Ganes C. Sen

Functional equivalents of interferon-mediated signals needed for induction of an mRNA can be generated by double-stranded RNA and growth factors.

In our earlier studies we demonstrated that in HeLaM cells, interferon‐alpha produces two functionally distinguishable signals, both of which are needed for induced transcription of mRNA 561 and other inducible mRNAs. Interferon‐gamma cannot induce mRNA 561 because it produces only signal 1. Here we report that platelet‐derived growth factor or epidermal growth factor could also produce signal 1. On the other hand, signal 2, which can be produced by interferon‐alpha but not by interferon‐gamma, could be elicited also by double‐stranded RNA. Several lines of evidence suggest that the production of signal 2 by double‐stranded RNA was not mediated through interferon. Interferon‐induced transcription of mRNA 561 in HeLaM cells or in human fibroblast GM2767 cells was transient. However, in interferon‐alpha‐treated GM2767 cells, which had ceased to synthesize mRNA 561, transcription of this mRNA could be induced effectively by double‐stranded RNA suggesting that this induction process could bypass the interferon‐mediated down‐regulation of induced transcription. Unlike HeLaM and GM2767 cells, in Daudi cells, induction of mRNA 561 by interferon‐alpha was not transient. Transcription of this and two other induced mRNAs continued at a high rate even after 18 h of interferon‐alpha treatment of these cells. The lack of down‐regulation of interferon‐induced gene expression may be responsible for interferons acute antigrowth effects on these cells.

Gene induction by interferons and double-stranded RNA: selective inhibition by 2-aminopurine.

Transcription of several interferon-inducible human genes is also induced by double-stranded RNA. The nature and the mechanism of action of signals generated by interferons and by double-stranded RNA which mediate the induction of these genes are under investigation. Here we report that 2-aminopurine, a known inhibitor of protein kinases, could selectively block this induction process. Induction of mRNAs 561 and 6-16 in HeLaM cells by double-stranded RNA was completely inhibited by 10 mM 2-aminopurine, whereas cellular protein and RNA syntheses as well as the induction of metallothionein mRNA by CdCl2 were unaffected by this inhibitor. In addition, 2-aminopurine blocked the induction of the same two mRNAs and of mRNAs 2-5(A) synthetase, 2A, and 1-8 by alpha interferon and of mRNAs 2A and 1-8 by gamma interferon in HeLaM cells. The observed inhibition was at the level of transcription, and for establishing efficient inhibition, the 2-aminopurine treatment had to begin at early stages of interferon treatment. In GM2767 cells, 2-aminopurine inhibited induction of mRNAs 561 and 6-16 by double-stranded RNA but not by alpha interferon. These results suggest that double-stranded RNA-induced signal 2 is distinct from the interferon-alpha-induced signal 2 (R. K. Tiwari, J. Kusari, and G. C. Sen, EMBO J. 6:3373-3378, 1987) and that 2-aminopurine can block the former but not the latter. Moreover, it appeared that 2-aminopurine could block the production of signal 1 by interferons. This was confirmed by experiments in which we separately tested the effects of 2-aminopurine on signal 1 and signal 2 production by interferons in HeLaM cells. Although no direct experimental evidence is available as yet, our results are consistent with the hypothesis that the functioning of a protein kinase activity may be necessary for transcriptional induction of genes by double-stranded RNA and for gene induction by interferons in those cells in which signal 1 production is needed.

Mechanism of interferon action: progress toward its understanding.

Publisher Summary This chapter describes the mechanism of action of interferons. Interferons are synthesized in vertebrate cells in response to various inducers. They are secreted out of the cells, interact with other cells, and thereby render them incapable of supporting virus replication. Studies on the interferon system can be classified broadly into two groups: studies on interferon biosynthesis and studies on interferon actions. The chapter discusses some aspects of the latter and describes the interferon proteins and interferon genes in detail. The chapter also discusses the biochemical changes brought about by interferon treatment of cells that are not infected by viruses. The chapter describes various antiviral effects as studied with intact virus-infected cells. Although much progress has been made in interferon research within the past few years, the mechanism of interferon actions in molecular terms is still not clear. The discovery of the dsRNA-dependent enzyme activities has implications beyond the interferon system. These are the first examples of enzymes activated by interaction with double-stranded ribonucleic acid (dsRNA).

Effects of interferon on the production of murine mammary tumor virus by mammary tumor cells in culture

A cell line established from the mammary tumor of a GR mouse produces murine mammary tumor virus upon stimulation- with dexamethasone in vitro. Treatment of these stimulated GR cells with 50 U/ml of partially purified mouse interferon inhibited the extracellular virus production by about 80% as measured by virus-associated reverse transcriptase activity or by metabolic labeling of the virus with [3H]uridine or 14C-amino acids. There was no inhibition of cellular RNA or protein synthesis under these conditions. The same effects were observed when the same dose of pure mouse interferon was used. Though the interferon-treated cells produced fewer extracellular virus particles, the intracellular levels of viral RNA as measured by hybridization analyses and the intracellular levels of the major viral envelope and core proteins as measured by radioimmunocompetition assays were not lowered in the interferon-treated cells. Electron microscopy of thin sections from the control and interferon-treated cells revealed no gross differences in any subcellular structures including that of the viruses. Scanning electron microscopy showed, however, an increased number of virus particles on the cell surface of the interferon-treated cells. These results suggest that the impairment of extracellular mammary tumor virus production by interferon treatment of GR cells is caused by an interferon-induced block at a very late stage of virus morphogenesis.

Synthesis of interferon-inducible proteins is regulated differently by interferon-α and interferon-γ

Abstract Both interferon-α and interferon-γ 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-α-treated cells, peaking around 6 hr after the beginning of interferon treatment and then declining to very low levels even if interferon-α was present continuously in the culture medium. In contrast, in cells treated with interferon-γ, their synthesis continued unabated even 22 hr after interferon treatment began. Constant presence of interferon-γ 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-α and interferon-γ were similar to those in cells treated with interferon-γ alone. Cells treated with interferon-α for 6 hr or with interferon-γ 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-α 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.

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.

Gene order of murine mammary tumor virus gag proteins and env proteins

Abstract The gene orders of murine mammary tumor virus (MuMTV) gag proteins and env proteins have been determined by pactamycin mapping techniques. A MuMTV producing cell line, Mm5mt, was pulse-labeled with [ 31 S]methionine in the presence or absence of 5 × 10 −7 M pactamycin, an inhibitor of initiation of protein synthesis. Both pactamycin and the labeled amino acid were removed after the pulsing period and cells were further incubated in normal growth medium. Virus was harvested from the medium after 24 hr, and the individual protein constituents of the virus were analyzed by SDS-gel electrophoresis. For each protein, the ratio of the radioactivity incorporation in the presence of pactamycin and in its absence was determined. The lower this ratio, the closer would be the protein to the amino terminal of the precursor polyprotein. The following gene orders were derived from these experiments: gag , NH, p10(p28,pp23)p14 COOH, and env , NH 2 gp52gp36 COOH.

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.

A High-Affinity Monoclonal Antibody (GIF-1) to Human Gamma-interferon: Neutralization of Interferon Mediated Inhibition of Retrovirus Production and 2’-5’ (A) Synthetase Induction

An hybridoma clone secreting an IgG1 monoclonal antibody (GIF-1) specific for human gamma-interferon (HuIFN-gamma) has been generated using HAT medium supplemented with insulin (HIAT) at the initial stage of cell fusion. This antibody is capable of neutralizing the antiviral activity of HuIFN-gamma, the ability of HuIFN-gamma to inhibit retroviral replication in RD-114 cells, and the ability of HuIFN-gamma to induce the 2-5 oligoadenylate (A) synthetase in RD-114 and HeLa cells. Eluate from an immunoaffinity column containing GIF-1 yielded two protein bands of molecular weight of 20 and 25 kd when subjected to SDS-PAGE.