Studies on the Mechanism of Interferon Action

Interferon does not inactivate viruses or viral RNA. Virus growth is inhibited in interferon-treated cells, but apart from conferring resistance to virus growth, no other effect of interferon on cells has been definitely shown to take place. Interferon binds to cells even in the cold, but a period of incubation at 37°C is required for development of antiviral activity. Cytoplasmic uptake of interferon has not been unequivocally demonstrated. Studies with antimetabolites indicate that the antiviral action of interferon requires host RNA and protein synthesis. Experiments with 2-mercapto-1(β-4-pyridethyl) benzimidazole (MPB) suggest that an additional step is required between the binding and the synthesis of macromolecules. Interferon does not affect the adsorption, penetration, or uncoating of RNA or DNA viruses, but viral RNA synthesis is inhibited in cells infected with RNA viruses. The main action of interferon appears to be the inhibition of the translation of virus genetic information probably by inhibiting the initiation of virus protein synthesis.     

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.     

Classification of interferons with antibody to immune interferon

Mouse immune Interferon, induced by the T-cell mitogen staphylococcal enterotoxin A (SEA), was partially purified and used to immunize rabbits. The resulting antiserum neutralized all immune interferon preparations tested, including interferon induced in vitro by SEA, concanavalin A, phytohemagglutinin P, and pokeweed mitogen, and in mixed lymphocyte cultures. Interferon produced in vivo with specific antigen was also neutralized. The antiserum was equally potent against all these interferon preparations. The serum did not neutralize any virus-type interferon preparation tested, but immune interferon induced by SEA in athymic nude mouse spleen cells was neutralized. The neutralizing activity was precipitable by 33% ammonium sulfate, and was not removed by absorption of the serum with mouse cells. The data suggest that immune interferons produced under diverse conditions are antigenically the same or closely related.     

Mechanism of Antiviral Activity <Emphasis Type="Italic">in vivo</Emphasis> of Polycarboxylates which induce Interferon Production

SYNTHETIC polycarboxylates have been reported to impart resistance to viral infection to experimental animals^1–8. Injection of these polyanions induces interferon^1–3,5–9, to which it therefore seemed logical to attribute the antiviral effect. The high degree and long duration of protection, however, are not in accord with the low and transitory levels of interferon induced, suggesting that mechanisms other than interferon are involved. Certain polyanions have been found directly to inactivate virus or to inhibit its adsorption to cells¹⁰in vitro. This may delay the development of viral infection in vivo. Stimulation of reticuloendothelial cell activity, as demonstrated by increased phagocytosis induced by pyran copolymer¹¹, may deviate virus from its target cells.     

Suppression of in vitro antibody response by ribosome-associated factor(s) from interferon-treated cells.

Initiation factor preparation (eIF-IF) from mouse L cells treated with virus-type interferon suppressed the in vitro plaque-forming cell (PFC) response to sheep red blood cells. The eIF-IF preparation had previously been shown to block formation of the ternary complex Met-tRNA_f-eIF-GTP. The formation of this complex is a necessary step in initiation of protein synthesis. Initiation factor preparation (eIF) from untreated L cells affected neither the PFC response nor the participation of eIF in the formation of ternary complex. The induced factor was shown not to be Interferon by antibody neutralization experiments with anti-interferon. The factor must be present in the PFC cultures during the early stages of antigen induction in order to suppress the immune response. Speculatively, eIF-IF may act at the level of the macrophage, perhaps entering the cell by pinocytosis. This may account for its inability to inhibit virus replication in L cells. The production of the inhibitory factor is blocked or partially blocked by actinomycin D. It is possible that this factor is a mediator of the immunosuppressive effects of virus-type interferon. This is the first report of biological activity on cells, which is associated with a ribosome-associated factor induced by interferon.     

Influenza virus-induced interferon production in mouse spleen cell culture: T cells as the main producer.

Interferon production by spleen cells from unimmunized C3H mice challenged in vitro with influenza virus AO/PR8 was investigated. Glass-nonadherent cells (lymphocytes) produced significant levels of interferon, although cocultivation of glass-adherent macrophages was needed for optimal production. Treatment of the cells with antithymocyte serum and complement markedly reduced the interferon production. When glass-nonadherent cells were fractionated on a nylon wool column, the T-cell-enriched fraction consistently produced more interferon than the B-cell-enriched fraction. It is concluded that T cells are an important producer of interferon in spleen cell cultures from normal mice upon challenge with influenza virus, although non-T cells (macrophages and B cells) also may produce interferon under suitable conditions.     

How does interferon inhibit tumour growth?

Interferon can inhibit tumour growth in experimental animals and in some patients with benign and malignant tumours. There is experimental evidence to suggest that several mechanisms may be involved: a direct effect on the tumor or an indirect effect via the host, or both. Thus, interferon may slow the rate of tumour cell multiplication and this may lead to cell death. Interferon may induce changes in the cell surface rendering tumour cells more sensitive to host defence mechanisms. Interferon may induce reversion in the phenotype of tumour cells. Interferon may stimulate specific and non-specific humoral and cellular host mechanisms. The relative importance of these different effects of interferon may vary depending on the host and the particular tumour.     

(2′–5′)An-dependent RNase: Enzyme levels are decreased in barrier-reared control and IFN-β or IFN-γ treated Balb/c mice

(2′–5′)A_n-dependent RNase functions as a translational regulatory protein which mediates interferon action. Levels of this enzyme are decreased in barrier-reared Balb/c (+/+), Balb/c (+/nu), and Balb/c (nu/nu) mice when compared to conventionally reared Balb/c (+/+) mice. This suggests that high levels of (2′–5′)A_n-dependent RNase in conventionally reared mice are maintained by continuous exposure to microbial flora which may induce interferons. Interferon treatment of barrier-reared mice does not, however, result in an increase in (2′–5′)A_n-dependent RNase levels. This suggests that responsiveness to interferons is decreased in barrier-reared mice. The high levels of (2′–5′)A_n-dependent RNase which are maintained in normal mice under physiological conditions may be important for rapid and effective defense against viral pathogens.     

Interferon induced in mouse spleen cells by Staphylococcus aureus

Interferon was produced in suspensions of mouse spleen cells treated with Staphylococcus aureus preparations (killed bacteria, culture supernatants, or purified enterotoxin) under a variety of cell culture conditions. The lysate of S. aureus was found to induce high levels of interferon (10^3.1 to 10^4.3 RU/ml) within 72 hr. The crude interferon was concentrated and partially purified by either ammonium sulfate precipitation or adsorption to silicic acid and elution by ethylene glycol-containing buffer. Sequential precipitation with 50 to 80% saturated ammonium sulfate resulted in a three- to seven-fold purification with 60% recovery of activity. Adsorption to silicic acid resulted in a 25- to 80-fold purification with 77% recovery. This material was further analyzed by gel filtration. The antiviral activity induced by S. aureus-treated spleen cells was characterized as due to interferon. Furthermore, the inhibitor was acidlabile and not neutralizable by antiserum against NDV-induced L-cell interferon, thus exhibiting properties of immune (γ) interferon. The partially purified interferon was used to prepare an antiserum in rabbits. This antiserum was able to neutralize mouse interferon induced by several T-cell mitogens, by antigens, and by mixed lymphocyte cultures, while remaining inactive against interferons induced in vitro by viruses or in vivo by Brucella abortus.     

The Antiviral Activities of ISG15

Post-translational protein modification is an important strategy for the regulation of the cell proteome independent of the need for new gene expression. Ubiquitin and ubiquitin-like modifiers mediate the regulation of protein levels, signaling pathways, vesicular trafficking, and many other cellular processes through their covalent conjugation to proteins. Interferon stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I interferon. In addition to conjugating to potentially hundreds of target proteins, ISG15 can be found in an unconjugated form both inside of the cell and released from interferon stimulated cells into the extracellular environment. Due to its robust expression after type I interferon stimulation and the broad panel of proteins that it targets, ISG15 has drawn much attention as a potential regulator of the immune response and has been shown to mediate protection in a number of different viral infection models. Here we will review the current state of the field of ISG15, the viruses against which ISG15 mediates protection, and the mechanisms by which ISG15 exerts antiviral activity.     

Effects of NV gene knock-out recombinant viral hemorrhagic septicemia virus (VHSV) on Mx gene expression in Epithelioma papulosum cyprini (EPC) cells and olive flounder (Paralichthys olivaceus)

To determine whether the NV gene of viral hemorrhagic septicemia virus (VHSV) is related to the type I interferon response of hosts, expression of Mx gene in Epithelioma papulosum cyprini (EPC) cells and in olive flounder (Paralichthys olivaceus) in response to infection with either wild-type VHSV or recombinant VHSVs (rVHSV-ΔNV-EGFP and rVHSV-wild) was investigated. A reporter vector was constructed for measuring Mx gene expression using olive flounder Mx promoter, in which the reporter Metridia luciferase was designed to be excreted to culture medium to facilitate measurement. The highest increase of luciferase activity was detected from supernatant of cells infected with rVHSV-ΔNV-EGFP. In contrast cells infected with wild-type VHSV showed a slight increase of the luciferase activity. Interestingly, cells infected with rVHSV-wild that has artificially changed nucleotides just before and after the NV gene ORF, also showed highly increased luciferase activity, but the increased amplitude was lower than that by rVHSV-ΔNV-EGFP. These results strongly suggest that the NV protein of VHSV plays an important role in suppressing interferon response in host cells, which provides a condition for the viruses to efficiently proliferate in host cells. In an in vivo experiment, the Mx gene expression in olive flounder challenged with the rVHSV-ΔNV-EGFP was clearly higher than fish challenged with rVHSV-wild or wild-type VHSV, suggesting that lacking of the NV gene in the genome of rVHSV-ΔNV-EGFP brought to strong interferon response that subsequently inhibit viral replication in fish.     

Action of Interferon: Kinetics and Differential Effects on Viral Functions

A highly purified rabbit interferon was tested for its capacity to inhibit various manifestations of infection of primary rabbit kidney (RK) cells with vesicular stomatitis (VS) virus. A kinetic analysis of the actinomycin-sensitive phase of interferon-induced cellular resistance revealed that RK cells could transcribe virtually all of the hypothetical antiviral messenger ribonucleic acid (mRNA) within 3 hr. Similar exposure to interferon reduced virus yield by 95 to 99% and markedly inhibited cytopathic effect on RK cells infected at a multiplicity of 10 or less. Interferon was less effective in blocking cytopathic effects when RK cells were infected at a multiplicity of 100. However, RK cells pretreated with the same amount of interferon and infected at a multiplicity of 100 failed to incorporate (3)H-amino acids into structural or nonstructural proteins of VS virus identified by polyacrylamide gel electrophoresis. Despite this inhibition of viral protein synthesis, interferon did not prevent the switch off by VS virus of cellular protein synthesis. The rapidity with which a high multiplicity of VS virus switched off cellular protein synthesis, even in cells rendered resistant to viral infection by interferon, is further evidence that this reaction is caused by an infecting virion component rather than by a newly synthesized viral product.     

Microassay for interferon, using [3H]uridine, microculture plates, and a multiple automated sample harvester.

A microassay for interferon is described which uses target cells grown in microculture wells, [3H]uridine to measure vesicular stomatitis virus replication in target cells, and a multiple automated sample harvester to collect the radioactively labeled viral ribonucleic acid onto glass fiber filter disks. The disks were placed in minivials, and radioactivity was counted in a liquid scintillation spectrophotometer. Interferon activity was calculated as the reciprocal of the highest titer which inhibited the incorporation of [3H]uridine into viral ribonucleic acid by 50%. Interferon titers determined by the microassay were similar to the plaque reduction assay when 100 plaque-forming units of challenge vesicular stomatitis virus was used. However, it was found that the interferon titers decreased approximately 2-fold for each 10-fold increase in the concentration of challenge vesicular stomatitis virus when tested in the range of 10(2) to 10(5) plaque-forming units. Interferon titers determined by the microassay show a high degree of repeatability, and the assay can be used to measure small and large numbers of interferon samples.     

The Pokeweed Antiviral Protein Specifically Inhibits Ty1-Directed +1 Ribosomal Frameshifting and Retrotransposition in Saccharomyces cerevisiae

Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the α-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed −1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M₁-dependent killer virus phenotype, suggesting that −1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.     

Time and dosage dependence of immunoenhancement by murine type II interferon preparations.

Preparations of Type II immune induced Interferon enhanced the plaque forming cell response of mice to sheep red blood cells both in vivo and in vitro. The enhancement of the antibody response was dependent on the dosage of interferon used and the time of administration of interferon. The expression of the antiviral and immuno-enhancing activities of Type II interferon preparations shared several physical-chemical properties, including pH 2 lability and heat stability. The plaque forming cell response to lipopolysaccharide, a T-independent antigen, could not be enhanced by treatment with Type II interferon. In addition, treatment of spleen cell cultures of nude thymic deficient mice with Type II interferon could not cause an enhancement of the plaque forming cell response to lipopolysaccharide. These data suggest that the immunoenhancing effect of Type II interferon on antibody responses is produced by an effect on T lymphocytes in contrast with the immunosuppressive effect which appears to be mediated through an effect on B lymphocytes.     

Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Purification and Characterization of Mouse L Cell Interferon

Interferon was produced in high yields in mouse L cell cultures infected with Newcastle disease virus, with a specific activity of the order of 10⁶ units per mg protein. It was partially purified by zinc acetate precipitation, carboxymethyl Sephadex chromatography, Sephadex gel filtration and pressure dialysis. On electrophoresis in polyacrylamide gel, it consisted of a fast-moving sharp component and a slow, broadly distributed component(s). The highest specific activity of the former component so far obtained was 8 × 10⁷ units per mg protein, numerically the highest value ever reported for interferon. It was considered likely, however, that the protein components in the purified samples, revealed by staining of the electrophoresis gel, still represented mostly impurities. Gel filtration experiment indicated some heterogeneity of interferon in molecular weight but the major component was estimated to be 30 000 in molecular weight. Interferon activity could be maintained without added preservatives for prolonged periods, provided that the protein concentration of the sample itself was high. One interferon unit as defined in this paper was found to correspond to 0.3 international unit.     

Temperature-Sensitive Mutants of Vesicular Stomatitis Virus: Synthesis of Virus-Specific Proteins

Viral proteins synthesized in L cells infected with temperature-sensitive (ts) mutants of vesicular stomatitis (VS) virus at permissive (31 C) and nonpermissive (39 C) temperatures were compared by polyacrylamide gel electrophoresis. Mutant ts 5, deficient in synthesis of viral ribonucleic acid (RNA⁻), failed to synthesize any of the five identifiable viral proteins at 39 C. Each of three RNA⁺ mutants, representing three separate complementation groups, showed distinctive patterns of viral protein synthesis at nonpermissive temperature. Equivalent amounts of ³H-amino acids were incorporated into the five viral proteins made in cells infected with RNA⁺ mutant ts 45 at 31 and 39 C. Complete virions of ts 45 could be identified by electron microscopy of infected cells incubated at the nonpermissive temperature; the defect in ts 45 appeared to be due in part to greater thermolability of virions as compared with the wild-type. RNA⁺ mutant ts 23 was deficient in synthesis of viral envelope protein S and failed to make detectable virions at the nonpermissive temperature. Infection of cells at 39 C with the third RNA⁺ mutant, ts 52, resulted in synthesis of all five viral proteins, but the peak of radioactivity representing the viral membrane glycoprotein migrated more rapidly on gels than coelectrophoresed authentic virion ¹⁴C-glycoprotein or viral ³H-glycoprotein extracted from cells infected at 31 C. These data and results of experiments on incorporation of radioactive glucosamine suggest that the primary defect in mutant ts 52 at nonpermissive temperature is failure of glycosylation of the viral glycoprotein. The viral structural proteins made in cells infected with ts 52 at the nonpermissive temperature did not assemble into sedimentable components as they did at permissive temperature; this observation indicates failure of insertion of the nonglycosylated protein (G′) into cell membrane. In support of this hypothesis was the finding that antiviral-antiferritin hybrid antibody did not detect VS viral antigen on the plasma membrane of L cells infected at 39 C with ts 52. In contrast, VS viral antigen localized in plasma membrane of L cells infected at 39 C with mutants ts 23 and ts 45 was readily detected by electron microscopy and fluorescence microscopy.