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Biomedical subjects

G C Sen

Publications and source records attributed to G C Sen.

At least 91 records · Page 5Linked to original sources
PubMed1987

Inhibition of vesicular stomatitis viral mRNA synthesis by interferons.

Interferon (IFN) treatment inhibited the replication of vesicular stomatitis virus (VSV) in human GM2767 and mouse JLSV-11 cells. The replication of this virus in either human RD-114 or mouse A402 cells was insensitive to IFN treatment. We analyzed various steps in the VSV life cycle as they occurred under different conditions of IFN treatment to identify the point(s) at which IFN was exerting its inhibitory effect. IFN treatment led to strong inhibition of viral protein synthesis and accumulation of viral RNA in both lines of IFN-sensitive cells. No such effect was observed in the IFN-resistant cells. Using a temperature-sensitive mutant (tsG41) and wild-type VSV that were not undergoing protein synthesis, we determined that the major site of action of IFN against VSV replication in JLSV-11 and GM2767 cells was at the level of primary viral transcription. The accumulation of primary viral transcripts was strongly inhibited in these cells by IFN treatment. This effect was not a consequence of any effect of IFN on virus entry and uncoating. Thus, it appears that IFN exerts a direct effect on the VSV transcriptional process in GM2767 and JLSV-11 cells.

Animals↗
PubMed1987

Expression of interferon-inducible genes in RD-114 cells.

RD-114 is a cell line which is partially responsive to interferon (IFN). Although both IFN-alpha and IFN gamma inhibit production of the resident retrovirus, they do not inhibit replication of other viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, in these cells. In the studies reported here, we studied the characteristics of induction of seven IFN-inducible mRNAs in RD-114 cells. We observed that mRNAs 561, 6-16, 1-8, 2A, and 6-26 have similar induction characteristics in RD-114 cells and in HeLa cells, a fully responsive line. mRNA 2'-5'-oligo-adenylate synthetase (2-5(A) synthetase), however, was induced more efficiently by IFN-alpha in HeLa cells than in RD-114 cells. The same was true for the induction of metallothionein II mRNA by IFN-gamma. However, the latter mRNA was induced equally strongly in both lines when ZnCl2 was used as the inducer, suggesting that the gene is not defective in RD-114 cells. Although IFN-alpha induced 2-5(A) synthetase mRNA poorly and IFN-gamma did not induce it at all in these cells, a mixture of IFN-alpha and IFN-gamma induced this mRNA quite effectively, to a level of induction comparable to that in HeLa cells. Only 1 U of IFN-gamma per ml was sufficient to elicit this synergism, and the data suggested that an IFN-gamma-inducible protein was needed for this process. Induction of mRNA 561 by IFN-alpha in RD-114 cells, unlike that in HeLa cells, did not need ongoing protein synthesis. Once induced, this mRNA turned over rapidly in both cell lines, and this turnover could be slowed down by inhibiting protein synthesis in either cell line. IFN-induced mRNAs, such as 561 and 1-8, were polysome associated in IFN-treated RD-114 cells, suggesting that they were actively translated. Therefore, it is unlikely that the products of these IFN-inducible genes, by themselves, mediate the inhibition of replication of those viruses which are insensitive to IFN action in RD-114 cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Clonal derivatives of the RD-114 cell line differ in their antiviral and gene-inducing responses to interferons.

The human rhabdomyosarcoma cell line RD-114 is partially responsive to interferons (IFNs). In these cells, alpha interferon (IFN-alpha) or gamma interferon (IFN-gamma) inhibits the replication of some viruses but not of others. Similarly, some of the IFN-inducible mRNAs are induced poorly, whereas others are induced well. Here we report the isolation of clonal derivatives of this line which display different spectra of responses to IFNs. Among the eight extensively characterized clonal lines, one, C10, did not respond to IFN-alpha or IFN-gamma at all. Retrovirus production by each of the seven other lines was inhibited by both IFN-alpha and IFN-gamma. Replication of vesicular stomatitis virus was inhibited strongly by IFN-alpha in clone B1 but not in others, whereas it was not appreciably affected by IFN-gamma in any clone. Replication of encephalomyocarditis virus was inhibited strongly by IFN-gamma in clones A1, A2, A3, B3, and B8 and by IFN-alpha in clone A2. Neither IFN inhibited the multiplication of these clones greatly, although their doubling times were slightly increased. Five mRNAs were induced by IFNs to varying degrees in the seven clones. mRNA 2A was most strongly induced by IFN-gamma in clone A3. mRNA 1-8 was strongly induced by IFN-alpha in clone A1 and by either IFN in clones A2 and A3. The highest concentrations of 2',5'-oligoadenylate synthetase mRNA, mRNA 561, and mRNA 6-16 were in IFN-alpha-treated clones A1 and A2. These results demonstrated the existence of clonal heterogeneity in IFN responses in a cell line and strengthened the view that IFN treatment of cells generates multiple signals leading to a variety of IFN-induced phenotypes.

Acetyltransferases↗
PubMed1987

Transcriptional analyses of interferon-inducible mRNAs.

Using nuclear runoff transcription assays we demonstrated that alpha interferon-mediated induction of transcription of four mRNAs in HeLa monolayer cells needed ongoing protein synthesis and that such a need could be obviated by pretreating the cells with gamma interferon which, by itself, did not induce transcription of these mRNAs. In another human cell line, RD-114, synthesis of alpha interferon-inducible mRNA 561 did not need ongoing protein synthesis. In this line, however, in which interferon inhibits replication of some viruses but not of others, transcription of two of the six interferon-inducible mRNAs that we examined was not appreciably enhanced by interferon.

HeLa Cells↗
PubMed1986

Regulation of synthesis and turnover of an interferon-inducible mRNA.

Regulation of synthesis and turnover of an interferon (IFN)-inducible mRNA, mRNA 561, in HeLa monolayer cells was studied. Cytoplasmic levels of this mRNA were estimated by hybridization analyses with a cDNA clone that we have isolated as a probe. IFN-alpha A induced a high level of this mRNA in a transient fashion, whereas no induction was observed in response to IFN-gamma. Surprisingly little mRNA 561 was induced in cells treated simultaneously with IFN-alpha A and an inhibitor of protein synthesis, suggesting that in addition to IFN-alpha A, an interferon-inducible protein was needed for induction of this mRNA. Apparently this putative protein could be induced by IFN-gamma as well. Thus, although little mRNA 561 was synthesized in cells treated either with IFN-gamma alone or with IFN-alpha A and cycloheximide, a large quantity of this mRNA was induced in cells which had been pretreated with IFN-gamma and then treated with IFN-alpha A and cycloheximide. Once mRNA 561 was induced by IFN-alpha A, it turned over rapidly. This rapid turnover could be blocked by actinomycin D or cycloheximide indicating that another IFN-inducible protein may mediate this process.

Cell Adhesion↗
PubMed1984

Synthesis of interferon-inducible proteins is regulated differently by interferon-alpha and interferon-gamma.

Both interferon-alpha and interferon-gamma induce the synthesis of several proteins in resting human fibroblasts. Synthesis of two such proteins of Mr 67,000 and 56,000 was transient in interferon-alpha-treated cells, peaking around 6 hr after the beginning of interferon treatment and then declining to very low levels even if interferon-alpha was present continuously in the culture medium. In contrast, in cells treated with interferon-gamma, their synthesis continued unabated even 22 hr after interferon treatment began. Constant presence of interferon-gamma in the culture medium was, however, necessary for the continued synthesis of these proteins. The kinetics of their synthesis in cells treated with both interferon-alpha and interferon-gamma were similar to those in cells treated with interferon-gamma alone. Cells treated with interferon-alpha for 6 hr or with interferon-gamma for 24 hr contained in vitro-translatable mRNAs for these proteins, whereas such mRNAs were below detectable levels in untreated cells. Surprisingly, cells which had been treated with interferon-alpha for 24 hr, and which were synthesizing little of these two induced proteins in vivo, contained substantial amounts of mRNAs for these proteins, as demonstrated by in vitro translation experiments. It appears, therefore, that synthesis of some interferon-inducible proteins is regulated both transcriptionally and translationally, and the nature of such regulations is different for different types of interferons.

Cell Line↗
PubMed1984

Antiviral and protein-inducing activities of recombinant human leukocyte interferons and their hybrids.

The antiviral activities of recombinant human leukocyte interferons IFN-alpha A and IFN-alpha D as well as five hybrids of these interferons against retroviruses, vesicular stomatitis virus, and encephalomyocarditis virus were studied in feline, human, and murine cells. Although these interferon species had widely different potencies, their activities against these viruses were, in general, proportional. The IFN-alpha A/D (Bgl) hybrid was the most potent species, and the IFN-alpha D/A (Bgl) hybrid was the least potent. However, the latter species did not interfere with the action of the former species. Like natural human leukocyte interferon, each of the seven species of recombinant interferons induced the synthesis of at least five proteins in human fibroblasts, whereas induction of only one such protein was readily detected in a feline fibroblast line in which these interferon species inhibited the replication of all three viruses.

Animals↗
PubMed1983

Interferon-mediated inhibition of production of Gazdar murine sarcoma virus, a retrovirus lacking env proteins and containing an uncleaved gag precursor.

HTG2 cells are murine sarcoma virus-transformed hamster cells. These cells continuously produce Gazdar sarcoma virus particles which are devoid of viral envelope proteins and which contain the uncleaved gag precursor polyprotein, Pr65, as their major protein constituent. Human interferon-alpha elicited an antiviral response in these cells as shown by the inhibition of replication of vesicular stomatitis virus in interferon-treated cells. Extracellular production of the retroviral particles by these cells was also inhibited by interferon in a dose-dependent manner and this inhibition was abolished by a specific antiserum to interferon. The intracellular level of Pr65 was not lowered in the interferon-treated cells, indicating that inhibition of viral protein synthesis was not responsible for inhibition of virus production. The present study suggests that interferon-mediated inhibition of retrovirus production, in general, is not a consequence of either a defective interaction between viral nucleoprotein cores and viral envelope proteins or a defect in the proteolytic processing of the gag polyprotein, since neither of these processes occurs during the morphogenesis of Gazdar particles and their production is nonetheless inhibited by interferon.

Animals↗
PubMed1983

Human interferon-alpha-and-gamma-mediated inhibition of retrovirus production in the absence of an inhibitory effect on vesicular stomatitis virus and encephalomyocarditis virus replication in RD-114 cells.

RD-114 line is a human sarcoma cell line chronically infected with RD-114 retrovirus. Treatment of these cells with increasing doses of human interferon-alpha or -gamma resulted in increasing inhibition of RD-114 virus production. Surprisingly, the replication of vesicular stomatitis virus and encephalomyocarditis virus, in these cells and in the parental RD cells which are not infected with the retrovirus, was insensitive to interferon treatment. Unlike reported differences in other properties of interferon-alpha and interferon-gamma, there were no differences in their antiretroviral properties such as dose response, kinetics of establishment of the antiretroviral state, and kinetics of its dissipation upon removal of interferon.

Dose-Response Relationship, Drug↗
PubMed1983

Effects of procaine and oxyphenylbutazone on interferon-mediated inhibition of murine leukemia virus production.

Treatment of murine NIH/MOL, C cells which are chronically infected with Moloney murine leukemia virus (MuLV), with mouse interferon (IFN), causes inhibition of extracellular MuLV production. We tested the effect of procaine, a drug that is known to expand cellular membranes and to increase their fluidity, on IFN-mediated inhibition of MuLV production. We observed that procaine did not alleviate this inhibition when IFN was present during procaine treatment. However, if IFN was removed from the culture medium before procaine treatment, the inhibition of MuLV production was partially reversed. We also observed that procaine treatment caused an increase in the amount of MuLV production by cells which had not been treated with IFN. Oxyphenylbutazone (OPB) is an inhibitor of cyclooxygenase, a key enzyme in prostaglandin biosynthesis. New synthesis of prostaglandins is thought to be needed for the antiviral actions of IFN. We observed that OPB did not prevent the antiretroviral action of IFN on NIH/MOL, C cells. OPB also failed to alleviate the IFN-mediated inhibition of replication of vesicular stomatitis virus either in NIH/MOL, C cells or in the L929 cells.

Animals↗
PubMed1983

Antiviral, anticellular and enzyme-inducing activities of interferons in RD-114 cells.

RD-114 is a human sarcoma-derived cell line which is chronically infected with the RD-114 retrovirus. In a previous study, we found that treatment of these cells with human interferon-alpha or human interferon-gamma causes a marked inhibition of RD-114 virus production, but that the replication of exogenous vesicular stomatitis or encephalomyocarditis virus is not impaired. In the present study, we report that neither type of interferon has strong inhibitory effects on DNA synthesis or on multiplication of the cells. We also failed to detect a double-stranded RNA-dependent protein kinase activity in extracts of both interferon-treated and untreated cells. However, a low level of 2',5'-oligoadenylate [2,5(A)] synthetase activity was detectable in extracts of interferon-treated cells. 2,5(A)-dependent endonuclease L activity was detectable in extracts of both untreated and interferon-treated cells. This was probably responsible for the inhibition of protein synthesis observed upon introduction of 2,5(A) to RD-114 cells. In many cells, interferon has been found to induce synthesis of several proteins demonstrable by autoradiographic analysis of slab gels on which extracts of interferon-treated and radiolabelled cells are separated. Using a similar method, no such induced protein synthesis was detectable in interferon-treated RD-114 cells. Our results indicate that RD-114 cells are resistant to most known actions of interferons except for the antiretroviral action to which they are as sensitive as any other cell line.

2',5'-Oligoadenylate Synthetase↗
PubMed1983

Differential antiviral effects of interferon in three murine cell lines.

Infectious leukemia virus production by two chronically infected NIH/MOL lines was strongly inhibited by interferon treatment of the cells. The corresponding degree of inhibition in JLSV-11 cells was much lower. Multiplication of encephalomyocarditis virus in all three cell lines was barely affected by interferon treatment. Replication of vesicular stomatitis virus, on the other hand, was highly sensitive to interferon in the JLSV-11 line and in one NIH/MOL line but was practically insensitive in the other NIH/MOL line. Anticellular actions of interferon were more pronounced in the JLSV-11 line than in the others. In response to interferon treatment, 2',5'-oligoadenylate synthetase activity was induced to a high level in JLSV-11 cells and to lower levels in the NIH/MOL lines. We failed to detect any 2',5'-oligoadenylate-dependent endonuclease activity in extracts of these cells. Double-stranded RNA-dependent protein kinase activity was present in extracts of interferon-treated NIH/MOL cells, but it was barely detectable in extracts of interferon-treated JLSV-11 cells. The above studies demonstrated that interferon could differentially affect the replication of three different viruses in three different cell lines, including two seemingly identical NIH/MOL lines, and that certain tentative conclusions can be drawn regarding the roles of different interferon-inducible enzyme markers in the different antiviral actions of interferons.

2',5'-Oligoadenylate Synthetase↗
PubMed1981

Synthesis of murine mammary tumor viral proteins in vitro.

The coding potential of murine mammary tumor viral genomic RNA was investigated by in vitro translation of various size classes of RNAs isolated from the virions. The major products of translation of full-size 35S polyadenylylated virion RNA were gag-related polyproteins of 75,000, 105,000, and 180,000 daltons (P75, P105, and P180, respectively). Studies on the kinetics of translation of these three proteins established that they were synthesized independently and that the smaller proteins were not post-translational cleavage products of the larger proteins. Tryptic peptide mapping showed that almost all of the P75 sequences were contained within P105 and almost all of the P105 sequences were contained within P180. The syntheses of all three proteins were inhibited by m7GTP, indicating that they were translated from capped mRNA's. Although a 24S polyadenylylated RNA had been identified as the intracellular mRNA for env precursor polyprotein, no such protein could be translated from the 24S polyadenylylated RNA isolated from the virions. However, translation of a 14S size class of polyadenylylated virion yielded four prominent proteins of about 36,000, 23,000, 21,000, and 20,000 daltons. These proteins were unrelated to murine mammary tumor viral structural proteins, as suggested from tryptic peptide mapping and immunoprecipitation data. They might be the products of an as-yet-unidentified gene located near the 3' terminus of the murine mammary tumor viral genomic RNA.

Amino Acid Sequence↗