Inhibition of mouse fibroblast interferon by gangliosides. Differential effects on biological activity and on induction of (2'--5')oligoadenylate synthetase

Gangliosides are potent inhibitors of the antiviral activity of mouse fibroblasts and other beta-interferons. We have compared the effects of gangliosides on antiviral and antigrowth activities of mouse fibroblast interferon and on the induction of (2'--5')oligoadenylate synthetase, one of the enzymes implicated in the antiviral state induced by interferon. Whereas both biological effects appear to be inhibited by gangliosides in an analogous fashion, inhibition of induction of (2'--5')oligoadenylate synthetase does not correlate with inhibition of vesicular stomatitis virus replication. Ganglioside concentrations that inhibit the interferon-induced (2'--5')oligoadenylate synthetase to levels close to those of uninduced cells, still allow for a 100--1000-fold reduction of viral yield. Significantly higher ganglioside concentrations are required to prevent completely the antiviral effect. This biphasic relationship between (2'--5')oligoadenylate synthetase levels and inhibition of viral yield suggests that no or very small increases in synthetase levels are involved in inhibition of virus by between two and three orders of magnitude.     

Vasoactive intestinal peptide induces 2',5'-oligoadenylate synthetase and antiviral state in cells of HT-29 colonic cancer]

Vasoactive Intestinal Peptide (VIP) is able at the concentration 10 to 100 nM to induce in HT-29 cells 2'5' oligoadenylate (2'5' A) synthetase activity. The kinetics of this induction show that the maximal effect is attained after 24 hrs. VIP induces 2'5' A synthetase parallel to inhibition of vesicular stomatitis virus growth. The levels of these two induced activities after VIP treatment are comparable to those induced by the poly (I).poly (C), an inducer of IFN beta/alpha in mammalian cells. Moreover the anti-IFN beta/alpha antibodies abolish the VIP-induced 2'5' A synthetase whereas anti-IFN gamma antibodies are ineffective. The fact that VIP establishes an antiviral state in HT-29 cells potentiates new pharmaceutical applications for this neuropeptide.     

Injection of mice with antibody to mouse interferon alpha/beta decreases the level of 2''-5'' oligoadenylate synthetase in peritoneal macrophages.

Injection of conventional or axenic weanling mice with potent sheep or goat antibody to mouse interferon alpha/beta resulted in a decrease in the basal level of 2-5A synthetase in resting peritoneal macrophages and rendered these cells permissive for vesicular stomatitis virus. There was a good inverse correlation between the level of 2-5A synthetase in peritoneal macrophages and the permissivity of these cells for vesicular stomatitis virus. The peritoneal macrophages of 1- and 2-week-old mice had low levels of 2-5A synthetase and were permissive for vesicular stomatitis virus, whereas at 3 weeks (and after) there was a marked increase in the level of 2-5A synthetase in peritoneal macrophages, and these cells were no longer permissive for vesicular stomatitis virus. We suggest that low levels of interferon alpha or beta or both are produced in normal mice, and that this interferon contributes to host defense by inducing and maintaining an antiviral state in some cells.     

Mx gene control of interferon action: different kinetics of the antiviral state against influenza virus and vesicular stomatitis virus.

The allele Mx regulates the extent to which interferon alpha/beta inhibits the growth of influenza viruses in mouse cells such as peritoneal macrophages. The time course of induction of the antiviral state against an influenza A virus is comparable in macrophages with and without Mx and is similar to that found with vesicular stomatitis virus. In contrast, the decay of the antiviral state against influenza virus is markedly slower in Mx-positive cells and slower than that against vesicular stomatitis virus observed in either Mx-positive or Mx-negative cells. Thus, after removal of interferon alpha/beta, Mx-positive cells remain protected against influenza virus at times when they have lost protection against vesicular stomatitis virus. These results suggest that interferon alpha/beta treatment activates different antiviral mechanisms, each acting against distinct groups of viruses and each independently controlled by host genes.     

Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon.

Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state.     

A mouse cell line, which is unprotected by interferon against lytic virus infection, lacks ribonuclease F activity

A mouse cell line, NIH 3T3, does not respond to some of the activities of interferon. Even after treatment with high concentrations of interferon the replication of lytic viruses, such as encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) is not inhibited in these cells. In contrast, interferon treatment of these same cells results in the inhibition of Moloney murine leukemia virus (MMuLV) production. We have analyzed enzymatic pathways which are induced by interferon in these cells. After interferon treatment, the level of the (2'-5')oligoadenylate [(2'-5)An] synthetase activity and the phosphorylation of the 67000-dalton protein (P1) are enhanced in NIH 3T3 cells to approximately the same level as interferon-sensitive mouse L-cells. Moreover, NIH 3T3 and L-cells, contain approximately the same levels of enzymes which inactivate (2'-5')An. Both exogenously added (2'-5')A3 or double-stranded RNA (dsRNA) failed to inhibit protein synthesis in NIH 3T3 extracts even though they were potent inhibitors of L-cell extract-directed protein synthesis. Direct measurements of the (2'-5')An-dependent ribonuclease F (RNase F) failed to detect such activity in NIH 3T3 cells. Our results, therefore, suggest that the presence of RNase F activity is necessary for the interferon-induced antiviral activity against EMCV and against VSV. The induction of protein kinase activity by interferon treatment of NIH 3T3 cells appears to have no direct effect on EMCV and VSV replication.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Decreased Epstein-Barr virus-induced transformation, and elevated 2-5A synthetase and RNase L activity in peripheral blood mononuclear cells from patients treated with recombinant interferon alfa 2b

Patients with cutaneous T-cell lymphoma (CTCL) were treated with recombinant alfa 2b interferon (rIFN alfa 2b) by intramuscular injection. Therapy-induced changes in Epstein-Barr virus (EBV) induced transformation of patient peripheral blood lymphocytes, 2',5' oligoadenylate (2-5A) synthetase levels and RNase L activation in peripheral blood mononuclear cells were monitored. Inhibition of EBV-induced transformation and elevation of 2-5A synthetase levels correlated with increased activation of RNase L, which provides evidence that intramuscular administration of rIFN alfa 2b induces a sustained anti-EBV state in CTCL patient peripheral blood mononuclear cells which can be detected in vitro.     

Differential antiproliferative activities of IFNs alpha, beta and gamma: kinetics of establishment of their antiproliferative effects and the rapid development of resistance to IFNs alpha and beta

Interferons have been recognized to have potent in vitro antiproliferative activities in mouse and human systems. To further investigate the kinetics of development of interferons' antiproliferative activities, mouse B-16 melanoma cells were treated with MuIFN-alpha, MuIFN-beta or MuIFN-gamma for various initial periods of time during an 8 day cloning assay. With MuIFN-alpha and MuIFN-beta treatments, maximal expression of antiproliferative activity was attained with 2 to 4 days of interferon treatment. In contrast, with MuIFN-gamma treatment, expression of antiproliferative activity increased with progressively longer periods of time of MuIFN-gamma treatment. These results suggested that B-16 melanoma cells were initially sensitive to all three of the interferons but rapidly became resistant to MuIFN-alpha and MuIFN-beta after 2 to 4 days of treatment. This suggestion was confirmed by cell growth kinetics experiments. The cells which were resistant to the antiproliferative activity of the MuIFN-alpha remained sensitive to the antiviral activity of MuIFN-alpha, suggesting that MuIFN-alpha and MuIFN-beta regulate their antiviral and antiproliferative responses via different mechanisms. The cells which were resistant to the antiproliferative activities of MuIFN-alpha and MuIFN-beta remained sensitive to MuIFN-gamma, suggesting that they were not generally resistant to antiproliferative effects. The cells which were resistant to the antiproliferative activities of the interferons gradually lost their resistance with a half-life of 11 days when they were cultured in the absence of interferons. The differential antiproliferative actions of alpha, beta and gamma interferons observed with murine B-16 melanoma were confirmed in the human system with G-361 melanoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of interferon on the production of HBsAg and induction of an antiviral state in human hepatoma cell line PLC/PRF/5

The effects of human alpha and beta interferons (IFN) on the production of HBsAG by PLC/PRF/5 cells, an HBsAg-producing human hepatoma cell line, were studied in the exponential and stationary phases of cell growth. When exponential phase cells were treated with 100 or 1,000 U of IFN per ml for 48 hr. the amount of HBsAg in the culture medium decreased. The number of cells and the synthesis of DNA and proteins were also reduced by the IFN treatment. These results suggested that IFN did not affect the production of HBsAg specifically in exponential phase cells. When cells in the stationary phase were similarly treated with IFN, HbsAg production was not inhibited nor did the number of cells decrease. To examine the antiviral state induced by IFN in PLC/PRF/5, induction of 2'5'-oligo (A) synthetase and susceptibility to two kinds of viruses were examined. The 2'5'-oligo (A) synthetase activity was increased in an IFN-dose dependent manner. Susceptibility to vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) was decreased by treatment with 10 and 100 U of IFN per ml for 20 hr. It was concluded that IFN-alpha and IFN-beta induce 2'5'-oligo (A) synthetase and the antiviral state, but do not inhibit HBsAg production by PLC/PRF/5 cells.     

Resistance to the antiproliferative activity of IFN-alpha: further characterization and demonstration of antagonistic effects of IFN-gamma

Mouse B16 melanoma cells have been shown to rapidly develop resistance to the antiproliferative effects of MuIFN-alpha or MuIFN-beta when exposed to these interferons. In cloning studies, the maximal antiproliferative effects of MuIFN-alpha were seen with 2-4 days treatment. This resistance has been further characterized. The level of resistance which develops in B16 melanoma cells is dependent upon the concentration of MuIFN-alpha to which the cells are exposed. In addition, B16 melanoma cells which are resistant to the antiproliferative effects of MuIFN-alpha have greatly elevated levels of the interferon-induced enzyme 2',5'-oligoadenylate (2-5A) synthetase. Since it has previously been shown that B16 melanoma cells do not develop resistance to the antiproliferative effects of MuIFN-gamma, several experiments studied the influence of MuIFN-gamma on the development of resistance to MuIFN-alpha. Combinations of IFN-gamma and IFN-alpha have previously been shown to result in a synergistic enhancement of the antiproliferative effects. Kinetic studies show that the response of the cells to the MuIFN-gamma antiproliferative effect appears to be dominant over the development of resistance since no resistance develops in response to combination treatment. Not only is MuIFN-gamma able to prevent development of resistance when it is present continuously, but also when it is used for the sequential treatment of the cells before their exposure to MuIFN-alpha. A 2-day pretreatment with MuIFN-gamma is sufficient to prevent the development of resistance during later exposure of the cells to MuIFN-alpha alone for up to 6 additional days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two inducers of cell differentiation enhance the 2'5' oligoadenylate synthetase activity in MSV transformed cells

The interferon induced 2′5′ oligoadenylate synthetase activity can be increased upon treatment of Moloney Sarcoma virus transformed cells with two inducers of cell differentiation: sodium n-butyrate and dimethyl sulfoxide. This effect does not seem to be the consequence of the inhibition of cell growth by butyrate since the basic level of the enzyme stayed the same in control cells whether growth was inhibited by the absence of serum in the medium or not. It did not seem either to be due to the induction of IFN by these compounds since we could not detect any antiviral activity in the supernatant of the treated cells. Treatment by interferon of the butyrate pretreated cells results in a higher enzyme activity and a higher antiviral state than in non-pretreated cells.     

Antiviral activity of a chemically stabilized 2-5A analog upon microinjection into HeLa cells

2-5A[ppp(A2'p)n5'A] has been implicated as a mediator in the antiviral action of interferon. Its direct evaluation as an indicator of virus replication is hampered by two limitations: its inability to penetrate intact cells, and its rapid intracellular degradation by (2'-5')phosphodiesterase. These problems could be overcome by using a microinjection technique whereby a phosphodiesterase-resistant analog of 2-A, in which the 2'-terminals adenosine residue is replaced by 2-(9-adenyl)-6-hydroxy-methyl-4-hexylmorpholine, was injected into individual HeLa cells before infection with mengovirus or vesicular stomatitis virus (VSV). This comparative assay with two representatives of different virus classes in a single experimental system pointed to the high sensitivity of VSV to inhibition by 2-5A oligonucleotides, in contrast with the low sensitivity of mengovirus. Microinjection of the hexylmorpholine 2-5A analog led to a much greater reduction in mengovirus yield than did microinjection of 2-5A itself.     

Characterization of the antiviral activity constitutively produced by murine conceptuses: absence of placental mRNAs for interferon alpha and beta

Antiviral activity has been found in conceptus and placental tissues in numerous species, including mice, pigs, sheep, cattle and humans. In sheep and cattle, the antiviral activity is due to an interferon alpha (IFN-alpha), but in other species the nature of the protein(s) responsible for placental activity is unknown. The objectives of this study were to determine if the constitutive antiviral activity associated with the mouse conceptus is produced as early as the peri-implantation period, and to determine if the activity is due to an IFN-alpha or -beta. Conceptus and placental tissue explants released antiviral activity from Day 4 through at least Day 16 of gestation as measured in an agar overlay bioassay employing CHO cells challenged with vesicular stomatitis virus. This activity was neutralized by antiserum against MuIFN-alpha/beta. The same antiserum failed, however, to immunoprecipitate radiolabeled proteins from medium collected from Day 4 blastocysts cultured in the presence of L-[35S]-methionine. S1 nuclease analysis of placental RNA and screening of ectoplacental cone and extraembryonic ectoderm cDNA libraries with MuIFN-alpha and -beta probes failed to detect IFN related mRNAs, even under relatively non-stringent conditions of hybridization. Thus, while antiviral activity is produced by peri-implantation conceptuses in several diverse mammalian species, it does not appear to be due to a conserved type of IFN in all these species.     

Antiviral state against influenza virus neutralized by microinjection of antibodies to interferon-induced Mx proteins.

In mouse Mx+ cells, interferon alpha/beta induces the synthesis of the nuclear Mx protein, whose accumulation is correlated with specific inhibition of influenza viral protein synthesis. When Mx+ mouse cells are microinjected with the monoclonal anti-Mx antibody 2C12, interferon alpha/beta still induces Mx protein, but no longer inhibits efficiently the expression of influenza viral proteins as visualized by immunofluorescent labeling. However, interferon inhibition of an unrelated control virus, vesicular stomatitis virus, remains unchanged. Proteins with homology to mouse Mx protein are found in interferon-treated cells of a variety of mammalian species. In rat cells, for instance, rat interferon alpha/beta induces three Mx proteins which all cross-react with antibody 2C12 but differ in mol. wt and intracellular location, and it protects these cells well against influenza viruses. However, when rat cells are microinjected with antibody 2C12, interferon alpha/beta cannot induce an efficient antiviral state against influenza virus infection, whereas protection against vesicular stomatitis virus is not altered. These results show that both mouse and rat cells require functional Mx proteins for efficient protection against influenza virus. They further demonstrate that microinjection of antibodies is a promising way of elucidating the role of particular interferon-induced proteins in the intact cell.     

M1204, a novel 2',5' oligoadenylate synthetase with a ubiquitin-like extension, is induced during maturation of murine dendritic cells.

A novel molecule expressed by spleen dendritic cells (DC) was isolated using a subtractive hybridization approach. The full-length M1204 clone has 3063 bp, with 1415 bp spanning a single open reading frame, coding for a protein of a predicted size of about 50 kDa. This sequence has strong homology to 2', 5' oligoadenylate synthetase and contains a ubiquitin-like domain. In Northern blot analyses the mRNA is strongly expressed in spleen DC, whereas, in bone marrow-derived DC, the amount of mRNA increases during the maturation process. None of the other leukocytes nor several hemopoietic cell lines tested express this mRNA, but clear expression occurs in many organs, the highest levels being in thymus, lung, and bone marrow. In situ hybridization, combined with immunocytochemical staining of tissue sections of lung and spleen, shows colocalization of M1204 with the 2A1 and NLDC DC markers. In Western blot experiments, an antiserum raised against the recombinant M1204 recognizes a single band in bone marrow-derived DC and in the lung. The expressed oligoadenylate synthetase domain is active in synthesizing 2',5' diadenylate, which by itself may inhibit viral protein synthesis and may also function as a substrate for 2',5' oligoadenylate synthetase. Since the oligoadenylate/RNase L system provides early protection against virus infection, we hypothesize that M1204 prevents virus-induced cell death in DC.     

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.     

The latent membrane protein 1 of Epstein-Barr virus establishes an antiviral state via induction of interferon-stimulated genes

Epstein-Barr virus (EBV) infection is associated with several human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. In this report, we show that LMP-1 is able to induce the expression of several interferon (IFN)-stimulated genes (ISGs) with antiviral properties such as 2'-5' oligoadenylate synthetase (OAS), stimulated trans-acting factor of 50 kDa (STAF-50), and ISG-15. LMP-1 inhibits vesicular stomatitis virus (VSV) replication at low multiplicity of infection (0.1 pfu/cell). The antiviral effect of LMP-1 is associated with the ability of LMP-1 to induce ISGs; an LMP-1 mutant that cannot induce ISGs fails to induce an antiviral state. High levels of ISGs are expressed in EBV latency cells in which LMP-1 is expressed. EBV latency cells have antiviral activity that inhibits replication of superinfecting VSV. The antiviral activity of LMP-1 is apparently not related to IFN production in our experimental systems. In addition, EBV latency is responsive to viral superinfection: LMP-1 is induced and EBV latency is disrupted by EBV lytic replication during VSV superinfection of EBV latency cells. These data suggest that LMP-1 has antiviral effect, which may be an intrinsic part of EBV latency program to assist the establishment and/or maintenance of EBV latency.     

Porcine conceptus proteins: antiviral activity and effect on 2',5'-oligoadenylate synthetase.

Interferon-like proteins synthesized by conceptuses of domestic ruminants inhibit luteolysis during early pregnancy. Although pig conceptuses secrete trophoblast interferons during the period of CL maintenance, estrogen is involved with maintenance of the CL. The principal purposes of this work were to confirm production of trophoblast interferons by porcine conceptuses and to compare the effect of trophoblast interferons on endometrium of pigs and cattle. When measured using Madin-Darby bovine kidney (MDBK) cells challenged with vesicular stomatitis virus, antiviral activity in uterine flushings from cyclic gilts was not detectable throughout the estrous cycle; however, in pregnant gilts, antiviral activity increased from undetectable amounts to 4-11 x 10(3) U on Days 14, 16, and 18. Porcine embryos in culture produced 1,100 U/embryo/ml/24 h. Porcine conceptus secretory proteins induced 2',5'-oligo(A) synthetase in MDBK cells and in endometrial explants of cows but had no measurable effect on 2',5'-oligo(A) synthetase activity of endometrial explants of pigs. Similarly, endometrial 2',5'-oligo(A) synthetase of pregnant pigs was unaffected in vivo during the period of maximal synthesis of conceptus secretory proteins. Porcine conceptus secretory proteins produced no detectable increase in serum antiviral activity or 2',5'-oligo(A) synthetase activity of blood mononuclear leukocytes in utero-ovarian venous blood. These results suggest that conceptus interferons of pigs play different roles in the establishment of pregnancy compared to their roles in ruminants.     

Multiple molecular forms of interferon display different specific activities in the induction of the antiviral state and 2'5' oligoadenylate synthetase

Both Hu IFN-alpha A and Hu IFN-alpha D, produced by two independent recombinant bacterial clones, are mixtures of monomers, dimers and trimers. These forms, when assayed individually in heterologous MDBK cells, induced different degree of antiviral and 2'5' oligoadenylate synthetase (2'5' A synthetase) activities: the antiviral activity of the monomer is greater than that of the dimer and the trimer, whereas the activity of 2'5' A synthetase induction is lower with the monomer than with the dimer or the trimer. Similar differences are also observed on human cells. Compared to the mononeric form, the dimeric and the trimeric forms of Hu IFN-alpha A show higher antiviral inducing activity on heterologous MDBK cells than on homologous WISH cells, whereas the 2'5' A synthetase inducing activity in these two cell lines is about the same. Thus for the same antiviral activity, the trimer or the dimer compared to the monomer are much better inducers of the 2'5' A synthetase on human than on MDBK cells.