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PubMed1985

Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon.

We have purified from Moloney murine leukemia virus (Mo-MuLV) a protease that has the capacity of accurately cleaving the polyprotein precursor Pr65gag into the mature viral structural proteins. Both the NH2- and COOH-terminal amino acid sequences have been determined and aligned with the amino acid sequence deduced from the DNA sequence of Mo-MuLV by other workers. The results show that: (i) the protease is located at the 5' end of the pol gene, and the first four amino acids are overlapped with the 3' end of the gag gene; (ii) the fifth amino acid residue is glutamine, which is inserted by suppression of the UAG termination codon at the gag-pol junction; and (iii) the protease is composed of 125 amino acids with calculated Mr = 13,315, and the COOH terminus of the protease is adjacent to the NH2 terminus of reverse transcriptase. The map order of the gag-pol gene is proposed to be 5'-p15-p12-p30-p10-protease-reverse transcriptase-endonuclease-3'.

Amino Acid Sequence↗

PubMed1996

The complete DNA sequence and genomic organization of the avian adenovirus CELO.

The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions.

Adenoviruses, Human↗

PubMed1993

HTLV-1 gene expression by defective proviruses in an infected T-cell line.

We have examined human T-lymphotropic virus type I (HTLV-I) gene expression in the human T-cell line, C8166-45 (C81), as a model to define the gene products expressed from defective proviruses. C81 cells contain one complete and two different deleted proviral genomes. The internal deletions of the latter encompass most of the gag to env region. All three proviruses are transcriptionally active as evidenced by the presence of three unspliced nuclear mRNAs. Unspliced genomic mRNAs and singly spliced env mRNAs were present in the nucleus, but were not detected in the cytoplasm, suggesting a defect in Rex function. Three small cytoplasmic mRNAs were observed and are likely to correspond to the normal 1.8-kb Tax1/Rex1 mRNA, a 1.6-kb mRNA formed by splicing the 5'LTR to the pX region, and a 2.1-kb mRNA of unknown origin. Consistent with the subcellular mRNA distribution pattern, the viral structural proteins encoded by gag and env genes were not detected. The transcriptional transactivator protein, Tax1 (p40), was abundantly expressed in C81 cells; in addition, a 42-kDa Tax1 protein, unique to this cell line, was also detected. Although Tax1 and Rex1 (p27) are translated from overlapping open reading frames in the same mRNA, Rex1 was not detected in C81 cells. The presence of a premature termination codon in the Tax1/Rex1 mRNA encoded by the full-length provirus was inferred from the presence of small Rex1-related polypeptides lacking C-terminal sequences and confirmed by sequence analysis. Furthermore, a p21X protein lacking the N-terminus of Rex1 was expressed at high levels; our data indicate that p21X is translated from the 1.6-kb mRNA which is derived primarily from deleted proviruses.

Blotting, Northern↗

PubMed2005

Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E.

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.

Animals↗

PubMed1979

Moloney lymphoma cells express a polyprotein containing the GAG gene-coded p15 and the Moloney leukemia virus-induced cell surface antigen (MCSA).

The relationship between MCSA and the viral structural proteins in YAC Moloney lymphoma cells was further investigated by using membrane immunofluorescence and immunoadsorbent columns. As previously observed, MCSA showed only minimal capping and thereby differed in behaviour from gp70, p30 and p12 virion antigens. Whereas antibody-induced capping of gp70 did not change the membrane distribution of MCSA, co-capping was observed between MCSA and p30 and p12 gag protein antigens. This indicated that, whereas MCSA is distinct from gp70, it is linked to p30 and p12 on the membrane of living cells. It was then attempted to isolate MCSA on anti-p30 and anti-p15 immunoadsorbent columns from solubilized YAC cells. Contrary to what was expected, no MCSA was found to the anti-p30 column. On the other hand, a part (10-15%) of the MCSA was bound to the anti-p15 column, indicating that MCSA is linked to the gag p15.

Animals↗

PubMed1990

Wing web or intravenous inoculation of chickens with v-src DNA induces visceral sarcomas.

We have previously found that sarcomas localized to visceral organs frequently arise following wing web inoculation of Rous sarcoma virus. This observation prompted an investigation of whether visceral sarcomas are also inducible by wing web inoculation of a subgenomic viral DNA fragment (v-src DNA) that includes v-src but lacks genes encoding viral structural protein. For this analysis, line SC chickens were inoculated with v-src DNA at 1-2 days posthatch and monitored for 9 weeks. Ninety percent of the chickens developed wing web (primary) tumors at the site of inoculation and, of these, about 30% exhibited visceral tumors. All tumors were histologically identifiable as sarcomas, and both the primary and visceral sarcoma cells were specifically reactive with two monoclonal antibodies elicited to different peptide fragments of pp60v-src. In a separate set of experiments, visceral sarcomas were also observed in about half of the line SC chickens that had been inoculated intravenously with v-src DNA. These results indicate that exogenous progeny virus production is not required for v-src-induced, visceral sarcoma formation. In addition, they demonstrate that intravenous inoculation of v-src DNA is a means to achieve rapid and widespread, disseminated sarcoma growth.

Animals↗

PubMed1989

Immunoblot analysis of the antibody response to ovine lentivirus infections.

The antibody response of sheep to maedi-visna virus (MVV) infection was studied, using an immunoblotting technique that identified the four major viral structural proteins: the envelope glycoprotein gp135, and internal proteins p25, p16 and p14. In sequential serum samples of two inoculated sheep, antibodies to p25 appeared first, soon followed by antibodies to p16. In two sheep with natural infections, which were sampled with longer intervals, antibodies to p25 and p16 appeared simultaneously. Antibody response to p14, which was weak and inconsistent in most cases, appeared after the p16 response. Antibodies to gp135 were detected in only one sheep and appeared 7 weeks after those to p25. Antibodies to p25 were particularly persistent. Antibody recognition patterns of 21 necropsied sheep with naturally occurring infections were compared. Sheep with lesions lacked antibodies to p14 and p16 and sometimes even p25, although these antibodies had been present in earlier stages of infection. Since the time of onset of lesions was unknown, we could not determine whether this decline in antibody activity occurred before, together with, or after the onset of lesion development.

Animals↗

PubMed2003

Specific HIV-1 env gene silencing by small interfering RNAs in human peripheral blood mononuclear cells.

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA) in the cell, and results in the silencing of homologous gene expression by the specific degradation of an mRNA containing the same sequence. dsRNA-mediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression. Synthetic 21-23 nucleotide (nt) small interfering RNAs (siRNAs) with 2-nt 3' overhangs were recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we show that synthetic siRNAs targeted against the viral structural Env proteins encoded by HIV-1 can specifically suppress the expression of HIV-1 genes. The siRNA-mediated RNAi also had advantages over antisense RNA-mediated inhibition, in terms of both the ease of designing effective antiviral agents and their potency. Especially, our best env-specific siRNAs, E7145 targeted to the central region of the V3 loop and E7490 targeted to the CD4 binding site of conserved regions on gp120, significantly inhibited the HIV-1 gene expression. Furthermore, E7145 and E7490 were effective against HIV-1(NL4-3) replication in PBMCs for a relatively long time (14 days). Therefore, the use of synthetic siRNAs provides a simple, rapid, and cost-effective tool for new anti-HIV-1 gene therapeutics.

Animals↗

PubMed1983

Respiratory syncytial virus polypeptides. III. The envelope-associated proteins.

Four proteins, GP1, VGP48, GP26 and VPM27, are associated with the envelope of respiratory syncytial (RS) virus. The status of GP1 has been uncertain, because a cellular glycoprotein migrates at the same position when Laemmli's discontinuous buffer system is used for PAGE, and because BSC-1 cells infected with the RSN-2 strain of RS virus appear not to contain GP1. However, additional evidence suggests that GP1 is a viral structural protein. (i) It is removed from cells by trypsin, while the cellular glycoprotein is not; (ii) it is separated from the cellular glycoprotein when the infected cells are analysed by neutral SDS-PAGE; (iii) it is present in the purified RSN-2 strain of RS virus produced by BSC-1 cells; (iv) it is also present in the purified Long strain of RS virus produced by either human or monkey cells. When purified Long strain virus is analysed by PAGE under non-reducing conditions, the glycoproteins VGP48 and GP26 migrate together, and VPM27 separates into two proteins, which one-dimensional peptide mapping suggests are not different proteins. These observations suggest that VGP48 and GP26 exist in the virion as a single molecule joined by disulphide bonds, and so resemble a paramyxovirus fusion protein, and that probably there are two forms of VPM27 which differ in either position or number of disulphide bonds.

Electrophoresis, Polyacrylamide Gel↗

PubMed1982

Candidate product of the FBJ murine osteosarcoma virus oncogene: characterization of a 55,000-dalton phosphoprotein.

Sera from rat bearing tumors induced by inoculation of FBJ murine osteogenic sarcoma virus (FBJ-MSV) nonproducer rat cells precipitate two proteins with molecular weights of 55,000 (p55) and 39,000 (p39) from FBJ-MSV-transformed cells. These proteins cannot be precipitated from uninfected cells or cells transformed by other strains of murine sarcoma virus, nor can they be precipitated by sera specific for the viral structural proteins. A methionine tryptic peptide mapping analysis showed that p55 and p39 have little or no homology and that they are not related to the helper virus gag and env gene products. p55 could also be detected among the in vitro translation products of 70S RNA from FBJ murine leukemia virus plus FBJ-MSV virions but not among those from FBJ murine leukemia virus alone. This suggests that p55 is encoded by the FBJ-MSV genome, whereas p39, which was not detected among the in vitro translation products, may not be virus encoded. Another difference between p55 and p39 is that p55 is phosphorylated, with most of the phosphate on a serine residue(s), whereas p39 is phosphorylated to a much lesser extent, if at all. No protein kinase activity was associated with p55 and p39 immune complexes under standard conditions. Our data suggest that p55 is a strong candidate for the FBJ-MSV oncogene product.

Animals↗

PubMed1991

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.

Amino Acid Sequence↗

PubMed2003

Choristoneura fumiferana Granulovirus p74 protein, a highly conserved baculoviral envelope protein.

A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3 non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV).

Amino Acid Sequence↗

PubMed1983

Preferential immune response to virion surface glycoproteins by caprine arthritis-encephalitis virus-infected goats.

Six months after inoculation with caprine arthritis-encephalitis virus, the serum and synovial fluid of virus-infected goats had antibodies to [35S]methionine-labeled viral proteins with apparent molecular weights of 125,000, 90,000, 28,000, and 15,000. The 125,000-, 90,000-, and 15,000-molecular-weight methionine-labeled proteins were identified as virion surface glycoproteins by lactoperoxidase iodination and galactose oxidase-boro[3H]hydride reduction labeling techniques. Radioimmunoassay antibody titers to purified p28, the most abundant viral structural protein, averaged 1:182 in synovial fluid and 1:67 in serum 6 months after inoculation. High dilutions of serum and synovial fluid reacted with gp90 and gp125 electroblotted onto nitrocellulose paper from polyacrylamide gels. Anti-gp90 activity was detected at dilutions with an immunoglobulin G content of 0.02 to 11 micrograms, whereas antibody to p28, when detectable on Western blots, was present in samples with an immunoglobulin G content of 0.1 to 2 mg, representing 100- to 1,000-fold-greater titers of antibody to the surface glycoprotein. Synovial fluids often contained more anti-gp90 antibody than did sera. Immunoprecipitation of lactoperoxidase-iodinated virus confirmed the presence of high antibody titers to the two virion surface glycoproteins. Because antiviral gp90 and gp125 antibody is abundant in the synovial fluid of infected goats, it probably contributes to the high immunoglobulin G1 concentrations seen at this site 6 months after caprine arthritis-encephalitis virus infection.

Animals↗

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↗

PubMed1992

DNA conformational change induced by the bacteriophage phi 29 connector.

Translocation of viral DNA inwards and outwards of the capsid of double-stranded DNA bacteriophages occurs through the connector, a key viral structure that is known to interact with DNA. It is shown here that phage phi 29 connector binds both linear and circular double-stranded DNA. However, DNA-mediated protection of phi 29 connectors against Staphylococcus aureus endoprotease V8 digestion suggests that binding to linear DNA is more stable than to circular DNA. Endoprotease V8-protection assays also suggest that the length of the linear DNA required to produce a stable phi 29 connector-DNA interaction is, at least, twice longer than the phi 29 connector channel. This result is confirmed by experiments of phi 29 connector-protection of DNA against DNase I digestion. Furthermore, DNA circularization assays indicate that phi 29 connectors restrain negative supercoiling when bound to linear DNA. This DNA conformational change is not observed upon binding to circular DNA and it could reflect the existence of some left-handed DNA coiling or DNA untwisting inside of the phi 29 connector channel.

Bacillus Phages↗

PubMed1983

High-frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells.

A protoplast fusion method was developed to stably transfect human cells with pSV2-derived plasmids at frequencies greater than 10(-3). This procedure made it possible to test the biological effect of a hepatitis B virus (HBV) gene independent of the viral structures required for infection. A pSV2gpt+ plasmid constructed to carry a subgenomic fragment of HBV that contained the core antigen gene (HBc gene) was transfected into human cells. A human epithelial cell line was stably transfected with the HBc+ gene by selecting recipient cells for expression of guanine phosphoribosyl transferase expression. With this gpt+/HBc+ cell line it was shown that growth in serum-free medium or treatment with 5'-azacytidine stimulates the production of the HBV core antigen. A hepatocellular carcinoma carrying the entire HBV genome was stimulated to produce the HBc gene product in response to the same factors that stimulated HBcAg production in the gpt+/HBc+ cell line constructed by transfection. The temporal relation between the cytopathologic response and HBc gene expression was similar for both cell types, indicating a primary role for HBc gene expression in the cytopathology of HBV-infected human liver.

Azacitidine↗

PubMed1992

T-cell hybridomas recognizing the envelope proteins of Semliki forest virus: their sensitivity to endo/lysosomal protease and the antigenicity.

Eight T-cell hybridomas were established from the draining lymph node of C3H mice immunized with Semliki forest virus (SFV). Six of them showed specificity toward viral-structure protein E2, while the remaining two clones included one with specificity to an other structural protein E1 and the other with specificity to C. The production of IL-2 by the E2 protein-specific T-cell hybridomas in the presence of SFV was suppressed by treating the antigen-presenting cells (APC) with ammonium chloride raising pH of the acidic compartments. It was found also that treatment of APC with a thiol protease inhibitor, leupeptin or E64, resulted in a reduced response of some of the E2-specific T-cell hybridomas. The E2 protein of SFV proved to be resistant at pH 7.0, and sensitive at pH 5.0 to in vitro cathepsin B treatment. In contrast, the E1 and C proteins proved to be resistant to both pH values. These results indicate that the thiol protease, probably cathepsin B, works as one of the enzymes group involved in antigen processing.

Ammonium Chloride↗

PubMed1999

Walleye dermal sarcoma virus: OrfA N-terminal end inhibits the activity of a reporter gene directed by eukaryotic promoters and has a negative effect on the growth of fish and mammalian cells.

Walleye dermal sarcoma virus (WDSV) is a fish retrovirus causing a skin tumor termed walleye dermal sarcoma, which develops and regresses on a seasonal basis. The WDSV genome contains three short open reading frames designated orfA, orfB, and orfC in addition to the viral structural genes, gag, pol, and env. orfA and orfB transcripts are detected in tumors by reverse transcription-PCR. Recently, OrfA, whose amino acid sequence is similar to that of cyclins A and D, has been shown to complement a cyclin-deficient yeast strain. We report that expression of the accessory gene orfA inhibited nonspecifically the activity of a reporter gene directed by various eukaryotic promoters. In addition, stable transfection with the wild-type orfA generated substantially fewer G418-resistant colonies in both fish and mammalian cells than the parent vector. An orfA mutant expressing only the first N-terminal 49 residues of the full-length protein had the same negative effect on the activity of the reporter gene and on the number of stably transfected colonies as the full-length OrfA. Thus, OrfA inhibits cell growth and/or causes cell death, and the first 49 N-terminal residues of this protein are sufficient to cause these negative effects.

Amino Acid Sequence↗