expressed sequence was chosen within a region which is highly divergent (49% amino acid identity) from the homologous sequence of another coltivirus, the Eyach virus. The pVP7 ELISA was evaluated with 368 serum samples from French blood donors and found to provide 98.1% specificity. Assays with the Calisher set of human serum samples, positive for anti-CTF virus antibodies (C. H. Calisher, J. D. Poland, S. B. Calisher, and L. A Warmoth, J. Clin. Microbiol. 22:84-88, 1985), showed that the pVP7 ELISA provided 100% sensitivity for the tested population. After elaboration of recombinant-protein-based ELISAs for diagnosis of infections with members of the viral genera Orbivirus, Orthoreovirus, and Rotavirus, it was shown that a recombinant protein could be used to detect antibodies to the human pathogen Colorado tick fever virus.     

Biochemistry of Theiler''s murine encephalomyelitis virus isolated from acutely infected mouse brain: identification of a previously unreported polypeptide

The WW strain of Theiler's murine encephalomyelitis virus (WW-TMEV) was purified from homogenates of acutely infected mouse brain. Infectious WW-TMEV was found to have an estimated sedimentation coefficient of 156 (s20,w) and a density of 1.35 g/cm3 in CsCl. Electron microscopy revealed a homogeneous population of 26-nm nonenveloped particles. Iodination of sodium dodecyl sulfate (SDS)-disrupted virions revealed four major capsid proteins with molecular weights of 58,000, 37,000, 34,000, and 27,000. A 6,000-dalton polypeptide was observed after long exposures of autoradiograms. The 37,000-, 24,000-, 27,000-, and 6,000-dalton polypeptides corresponded to picornaviral VP1, VP2, VP3, and VP4 capsid polypeptides, respectively. Comparison of autoradiograms of virions radiolabeled before and after SDS disruption indicated that the 58,000-dalton protein, VP2, and VP3 preferentially bound 125I under the labeling conditions used. Direct evidence was obtained that VP2 and VP3 were derived from the 58,000-dalton polypeptide by isolation of the 58,000-dalton polypeptide from polyacrylamide gels run under nonreducing conditions and subjecting it to reelectrophoresis under reducing conditions. The effect of trypsin on purified virions and their polypeptides was also investigated. Trypsin-sensitive sites were found in the 58,000-dalton protein, VP1, and VP2. Our results indicate that, in addition to the four typical picornaviral capsid polypeptides, there is a 58,000-dalton polypeptide present in WW-TMEV, which is sensitive to trypsin and can be reduced into two of the capsid proteins, VP2 and VP3.     

Sequence Analysis of the VP4, VP6, VP7, and NSP4 Gene Products ofthe Bovine Rotavirus WC3

The bovine rotavirus (BRV) WC3 serves as the background strain in the development of a multivalent reassortant vaccine against rotavirus gastroenteritis in infants. The genes encoding the outer capsid spike protein VP4, the inner capsid protein VP6, the outer capsid glycoprotein VP7, and the viral enterotoxin NSP4 of BRV WC3 were sequenced. Comparative analysis of the deduced amino acids of the sequenced genes indicated that the BRV WC3 strain shares a high degree of amino acid identity with serotype P7[5] VP4 (93–96%), serotype G6 VP7 (91–97%), subgroup (SG) I VP6 (96–99%), and NSP4 genogroup A (96–98%) BRV strains. Our results confirm and extend previous studies which suggested that the VP4 of BRV WC3 was closely related to that of the P7[5] prototype, BRV UK. In addition, the VP6 and VP7 of BRV WC3 were very similar to the VP6 and VP7 of both SG I and G6 BRV NCDV and UK strains. However, the NSP4 of BRV WC3 was more closely related to that BRV NCDV, the P6[1] prototype, than to that of BRV UK.     

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS–PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.     

VP2 is the major exposed protein on orbiviruses

Summary Iodination of African horsesickness virus and epizootic hemorrhagic disease virus resulted in labeling of VP2 and not of the other capsid protein, VP5, suggesting that VP2 is the major surface exposed protein of these orbiviruses.