Virus-induced Polypeptides Research Articles

African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: Two-dimensional gel analysis

High-resolution two-dimensional electrophoresis followed by computer analysis has been used to study quantitatively the patterns of protein synthesis produced in porcine alveolar macrophages and in Vero cells infected with African swine fever virus (ASFV). Initially, a protein database for each cell type was constructed. The porcine alveolar macrophage database includes 995 polypeptides (818 acidic, isoelectric focusing (IEF) and 177 basic, nonequilibrium pH gradient electrophoresis (NEPHGE)) whereas the Vero database contains 1,398 polypeptides (1,127 acidic, IEF and 271 basic, NEPHGE). Taking these databases as reference, ASFV highly virulent strain E70 induces 57 acid and 43 basic polypeptides in porcine alveolar macrophages, which account for most of the information content of the virus DNA. The kinetics of synthesis of the virus-induced polypeptides showed the existence of three classes of proteins: one whose synthesis starts early after infection, continues for a period and then switches off; another whose synthesis also starts early but continues for prolonged periods; and a third which requires DNA replication. The attenuated, cell adapted, strain BA71V induces 92 acidic and 37 basic proteins in Vero cells. Significant differences were observed when comparing the patterns of polypeptides induced by the two viral strains. In both cell systems studied, ASFV infection produces a general shutoff of protein synthesis that affects up to 65% of the cellular proteins. Interestingly, 28 proteins of porcine alveolar macrophages and 48 proteins of Vero cells are stimulated at least two times by ASFV infection.

African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: Two-dimensional gel analysis

African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: Two-dimensional gel analysis

Prevention of bovine viral diarrhea virus transmission during in vitro fertilization including oocytes with follicular epithelial cells from a persistently infected heifer

Intracellular virus-induced polypeptides from 3 cytopathogenic and 2 non-cytophthogenic bovine viral diarrhea (BVD) virus reference strains were analyzed by radioimmunoprecipitation and polyacrylamide gel electrophoresis, using a specific bovine multivalent antiserum and a neutralizing BVD-virus monoclonal antibody. Electrophoretic patterns of major proteins demonstrate extensive variation between strains. Most notably, a major 80 000 (80K) polypeptide was present in all cytopathogenic strains but absent in both non-cytopathogenic strains. Furthermore, a neutralizing monoclonal antibody produced against the NADL strain immunoprecipitated a 53K glycoprotein indicating that this protein carries an important neutralization epitope that is not present in all strains tested.

Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant.

Modified vaccinia virus Ankara (MVA), having acquired genomic deletions during passage in chicken embryo fibroblasts, is highly attenuated and unable to productively infect most mammalian cell lines. Multiplication in rabbit kidney-derived RK13 cells, but not other nonpermissive cells, can be restored by insertion of the vaccinia virus K1L gene into the MVA genome. During nonproductive infection of RK13 cells by MVA, transcription of representative viral early genes was revealed by Northern (RNA) blotting, whereas synthesis of an intermediate mRNA and replication of viral DNA could not be detected. Despite the persistence of viral early mRNA for at least several hours, synthesis of virus-induced polypeptides occurred only during the first hour and was followed by abrupt inhibition of all protein synthesis. Transfection of RK13 cells with a eukaryotic expression plasmid that contained the K1L gene allowed MVA infection to proceed to late stages of viral protein synthesis. Moreover, RK13 cell lines that stably expressed the K1L gene were permissive for MVA as well as a K1E deletion mutant of the WR strain of vaccinia virus. This is the first description of the complementation of a poxvirus mutant by cells that stably express a viral gene.

Comparative Analysis of Virus-Induced Polypeptides of an Avirulent and a Virulent Strain of Avian Reovirus

Ten avian reovirus-specific polypeptides, ranging in molecular weight from 32,000 to 145,000, were identified in chicken embryo fibroblasts infected with virulent and attenuated strains. Time-course studies on the viral polypeptides indicated that all of these proteins could be detected as early as 16 hr postinfection (PI) for both strains of avian reoviruses. With the virulent strain 1733, protein production reached a plateau 24 hr PI, whereas with an attenuated strain S1133, production plateaued at 48 hr PI. Immunoprecipitation of labeled polypeptides from S1133 and 1733 strains with homologous and heterologous sera revealed that sera against strain 1733 recognizes sigma NS protein in both strains 1733 and S1133, whereas sera against strain S1133 recognizes this protein only in 1733, suggesting a difference in the primary structure or the conformation of that particular protein in both viruses. None of the polypeptides of avian reovirus were glycosylated or phosphorylated.

Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting.

The structural and non-structural proteins induced by African horsesickness virus serotype 4 (AHSV-4) in infected Vero cells were analysed by SDS-PAGE. Twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (VP2, VP3, VP5 and VP7) and three minor (VP1, VP4 and VP6) structural proteins and four non-structural proteins (P58, P48, P21 and P20) were shown to be virus-coded, as deduced from electrophoretic and antigenic studies of purified virions and infected cells. The proteins that elicit the major antibody responses both in vaccinated and naturally or experimentally infected horses were shown to be three structural proteins, VP2, VP5 and VP7, and the four major non-structural proteins, P58, P48, P21 and P20, as deduced by radioimmunoprecipitation and immunoblotting assays. The cross-reactivity between AHSV-4 and sera obtained from horses experimentally infected with seven other serotypes was also determined. The results showed that VP5, VP7, P48, P21 and P20 are conserved and can be used to diagnose the infection of any of these eight serotypes.

Protein synthesis in BM-N cells infected with Bombyx mori nuclear polyhedrosis virus

Polypeptide synthesis in the BM-N cells infected with Bombyx mori nuclear polyhedrosis virus (BmNPV) was examined. At least 26 virus-induced polypeptides were sequentially detected during BmNPV infection when cell extracts pulse-labeled with [ 35S]methionine for 3 hr were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography. Three virus-induced polypeptides were first detected at 6 hr postinfection (pi) and the synthesis of the majority of virus-induced polypeptides (18 of 26 polypeptides) commenced at 12–18 hr pi. No additional virus-induced polypeptides were detected at 30 hr pi or later. Immunoprecipitation experiments showed that at least 20 virus-induced polypeptides, two of which being unidentified in the pulse labeling experiment without immunoprecipitation, were related to the structural polypeptides of BmNPV virions. Pulse-chase experimets initiated at various times pi showed that at least 12 virus-induced polypeptides disappeared or reduced their radioactivity and at least 16 polypeptides appeared or increased their radioactivity during the chase periods up to 48 hr, suggesting that a number of virus-induced polypeptides underwent post-translational modification. Pulse labeling with N-acetyl-[ 14C]glucosamine and [ 32P]orthophosphate revealed that 6 virus-induced polypeptides were glycosylated and 10 virus-induced polypeptides phosphorylated. In agreement with the results in other NPV species, the polyhedrin of BmNPV were characterized to be a glycophosphoprotein.

Proteins of Palyam serogroup viruses

The replication and polypeptide synthesis of Abadina virus, a member of the Palyam serogroup of orbiviruses, was studied. The first virus-specified proteins could be demonstrated 2 to 4 h post-infection (p.i.) by immunoprecipitation. The rate of synthesis increased rapidly until 12 h p.i. after which it remained fairly constant until 18 h p.i. when it began to decline. Host cell protein synthesis shutoff was incomplete. Twelve virus-induced polypeptides were identified in infected cell lysates, ranging in Mr from 36K to 143K. Three small polypeptides (Mr 15K to 20K) identified in homologous immunoprecipitation studies are also thought to be virus-specified. Nine structural proteins were identified, four being major components of the purified virion. Partial proteolysis was used to demonstrate homology between some proteins. Pulse-chase experiments provided no evidence for a precursor-product relationship between any of the Abadina virus proteins. A non-structural protein was found to be phosphorylated.

Intracellular virus-induced polypeptides of pestivirus border disease virus

Intracellular virus specific polypeptides of pestivirus, border disease virus (BDV) in bovine turbinate cells were analysed by radio-immunoprecipitation with specific antisera. Eleven viral polypeptides with molecular weights of 220, 165, 118, 84, 66, 58, 55, 53, 45, 37 and 31 kDa, respectively, were detected in infected cells. Of these, the 165, 118, 84, 66, 58, 55, 53, 45 and 31 kDa proteins were found to be glycosylated. Comparative studies indicated that the polypeptides induced by BDV share many antigenic epitopes with those of the polypeptides induced by bovine viral diarrhea virus (BVDV), a serologically related virus of the same genus, pestivirus. The polypeptide profile of BDV appeared to be more similar to that of the noncytopathic BVDV strain NY1 compared to that of cytopathic BVDV strains NADL and Singer. Peptide mapping analysis of homologous polypeptides from BVDV and BDV confirmed their structural relatedness.

Amphibian and Piscine Iridoviruses Proposal for Nomenclature and Taxonomy Based on Molecular and Biological Properties

We have compared a number of properties of the well-characterized iridovirus, frog virus 3, with two other iridoviruses from amphibia, bullfrog edema virus and Lucké triturus virus, and with a piscine iridovirus, goldfish virus (GFV), to provide information for developing taxonomic classification of these viruses and establishing their ecological niche. Purified virions had similar size and shape (icosahedral) for each virus, and the genomic DNAs of each virus were methylated by a virus-induced DNA methyltransferase. The three amphibian viruses replicated equally well in fish (FHM), hamster (BHK), and human (WI-38) cell monolayer with identical cytopathology, while GFV failed to replicate in these cell lines. However, GFV replicated albeit at a slow rate, in a goldfish cell line; there was no detectable replication by the amphibian viruses in these cells. The amphibian iridoviruses had virtually similar DNA sequences, while those of GFV were markedly different. Analyses of virus-induced polypeptides in infected cells corroborated the DNA analyses; the polypeptides of the amphibian viruses were similar and distinct from those of the fish virus. Nongenetic reactivation could only be accomplished between the three amphibian viruses but not with the piscine virus. Based on these data, we suggest taxonomic and nomenclature designations of amphibian and piscine iridoviruses.

Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI.

Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70 (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987). Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, we have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI (R. Longnecker, S. Chatterjee, R. J. Whitley, and B. Roizman, Proc. Natl. Acad. Sci. USA 84:147-151, 1987). Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG.

Turkey rhinotracheitis virus: in vivo and in vitro polypeptide synthesis.

Turkey rhinotracheitis (TRT) virus is a paramyxovirus associated with recent outbreaks of acute respiratory disease in turkeys. On morphological criteria it resembles the pneumoviruses more than the other members of the paramyxovirus family. In this communication we report the identification of five virus-induced polypeptides in TRT virus-infected BS-C-1 cells. The Mr of these polypeptides were estimated as 38K, 35K, 30K, 19K and 15K from their electrophoretic mobility in SDS-polyacrylamide 6 to 15% gradient gels. The virus specificity of four (the 38K, 35K, 30K and 19K Mr polypeptides) of these five was confirmed by their predominance as products of in vitro translation of mRNA from TRT virus-infected BS-C-1 cells. An additional virus-specific polypeptide with an estimated Mr of 22K was revealed by in vitro translation and may be either a non-structural polypeptide or a minor structural protein. All six polypeptides were immunoprecipitated by a murine antiserum. Three other polypeptides (129K, 57K and 45K) were also immunoprecipitated. The 57K, 45K and 15K Mr polypeptides were glycosylated, and the 57K glycopolypeptide may be a disulphide-bonded dimer of the 45K and 15K glycopolypeptides. Another major glycopolypeptide of 83K Mr was observed but its virus-specificity could not be confirmed. Taken together our results tentatively identify eight or nine TRT virus-specified polypeptides and are consistent with the classification of TRT virus as a new member of the genus Pneumovirus.

Bovine viral diarrhea virus proteins: Heterogeneity of cytopathogenic and non-cytopathogenic strains and evidence of a 53K glycoprotein neutralization epitope

Intracellular virus-induced polypeptides from 3 cytopathogenic and 2 non-cytophthogenic bovine viral diarrhea (BVD) virus reference strains were analyzed by radioimmunoprecipitation and polyacrylamide gel electrophoresis, using a specific bovine multivalent antiserum and a neutralizing BVD-virus monoclonal antibody. Electrophoretic patterns of major proteins demonstrate extensive variation between strains. Most notably, a major 80 000 (80K) polypeptide was present in all cytopathogenic strains but absent in both non-cytopathogenic strains. Furthermore, a neutralizing monoclonal antibody produced against the NADL strain immunoprecipitated a 53K glycoprotein indicating that this protein carries an important neutralization epitope that is not present in all strains tested.

Selective inhibition of protein synthesis by synthetic and vaccinia virus-core synthesized poly(riboadenylic acids)

Studies were undertaken to compare the effect of poly(A)s from various sources on selective inhibition of protein synthesis in the reticulocyte lysate system programmed with viral and cellular mRNAs. RNA synthesized in vitro by vaccinia virus cores in the presence of only ATP inhibited overall HeLa cell polypeptide synthesis by over 80% with a minimal effect on translation of vaccinia virus mRNAs. Hybridization of the [α- 32P]AMP-labeled RNA made in vitro by vaccinia virus cores in the presence of only ATP, showed no complementarity to Hindlll restriction fragments of vaccinia virus DNA indicating that the in vitro product was poly(A). Fractionation of synthetic and core-synthesized poly(A) into three size classes showed that the larger the size of poly(A), the greater its inhibitory activity of protein synthesis in the cell-free system. Inhibition of translation of mRNAs from vaccinia virus-infected HeLa cells was also observed in the presence of poly(A). However, virus-induced polypeptide synthesis was more resistant to the effect of poly(A) than were cellular polypeptides. Oligo(dT) when added to the reticulocyte lysate system was capable of reversing the inhibition of protein synthesis caused by both core-synthesized poly(A) and core-transcribed RNAs. These results indicate that poly(A) synthesized by the virion-associated enzyme has inhibitory properties similar to those of synthetic poly(A).

Characterization of herpesvirus sylvilagus glycoproteins released into the culture medium of infected cells: antisera to gp13 and gp32 neutralize viral infectivity in vitro and identify antigens on plasma membranes of infected cells.

Polypeptides released into the culture medium of herpesvirus sylvilagus-infected cells were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracellular fluid from [35S]methionine- and [3H]glucosamine-labeled cell cultures. Virus-induced glycoproteins 31, 32, and 33 (molecular weights of 62,000, 59,000, and 54,000, respectively) were the most abundant species and appeared predominantly in the culture medium. This observation, together with the known cell-associated nature of herpesvirus sylvilagus, suggested that virus-induced glycoproteins 31, 32, and 33 were specifically released. Immunization of rabbits with virus-induced glycoproteins 13 (molecular weight of 130,000) and 32 resulted in the production of antibodies that neutralized viral infectivity in vitro. Both antiserum to gp13 and antiserum to gp32 immunoprecipitated gp13, gp26, gp33a, gp45, and virus-induced polypeptide 39 (molecular weights of 130,000, 77,000, 49,000, 27,000, and 36,000, respectively) from [35S]methionine-labeled cell extracts as well as virus-induced glycoproteins 31, 32, and 33 from the culture medium. In addition, membrane immunofluorescence assays indicate that an antigen(s) reactive with anti-gp13/32 serum was located on the plasma membrane of infected cells.

Orf virus replication in bovine testis cells: kinetics of viral DNA, polypeptide, and infectious virus production and analysis of virion polypeptides.

The replication of orf virus in bovine testis cells was analysed in one-step growth experiments. Newly replicated viral DNA was detected 4 to 6 hours post-infection (p.i.), accumulated rapidly between 8 and 16 hours p.i. and reached a plateau between 25 and 30 hours p.i. Most virus-induced polypeptides were first detected in a two hour period beginning 10 hours p.i., reached a peak rate of synthesis between 14 and 16 hours p.i., and continued at that rate for at least 10 hours. Host polypeptide synthesis declined to very low levels by 20 hours p.i. From these results, the transition between early and late events appears to occur between 8 and 10 hours p.i. Infectious virus was first detected between 16 and 18 hours p.i. and continued to be produced at a steady rate till 40 hours p.i. Up to 35 polypeptides were detected in SDS-polyacrylamide gels of purified orf virions disrupted in SDS/2-ME. Virions treated with NP40/2-ME were separable into soluble and insoluble components by centrifugation. Some 13 polypeptides were found in the soluble fraction and a polypeptide of molecular weight 38,500 believed to be the basic subunit of the virion surface tubule structure. Little difference was found between polypeptide profiles of five independently isolated NZ orf virus strains.

Glycoproteins of Bovine Viral Diarrhoea-Mucosal Disease Virus in Infected Bovine Cells

Bovine cell cultures infected with bovine viral diarrhoea-mucosal disease (BVD) virus were radiolabelled with L-[35S]methionine or D-[2-3H]mannose followed by analysis of the labelled polypeptides by radioimmunoprecipitation and polyacrylamide gel electrophoresis in one and two dimensions. Six glycoproteins were detected in infected cells. Two abundant species had Mr of 48K and 56K to 58K while the less abundant species had Mr of 118K, 75K, 65K and 25K. When cells were radiolabelled with L-[35S]methionine in the presence of tunicamycin 56K to 58K migrated with apparent masses of 54K (a minor species) and 48K to 50K (the major molecular species) in PAGE. Endoglycosidase F digestion of virus-induced polypeptides caused a 4K to 6K reduction in the apparent molecular mass of 56K to 58K yielding a single 52K digested product, indicating that the heterogeneity of 56K to 58K was due to differences in the oligosaccharide moieties. Tunicamycin caused a drastic reduction in the yield of infectious virus indicating that the carbohydrate moieties serve a critical role in the infectious cycle of BVD virus.

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.

Differences in virus-induced polypeptides in cells infected by cytopathic and noncytopathic biotypes of bovine virus diarrhea-mucosal disease virus

Two biotypes of bovine viral diarrhea-mucosal disease virus are present in nature: one that induces cytopathology in infected bovine cells and the other that infects cells without overt cytopathology. Infections with both types of virus yield similar amounts of infectious progeny virus. Field and laboratory isolates of both biotypes of bovine viral diarrhea (BVD) virus were analyzed by radioimmunoprecipitation and polyacrylamide gel electrophoresis of infected cell extracts. The noncytopathic biotype BVD (NCB-BVD) virus isolates can be differentiated from cytopathic biotype BVD (CB-BVD) isolates on the basis of peculiar polypeptide profiles they induce in the infected cell. The most abundant polypeptide in CB-BVD infected cells is the 80K polypeptide. NCB-BVD virus-infected cells lack the 80K polypeptide and induce a predominant 118K polypeptide. d-[2- 3H]Mannose labeling of cells infected with NCB-BVD indicated that at least three polypeptides are N-glycosylated: 75K, 56K-58K, and 48K. In addition the sizes and ratios of the glycoproteins induced by all virus isolates showed a marked variation. We present evidence indicating that there is remarkable heterogeneity among the field viral isolates of BVD and this methodology is of potential value for molecular epidemiology studies.

Reactions of sera from patients with rheumatoid arthritis, systemic lupus erythematosus and infectious mononucleosis to Epstein-Barr virus-induced polypeptides.

P3HR-1 and Ramos cells induced with sodium butyrate and 12-O-tetradecanoylphorbol 13-acetate were used in the protein immunoblot technique to identify Epstein-Barr virus (EBV)-specific antibodies present in sera from clinically normal individuals and patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and infectious mononucleosis (IM). Sixteen EBV-specific polypeptides were detected ranging in mol. wt. from 22,000 (22K) to 140K. Many of the sera contained antibodies to different subsets of these antigens, and a high proportion expressed autoantibodies which reacted with cellular components from an EBV genome-negative cell line. About 50% of the sera from each category reacted with the 44K to 48K and 36K and 38K early antigen (EA) components. A high proportion of the SLE sera (64%) were found to contain anti-EA antibodies, suggesting an association between EBV and SLE. Almost all of the EBV-seropositive sera examined contained antibodies against a 22K late antigen, but none of the sera from IM patients reacted with this polypeptide.