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.     

Glycoprotein enrichment in Moloney leukemia virus structural proteins released from interferon-treated cells

Interferon treatment of Moloney-leukemia-virus-infected cells (3T3/MLV) leads to the formation of virus particles enriched with viral structural glycoproteins, in addition to the inhibition of virus production. A preferential inhibitory effect on incorporation of RNA and proteins rather than glycoproteins was found in the released virus particles from interferon-treated cells. Enrichment in 70,000- and 45,000-dalton glycoprotein (gP-70, gP-45) in these particles was further demonstrated by polyacrylamide analysis of viral proteins pulse-labeled with [3H]-leucine. Viral glycoproteins released as soluble antigens were also determined. A 40% reduction was found in gP-70 and gP-45 released from interferon-treated cells. Radioimmunoprecipitation of pulse-chase-labeled cellular viral proteins showed no effect of interferon on the formation of viral structural 30,000-, 15,000- to 12,000-dalton proteins, and gP-70 and gP-45 from their respective precursors. The uncoordinate effect of interferon inhibition on viral 30,000-dalton protein and gP-70 is discussed.     

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.     

Synthesis of type C virus particles from murine-cultured cells induced by iododeoxyuridine. V. Effect of interferon and its interaction with dexamethasone.

Previous studies have shown that in certain cell systems dexamethasone may enhance the production of type C viruses. Conversely, interferon has been shown to inhibit their production. Both appear to exert their influence late in the viral replication cycle rather than on the synthesis of viral-specific RNA. In this report dexamethasone and interferon have been used to study some aspects of the mechanisms involved in the synthesis of type C viruses in murine K-BALB cells following induction of virus production by iododeoxyuridine. Interferon inhibited production of xenotropic type C virus induced by iododeoxyuridine from K-BALB cells both in the absence and presence of dexamethasone, but it did not affect production of N-tropic type C virus. Exposure of the cells to interferon for longer than 12 h was required for maximum effect. Two types of inhibitory effects were observed: one diminished by dexamethasone when the steroid was added 24 h after interferon removal, and the second resistant to dexamethasone. The concentration of intracellular group-specific antigen was diminshed after interferon and increased after dexamethasone exposure. When induced cells were treated with both interferon and dexamethasone, the intracellular group-specific protein concentration was slightly increased, but virus production was reduced 10-fold compared with induced cells treated with dexamethasone alone. We conclude that interferon and dexamethasone may affect both the synthesis of viral proteins and the assembly or release of virus particles and that dexamethasone can partially nullify the inhibitory activity of interferon. The results also support previous conclusions that the regulatory mechanisms for synthesis of viral proteins and for the release of viral particles may differ and that controls for xenotropic and ecotropic virus formation may not be identical.     

Intracellular production of virus particles and viral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect of interferon.

The effect of interferon on the biochemical properties and the maturation process of intracellular viral particles isolated from the cytoplasmic fraction of NIH/3T3 cells chronically infected with Moloney murine leukemia virus was investigated. By labeling these virions with either [35S]methionine or [3H]glucosamine, we demonstrated that they contain the same viral proteins and glycoproteins found in extracellular virions. Interferon treatment was found to reduce the rate of intracellular virus assembly. This effect was not a consequence of an interferon inhibition of viral RNA synthesis or its translation or a consequence of an interference with the posttranslational cleavage processing of viral precursor proteins, since all of these steps were not affected by interferon. However, the reduced rate of virus assembly could be attributed to the inhibition of viral protein glycosylation observed in interferon-treated cells. Nevertheless, despite this reduced rate, virus particles accumulated in interferon-treated cells. This accumulation was probably due to the strong inhibition of their final release from such cells.     

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-alpha (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon-treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.     

Accumulation of cystine auxotrophic thymocytes accompanying type C viral leukemogenesis in the mouse.

Certain continuous lymphoid and myeloid tumor cell lines of rodent origin are unable to grow in tissue culture in the absence of pre-formed L-cystine (CYS). In contrast, three NZB murine lymphoid cell lines obtained from NZB mice free of hematopoietic neoplasm can grow as well in cystine-deficient media containing L-cystathionine (CSN), the immediate precursor of CYS in the biosynthetic pathway, as in cystine sufficient medium. The former class of cells is, therefore, CYS auxotrophs (CYS-) and the latter CYS prototrophs (CYS+). Compared to CYS+ cells, the CYS- lines appear to be relatively deficient in the enzyme cystathionase, which catalyzes the cleavage of CSN to CYS and alpha-ketobutyrate. Using protein synthetic capacity as a criterion, normal thymocytes from mixed-bred Swiss mice behave like CYS prototrophs, while those from littermates bearing Moloney type C virus-induced thymic tumors behave like CYS auxotrophs. The former are also characterized by substantially higher levels of cystathionase than the latter. Extracts of thymocytes from tumor-free AKR mouse thymus are also characterized by higher levels of cystathionase activity than extracts of spontaneous AKR thymomas. Exogenous in vitro type C virus infection of a CYS+ cell results in vigorous virus production but no concomitant reduction in cystathionase activity. Thus viral replication alone in any random lymphoid cell is not sufficient to alter the enzyme level. The data therefore suggests that CYS auxotrophy may closely accompany neoplastic transformation of certain hematopoietic cells in vivo, including that induced by certain "thymic" type C viruses.     

Inhibition of Arbovirus Protein Synthesis by Interferon

Infection of cells treated with guanidine and actinomycin D and then washed to remove the guanidine inhibition of virus growth had no effect on antiviral activity already established by interferon. Protein synthesis in interferon-treated cells infected under these conditions was decreased as compared to control cells similarly treated but not exposed to interferon. In these control cells, analysis by polyacrylamide gel electrophoresis indicated that six proteins were produced during the first hour after guanidine reversal. Five of these proteins have been previously identified as probably being viral in origin. In interferon-treated cells, only a single major protein was produced. Ribonucleic acid (RNA) synthesis by Semliki Forest virus during the first hour after guanidine reversal was markedly depressed by incubation at 42 C, but no inhibition of total virus protein synthesis was seen; this finding suggested that much of the virus protein produced in the first hour after guanidine reversal was carried out by input virus RNA. Interferon was fully active in cells incubated at 42 C. The results suggested that interferon inhibits the production of Semliki Forest virus proteins ordinarily produced under the direction of the virus genome.