Virus-specific Polypeptides Research Articles

High-performance liquid chromatographic separation and immunological characterization of soluble bovine viral diarrhea virus antigen

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis of the protein extracts from bovine viral diarrhea virus (BVDV, Singer strain) infected primary calf testicle cells (soluble antigen) showed the presence of four virus specific polypeptides of 105, 90, 84 and 67 kiloDaltons (kD) the 84-kD being the most abundant. Anion-exchange high-performance liquid chromatography (HPLC) of soluble antigen separated the virus specific polypeptides in individual peaks while the gel permeation HPLC collected all of them in a single protein aggregate peak of 290 kD. Except for the 84-kD polypeptide peak in anion-exchange HPLC, all peak fractions were found to be heterogeneous in nature having more than one polypeptide. Analysis of the antisera raised against the peaks having antigen activity showed that antisera against the 84-kD polypeptide peak did not neutralise BVDV while those against the fractions containing the 90- and 105-kD polypeptides neutralised the virus.

Phospholipase A2 activity is copurified together with herpes simplex virus-specified Fc receptor proteins.

We purified Fc-binding proteins from herpes simplex virus (HSV)-infected Vero cells by using an inverse immunoaffinity column chromatography. Polyacrylamide gel electrophoresis analysis revealed a single, virus-specific 65-kd polypeptide both in HSV type 1- and HSV type 2-infected cell-derived preparations. A Ca2+-dependent phospholipase A2 activity was demonstrated to be associated with the viral Fc-binding proteins.

Structural polypeptides of feline parvovirus subspecies viruses.

Structural polypeptides of feline parvovirus (FPV) subspecies viruses, that is, feline panleukopenia virus (FPLV), mink enteritis virus (MEV) and canine parvovirus (CPV), were comparatively investigated. Viruses propagated in the Crandell feline kidney (CRFK) cell culture were purified by equilibrium density gradient centrifugation in CsCl and were examined by electron-microscopy and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Virus infected CRFK cells were also analyzed by SDS-PAGE to determine virus-specific polypeptides. The purified virion of each subspecies contained three size classes of polypeptide which were almost identical for all three viruses: VP 1 (84, 000), VP 2 (64, 000) and VP 3 (63, 000). The characteristics of FPV polypeptides revealed in the present study were very similar to those of other members in genus Parvovirus and the results indicate that FPLV, MEV and CPV can not be distinguished by the SDS-PAGE analysis of purified virions.

Synthesis of the infectious pancreatic necrosis virus polyprotein, detection of a virus-encoded protease, and fine structure mapping of genome segment A coding regions.

Full-length and truncated genome segment A-specific infectious pancreatic necrosis virus cDNA was subcloned into plasmid transcription vectors, and runoff transcripts were produced in vitro. These transcripts were translated in cell-free rabbit reticulocyte lysates and the translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Virus-specific polypeptides were gel purified and mapped by partial proteolysis with N-chlorosuccinimide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide profiles were compared with those of the corresponding polypeptides purified from infectious pancreatic necrosis virus-infected cells or prepared by in vitro translation of denatured genomic RNA. The cDNA directed the synthesis of authentic pVP2, VP3, and NS polypeptides as well as a number of previously undescribed polypeptides. A 101,000-molecular-weight polypeptide was isolated and shown to be the unprocessed infectious pancreatic necrosis virus polyprotein. The NS polypeptide appears to be a virus-encoded protease responsible for the cleavage of pVP2 from the polyprotein. The carboxy terminus of NS was mapped to within three or four amino acids on the polyprotein. The most likely internal translation start sites responsible for NS and VP3 production in vitro were also mapped.

The polypeptides of human parainfluenza type 2 virus and their synthesis in infected cells

We have studied the structural components of three strains of human parainfluenza virus type 2 (HPI-2) and identified the virus-specific polypeptides. Molecular weight of P and F1 polypeptides determined by us, when compared with that reported previously, was in reversed order. HN polypeptide existed chiefly as disulfide-linked dimers in cells infected with Toshiba strain, while as monomers and dimers in nearly equal proportion in cells infected with two other strains. Similar disulfide-linked NP oligomers were found in cells infected with all three strains. F1 and F2, cleaved forms of F protein, could be detected in cells infected with all three strains, but the ratio of cleaved (F1 and F2) to uncleaved (F0) forms was markedly lower in 62-M 786- and 63-M 1-infected than in Toshiba strain-infected cells. However, there was no difference of oligopeptide mapping patterns and isoelectric point of F polypeptide between Toshiba and 62-M 786 strains. By contrast, oligopeptide mapping patterns of HN protein of Toshiba strain differed from those of the two other strains. Furthermore, the HN polypeptide of Toshiba strain was phosphorylated in the infected Vero cells, but that of the other two strains was not.

Properties of RNA polymerase activity associated with infectious bursal disease virus and characterization of its reaction products

An RNA-dependent RNA polymerase activity of infectious bursal disease virus (IBDV) could be demonstrated without any special treatment of the virus particles. Ca 2+ ions had to be removed from the reaction mixture. Mg 2+ (4 mM) was essential for the polymerase activity which was optimal at pH 8.5 and 40 °C. The RNA products synthesized in vitro were 24S single-stranded (ss) RNA and 14S double-stranded (ds) RNA which remained closely associated with IBDV particles and which could only be released by proteolytic degradation of the virus. The positions of the two bands in polyacrylamide gels and hybridization with virion RNA identified the 14S RNA as the two genomic dsRNA segments. The 24S ssRNA also formed two bands, did not self-anneal, hybridized with virion RNA, and induced in vitro translation of virus-specific polypeptides. Therefore, this product was considered to be newly transcribed mRNA.

Characterization of bovine viral diarrhoea-mucosal disease virus-specific proteins in bovine cells.

The presence of virus-specific polypeptides in bovine viral diarrhoea-mucosal disease (BVD) virus-infected bovine cells was studied by radiolabelling in the presence of a hypertonic initiation block (HIB) and by analysis by SDS-PAGE. These experiments were complemented by radioimmunoprecipitations with anti-BVD hyperimmune serum of infected cells labelled under isotonic conditions. A total of 12 polypeptides (Mr 165, 135, 118, 80, 75, 62, 56 to 58, 48, 37, 32, 35 and 19, all X 10(-3)) were identified in infected cells. Time course analysis of the induction of the viral polypeptides indicated that they could be detected as early as 4 h post-infection and their synthesis reached a plateau between 12 and 20 h post-infection. The most abundant polypeptides were the ones that could be detected earliest. HIB was found to be an excellent adjunct to existing techniques in the identification of viral polypeptides. Seven of these polypeptides had not been reported previously. This is the first report of the direct detection of BVD virus-induced polypeptides in infected cells without the aid of immunoprecipitation. The sum of the molecular masses of these polypeptides is greater than the coding capacity of the genome; therefore precursor-product relationships must exist between these polypeptides.

Induction of cell fusion in Newcastle disease virus-infected L929 cells by anti-L929 cell antisera.

Infection of L929 cells by Newcastle disease virus (NDV) did not induce cell fusion, whereas addition of anti-L929 cell antiserum to the culture medium did result in cell fusion. When the antiserum was added to the culture medium of NDV-infected cells at the start of incubation, polykaryocytes appeared after 18 h post-infection (p.i.). On the other hand, when the antiserum was added to the culture medium at 18 h p.i., no polykaryocytes appeared, suggesting that the presence of the antiserum at the stage of NDV replication was necessary for the antiserum-induced cell fusion. Since anti-F polypeptide antiserum inhibited the cell fusion, functional F protein was essential for the antiserum-induced cell fusion. All the anti-L929 cell xenogenic sera tested were able to induce the cell fusion, whereas anti-L929 cell allogenic sera did not induce cell fusion. Anti-MCA cell serum also induced the cell fusion. Little replication of NDV was observed in the presence of anti-L929 cell serum and synthesis of virus-specific polypeptides, especially of M protein, was suppressed. However, there was no difference in expression of virus-specific glycoproteins on the surface membranes between cells treated with normal rabbit serum and those treated with anti-L929 cell serum. On the basis of the above results, the mechanism of induction of cell fusion by anti-host cell serum is discussed.

Identification with monoclonal antibodies of virus-specific DNA-binding proteins in the nuclei of cells infected with three serotypes of Marek's disease virus-related viruses.

Two groups of virus-specific polypeptides were identified in the nuclei of infected cells by cross-reacting monoclonal antibodies with three serotypes of Marek's disease virus. Of these, a 135,000-molecular-weight polypeptide common to all three serotypes was found to bind to both double-stranded and single-stranded DNAs.

African Swine Fever Virus Gene Expression in Infected Vero Cells

Polypeptides synthesized in Vero cells infected with African swine fever virus (ASFV) can be divided into early and late classes on the basis of their sensitivity to cytosine arabinoside. As ASFV does not inhibit cell protein synthesis until late in infection, immunoprecipitation was used to identify virus-specific polypeptides. Eighteen early and 15 late polypeptides were detected by polyacrylamide gel electrophoresis. Early polypeptides can be further divided into those which are transiently expressed at early times and the majority which are synthesized throughout infection. In vitro translation products of RNAs from infected cells at different stages of infection were compared with in vivo products after immunoprecipitation. A good correspondence was observed between the in vivo and in vitro patterns. All early RNAs translated in vitro were synthesized in the presence of cytosine arabinoside and cycloheximide and can therefore be classified as immediate early RNAs. Only two polypeptides transcribed from early RNA were not present among late RNA products. No evidence was obtained for a discrete class of delayed early RNAs.

Human antibody response to herpes simplex virus-specific polypeptides after primary and recurrent infection

Human antibody responses to specific polypeptides of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) were assessed in serial serum specimens from 18 infected patients by immunoblot technology. Nine patients had HSV-1 infections (six genital and three oral) and nine had HSV-2 genital infections. Antibodies to homologous and heterologous HSV antigens were studied and correlated with total microneutralization and enzyme-linked immunosorbent assay antibodies as well as correlated directly to purified glycoproteins. The data indicated a sequential appearance of antibodies to specific polypeptides, according to virus type and site of infection. After HSV-1 infection, the initial response was to glycoprotein B, but the same was not true for HSV-2 infection, where the initial response appeared to be to the type-specific glycoprotein G. A difference in sequential appearance of antibodies for the two viruses indicated greater reactivity to lower-molecular-weight polypeptides after genital infection, irrespective of type, in contrast to nongenital HSV-1 infections. The antibody responses for selected sera to purified glycoproteins B and D were verified by enzyme-linked immunosorbent assay antibody determinations.

Isolation and characterisation of herpes simplex virus type 1 mutants which fail to induce dUTPase activity

The herpes simplex virus (HSV) type 1 dUTPase gene was inactivated by insertion of HindIII oligonucleotide linker sequences into the KpnI site within the coding region of the cloned gene. The mutated gene was introduced into wild type herpes simplex virus by marker rescue and the recombinants were identified by the acquisition of a HindIII site within genome map coordinates 0.69 to 0.70 and the failure to induce virus-specific dUTPase activity. A spontaneous dUTPase deficient mutant, which had an identical restriction endonuclease DNA pattern to wild type virus, was also isolated from this transfection experiment. Both types of dUTPase-negative mutants failed to induce a virus-specific 39,000 mol wt polypeptide. Cells infected with the insertional mutant contained instead a novel polypeptide about 40,000 mol wt. No abnormal virus specific polypeptide was detected in cells infected with the spontaneous mutant. We conclude that the 39,000 mol wt polypeptide induced by wild type HSV-1 is the virus-coded dUTPase. Since both types of mutants grew well in exponentially growing and serum-starved tissue culture cells in the absence of wild type helper virus, the dUTPase is not required for virus replication under these conditions. Thymidine kinase deficient, dUTPase deficient double mutants were constructed by recombination of a thymidine kinase insertional mutation into dUTPase deficient virus. These mutants also grew as well as wild type virus both in normal tissue culture cells and cells lacking the cellular thymidine kinase.

Vaccinia virus proteins on the plasma membrane of infected cells III. Infection of peritoneal macrophages

Primary macrophage cultures were prepared from the peritoneal exudate cell population harvested from mice challenged intraperitoneally with saline, thioglycollate, or vaccinia virus. Vaccinia virus was adsorbed and penetrated into primary macrophages and L-cells with similar kinetics. As evidenced by the expression of some “early” virus-specified proteins, partial uncoating and activation of the virion-associated DNA-dependent RNA polymerase occurred in the infected macrophages. Subsequently, the viral replication cycle in macrophages was aborted; with time after infection, viral DNA and virion proteins initially associated with infected cells could be detected in an acid-soluble form in the medium harvested from infected macrophage cultures. The results suggest that at the time that the final stages of virus uncoating should have occurred, intracellular subviral particles were, instead, degraded in the infected, primary macrophages. Viral DNA synthesis could not be measured in vaccinia virus-infected macrophages, no “late” virus functions were expressed, and progeny virions were not assembled. As measured by the binding of antiviral-antibody- 125I-protein A complexes to the surface of vaccinia virus-infected cells, the expression of virus-specified antigens on the surfaces of infected macrophages was significantly reduced and never exceeded that measured at 2 hr after infection on the surfaces of infected L-cells. The expression of virus-specified polypeptides with mol mass of 48–50, 45–46, 36–37, and 25 kDa on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages, rendered the infected macrophages susceptible to lysis by vaccinia virus-specific cytotoxic T-cells.

Expression of High Molecular Weight Polypeptides by Carnation Mottle Virus RNA

SUMMARY In vitro translation of virion RNA from carnation mottle virus (CarMV) in a messenger RNA-dependent rabbit reticulocyte lysate (MDL) resulted in the synthesis of four virus-specific polypeptides of apparent M r 100000 (P100), 77000 (P77), 38000 (P38) and 30000 (P30). Partial peptide mapping experiments and in vitro translation in the presence of partially purified calf liver amber suppressor tRNA demonstrated that P30, P77 and P100 are a series of overlapping polypeptides generated by a double readthrough mechanism. In addition we report the infection of Chenopodium quinoa protoplasts with CarMV. The viral coat protein was detected in virus-infected protoplasts. Only one other infection-specific protein with an apparent M r of 100000 corresponded in size to any of the other in vitro translation products.

Wound tumor virus polypeptide synthesis in productive noncytopathic infection of cultured insect vector cells

Inoculation of the leafhopper cell line AC-20 with wound tumor virus resulted in a productive noncytopathic infection with no detectable alteration of cellular protein synthesis. Virus-specific polypeptide synthesis, detectable by 8 h postinoculation, increased in a linear fashion, reaching a peak (approximately 10 to 15% of total protein synthesis) by 48 h postinoculation. The rate of viral protein synthesis continued at this level for several days but declined, relative to cellular protein synthesis, as infected cells were passaged. By passage 10, the synthesis of viral polypeptides was reduced to a level approximately 5% of that observed at 48 h postinoculation. Viral protein synthesis was not stimulated by superinfection. Viral antigens and infectious virus persisted in the majority (greater than 90%) of cells in an infected culture even after more than 100 passages. The synthesis of wound tumor virus polypeptides in infected insect vector cells appears to be regulated in a coordinated and selective manner.

Homogeneity among Senegalese strains of yellow fever virus.

A series of 16 yellow fever (YF) viruses isolated from mosquitoes, monkeys and humans in different epidemiological contexts in Senegal and The Gambia between 1976 and 1983, was analyzed by T1 RNase oligonucleotide fingerprints of the genomic 32P-labeled RNA, by SDS-polyacrylamide gel electrophoresis of the intracellular virus-specified polypeptides, by peptide mapping of the envelope E glycoprotein and by immunological reactivities with monoclonal antibody fluids (MAF's) against the E glycoprotein. These strains had not been passed in suckling mice and were isolated in Aedes pseudoscutellaris Mos 61 cultured cells. These strains showed no virulence in three-week-old Swiss mice when injected intraperitoneally. Direct comparison of the large T1 RNase-resistant oligonucleotide maps indicated a relative genetic stability (92%-100%). A greater change was observed when these strains were compared with an epidemic YF strain isolated in 1965 with an oligonucleotide fingerprint map sharing 82%-88% similarity. The YF-specified proteins were identical in their molecular weight, and the fragments obtained after limited proteolysis of the envelope protein using protease V8 or alphachymotrypsine indicated that the strains were chemically similar. Only a few differences were observed when the strains were seroneutralized with MAF's, but no relation could be made with genetic or biological data. This suggested that the YF virus strains isolated from the same geographic area and during a short period of time had evolved slowly. Moreover, all the viruses were closely related and no correlation could be established with the apparent variations in virulence in nature.

Effect of tacaribe virus infection on host cell protein and nucleic acid synthesis.

Tacaribe virus stocks were prepared which induced definite lytic responses in Vero cells infected at multiplicities giving synchronous infection. Under these conditions, the first signs of cytopathic effect (c.p.e.) appeared at about 30 h post-infection and cell lysis occurred after 40 h. Before the onset of cytopathic changes, the virus induced inhibition of host cell protein, DNA and RNA (primarily rRNA) synthesis. These were designated c.p.e. (+) virus stocks. The effect of virus on host cell macromolecular synthesis and development of c.p.e. were not related to the virus isolate, but to the conditions under which the virus was produced. Thus, from a single virus clone, working stocks were derived which could or could not induce inhibition of host cell functions and c.p.e. development. The virus stocks that did not induce inhibition are defined as c.p.e. (-). Analysis of [3H]leucine-labelled proteins from Vero cells infected with either the c.p.e. (+) or the c.p.e. (-) virus stocks revealed synthesis of two virus-specific polypeptides migrating with mobilities corresponding to mol. wt. 68 000 and 79000. These are presumed to correspond, respectively, to the nucleoprotein and to the minor polypeptide p79. In cells infected with the c.p.e. (+) virus stock, the virus-specific polypeptides were synthesized at times when there was a drastic inhibition of host cell protein synthesis. The yield of infectious progeny during the first 24 h of infection is similar in Vero cells infected with either the c.p.e. (+) or the c.p.e. (-) virus stocks. The proportion of defective interfering particles was much higher in the c.p.e. (-) than in the c.p.e. (+) virus stocks. The results presented here are the first demonstration that an arenavirus affects the biosynthetic machinery of the host cell.

Intracellular synthesis of human parainfluenza type 3 virus-specified polypeptides.

The intracellular synthesis of human parainfluenza type 3 virus-specified polypeptides was examined by polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts under reducing conditions. All of the virion structural proteins were detected in cell extracts, including: L, 180,000 molecular weight (180K); P, 83K; HN, 69K; NP, 66K; F0, 60K; F1, 51K; and M, 38K. P and NP were phosphorylated. HN and F were glycosylated. The kinetics of intracellular viral protein synthesis did not detect any early or late proteins. Pulse-chase experiments failed to detect any precursor-product relationships. No nonstructural proteins were detected.

Differential effects of defective interfering Semliki Forest virus on cellular and virus polypeptide synthesis

Defective interfering Semliki Forest virus (DI SFV) inhibited virus RNA and virus polypeptide synthesis in cells coinfected with standard virus but did not delay or alter kinetics of RNA synthesis. Inhibition of polypeptide synthesis was 20-fold greater than that of RNA synthesis which presumably reflected the amplification resulting from cumulative translation of mRNAs. At high concentration, DI virus p12e inhibited the shutoff of host protein synthesis and allowed no synthesis of structural or nonstructural polypeptides. Dilution of DI virus restored the inhibition of host protein synthesis but further dilution was necessary before virus-specified polypeptide synthesis could be demonstrated. Another DI virus (p20a) with the same interference titre as p12e also inhibited shutoff of host protein synthesis but synthesis of virus-induced polypeptides was inhibited differentially: significant amounts of polypeptides comigrating with the structural precursor polypeptide p62 and the nonstructural polypeptide nsp63 were present and the synthesis of nsp90 was little affected at any concentration of DI virus p20a tested. None of the DI viruses tested induced the synthesis of any viral or novel polypeptide. It was concluded that DI SFV preparations have qualitatively different interfering activities in relation to their effects on virus and host cell polypeptide synthesis.

Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus.

A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to immunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA.