Development of the Antiviral State in Response to Interferon

The development of resistance in response to interferon depends on cellular RNA synthesis and probably also on cellular protein synthesis. The evidence for these requirements is reviewed, as well as the proposal that this evidence indicates the existence of a specific response of the cell to interferon, involving the induced synthesis of an antiviral protein. Direct evidence for such an interpretation has not been obtained, and alternative explanations are discussed which do not require quantitative or qualitative differences in the RNA and protein made in cells exposed to interferon. The possible role of the ribosome in the antiviral action of interferon is also discussed.     

Origin and Possible Mode of Action of a Tissue Antagonist of Interferon

WE have presented data¹ supporting the hypothesis proposed by Taylor² and by Friedman and Sonnabend³ that interferon acts through a protein which confers the antiviral state on the cell. We postulated two separate regulatory mechanisms: one governing interferon synthesis and the other the production and action of the antiviral protein in the cell⁴. Our studies on the regulation of the antiviral state led us to the demonstration of a substance in human amniotic and chorionic membranes which, when added to the cell 4–18 h after interferon, decreased the antiviral state⁵. The extraction and purification of this tissue antagonist of interferon (TAI) have been previously described. TAI was resistant to proteases (trypsin, pepsin) and to nucleases (RNAase, DNAase). It was also resistant to heating at 56° C or 75° C for 1 h and was only inactivated at 95° C. Although the biologically active component was not chemically defined, its properties were reminiscent of mucopolysaccharides. It is unknown at present whether the TAI represents a substance or a group of substances.     

INHIBITION OF PROTEIN SYNTHESIS IN NONINFECTED L CELLS BY PARTIALLY PURIFIED INTERFERON PREPARATIONS

Partially purified interferon preparations, obtained from L-cell monolayers infected with Newcastle disease virus (NDV), were shown to inhibit protein synthesis in noninfected L cells. The incorporation of several amino acids-¹⁴C was equally sensitive to the pretreatment of the cells with the interferon preparation. Treatment of L-cell monolayers for 24 hr with 800 units of interferon resulted in a 50% decrease in amino acid incorporation. The degree of inhibition was found to be a function of the interferon concentration and the time of exposure of the cells to the partially purified preparations. No inhibitory effect was detected in medium obtained from noninfected cells and purified in an identical manner. The inhibitory effect was shown to be cell specific in that the partially purified interferon from L cells did not reduce amino acid incorporation in heterospecific cell lines. Heating the interferon preparations at 60°C destroyed their antiviral activity and their ability to inhibit valine-¹⁴C incorporation in L cells.     

ADAR1 Facilitates HIV-1 Replication in Primary CD4+ T Cells

Unlike resting CD4⁺ T cells, activated CD4⁺T cells are highly susceptible to infection of human immunodeficiency virus 1 (HIV-1). HIV-1 infects T cells and macrophages without activating the nucleic acid sensors and the anti-viral type I interferon response. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that displays antiviral activity against several RNA viruses. Mutations in ADAR1 cause the autoimmune disorder Aicardi-Goutieères syndrome (AGS). This disease is characterized by an inappropriate activation of the interferon-stimulated gene response. Here we show that HIV-1 replication, in ADAR1-deficient CD4⁺T lymphocytes from AGS patients, is blocked at the level of protein translation. Furthermore, viral protein synthesis block is accompanied by an activation of interferon-stimulated genes. RNA silencing of ADAR1 in Jurkat cells also inhibited HIV-1 protein synthesis. Our data support that HIV-1 requires ADAR1 for efficient replication in human CD4⁺T cells.     

Viperin is an iron‐sulfur protein that inhibits genome synthesis of tick‐borne encephalitis virus via radical SAM domain activity

Viperin is an interferon‐induced protein with a broad antiviral activity. This evolutionary conserved protein contains a radical S‐adenosyl‐l ‐methionine (SAM) domain which has been shown in vitro to hold a [4Fe‐4S] cluster. We identified tick‐borne encephalitis virus (TBEV) as a novel target for which human viperin inhibits productionof the viral genome RNA. Wt viperin was found to require ER localization for full antiviral activity and to interact with the cytosolic Fe/S protein assembly factor CIAO1. Radiolabelling in vivo revealed incorporation of ⁵⁵Fe, indicative for the presence of an Fe‐S cluster. Mutation of the cysteine residues ligating the Fe‐S cluster in the central radical SAM domain entirely abolished both antiviral activity and incorporation of ⁵⁵Fe. Mutants lacking the extreme C‐terminal W361 did not interact with CIAO1, were not matured, and were antivirally inactive. Moreover, intracellular removal of SAM by ectopic expression of the bacteriophage T3 SAMase abolished antiviral activity. Collectively, our data suggest that viperin requires CIAO1 for [4Fe‐4S] cluster assembly, and acts through an enzymatic, Fe‐S cluster‐ and SAM‐dependent mechanism to inhibit viral RNA synthesis.     

Interferon- and sarcolectin-dependent cellular regulatory interactions

Interferons, via specific membrane-bound receptors, induce various cellular functions of which antiviral protection is the most extensively studied. We have previously reported the existence of interferon antagonists (referred to as sarcolectins) in various tissue extracts from placental blood, cartilage, brain, muscle, or from sarcomas. These sarcolectins have been fully characterized and purified to homogeneity. In interferon-treated cells, they restore virus sensitivity 4-6 h after the establishment of antiviral protection. In the present study we investigate the effect of sarcolectins on the steady state levels of two double-stranded RNA dependent enzymes, 2-5A (p chi (A2'p)nA) synthetase and protein kinase. Several authors have previously emphasized the role of these enzymes in the mechanism of interferon's antiviral action. Interferon promotes a 4-8 fold increase in protein kinase and 2-5A synthetase in cells. Addition of sarcolectin 5 h after interferon results in a dramatic reduction in the steady state levels of both these enzymes, as shown by their decreased activity and yield observed in Western blot assays. The degradation of the antiviral response in sarcolectin-treated cells might therefore be at least partially attributed to a reduced synthesis of protein kinase and 2-5A synthetase. Since there are no direct interactions between sarcolectins and interferon or its receptors, it can be postulated that sarcolectins exert their effect through these interferon-dependent proteins. We postulate that the opposing biological effects of interferon and sarcolectins strike a balance which may, however, be modified in one direction or the other, depending on their respective concentrations.     

Interferon inhibits DNA Synthesis induced in Mouse Lymphocyte Suspensions by Phytohaemagglutinin or by Allogeneic Cells

IN addition to its well known antiviral activity, interferon has recently been shown to inhibit the multiplication of tumour and mammalian cells in cell culture^1–6. We report here the inhibition by interferon of DNA synthesis induced in mouse spleen lymphocytes by the non-viral stimuli phytohaemagglutinin (PHA) and allogeneic lymphocytes. These findings are in accord with our contention that interferon affects cell function and, furthermore, they suggest that by acting on lymphocytes, interferon plays a role in the immunological response of the host.     

Construction of a novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain and enhancement of its antitumor activity

A novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain was constructed with the method of genetic and protein engineering. The fusion protein kept complete antiviral activity with the titer of 10⁸ IU per liter of culture. The EGF-RBD of the fusion protein exhibited competitive binding activity against¹²⁵I-mEGF for mEGF receptors on A431 cells. The fusion protein was shown to be more potent in inhibiting the growth of cultured mouse breast carcinoma cells than interferon γ. Experimental data on mouse B16 malignant melanoma model indicated that the tumor weight of fusion protein-treated group was statistically significantly smaller than that of interferon γ -treated group. The work here provides a necessarily reliable clue for the upcoming clinical employment of a novel class of targeting interferons.     

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.     

Effect of Antilymphocytic Serum on Circulating Interferon in Mice as a Function of the Inducer

MOST investigators concerned with interferon synthesis in vivo have used the experimental procedure described by Baron and Buckler¹, in which circulating interferon is induced by intravenous administration of viruses. When interpreting results, however, it is difficult to know which cells are responsible for circulating interferon synthesis in the animal. Using a radiobiological approach, we have shown that after an intravenous injection of virus, interferon released into the blood stream of mice originates in cell populations of varying radiosensitivities, depending on the virus inoculated². Myxo-virus-induced circulating interferon production is characterized by high radiosensitivity, for serum interferon titres are decreased by more than 90% in C3H/He mice after one total body X-irradiation of 250 r. Moreover, the species specificity of interferon has enabled us to show that circulating interferon induced by Newcastle disease virus (NDV) is of donor type in xenogeneic radiochimaeras, from which we concluded that cells responsible for interferon synthesis with this virus originate from haemopoietic stem cells^3,4. Both granulocytes and lymphocytes fulfil the criteria of very radiosensitive elements derived from haemopoietic stem cells^5,6. We wish to report that myxovirus-induced circulating interferon production is selectively depressed after administration of antilymphocyte serum (ALS).     

Protein synthesis in extracts from interferon-treated Mengo virus infected cells

We previously showed in intact L cells that interferon treatment did not modify the shut-off of cellular RNA and protein synthesis induced by infection with Mengo virus although viral replication is inhibited (1,2). We have also demonstrated that inhibition of host protein synthesis was not due to degradation of messengers since cellular mRNA could be extracted from interferon-treated infected cells and efficiently translated in a reticulocyte lysate(2). Cellular mRNA was not degraded although 2–5A was present as reported here. We prepared cell-free systems from such cells at a time when cellular shut-off was fully established. The undegraded messengers remained untranslated under cell-free protein synthesis conditions and almost no polysomes were detected. The decreased amount of [³⁵S]Met-tRNA-40S complex observed in these lysates might account for the inhibition of protein synthesis at the level of initiation.     

Cytotoxicity of pokeweed antiviral protein

Pokeweed antiviral protein, a plant protein which inactivates eukaryotic ribosomes, was found to be cytotoxic to both HeLa and Vero cells. Cellular protein synthesis was inhibited by exposure of the cells to microM concentrations of the antiviral protein for 24 h periods or longer. The extent of the inhibition of cellular protein synthesis was dependent upon the time of exposure to pokeweed antiviral protein and was partially reversed by washing the cells at various times prior to the measurement of protein synthesis. The antiviral protein was also observed to bind nonspecifically to cells at both 4 degrees and 34 degrees C. The data indicate that the pokeweed antiviral protein is capable of slowly entering mammalian cells which results in the inhibition cellular protein synthesis.     

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.     

Herpes simplex virus triggers and then disarms a host antiviral response

Virus infection induces an antiviral response that is predominantly associated with the synthesis and secretion of soluble interferon. Here, we report that herpes simplex virus type 1 virions induce an interferon-independent antiviral state in human embryonic lung cells that prevents plaquing of a variety of viruses. Microarray analysis of 19,000 human expressed sequence tags revealed induction of a limited set of host genes, the majority of which are also induced by interferon. Genes implicated in controlling the intracellular spread of virus and eliminating virally infected cells were among those induced. Induction of the cellular response occurred in the absence of de novo cellular protein synthesis and required viral penetration. In addition, this response was only seen when viral gene expression was inhibited, suggesting that a newly synthesized viral protein(s) may function as an inhibitor of this response.     

Differentiation of the immunosuppressive and antiviral effects of interferon.

Virus-induced (virus-type) interferon suppression of the in vitro antibody response of mouse (C57B1/6) spleen cells to sheep red blood cells was blocked by 5 × 10^?5M 2-mercaptoethanol (2-ME). The blockade was not due to a direct effect on interferon since 2-ME was capable of blocking the suppression when added to cultures up to 48 hr after interferon. 2-ME blockade of virus-type interferon immunosuppression was not due to the immunoenhancing property of 2-ME. Similar protective effects of 2-ME were observed during immunosuppression by virus-type interferon inducers, but not T-cell mitogen inducers of interferon (immune interferon). The data suggest that the immunosuppressive properties of virus-type and immune interferon preparations involve different mechanisms. Virus-type interferon inhibited DNA synthesis in unstimulated spleen cell cultures and in 2-ME stimulated cultures, and the degree of inhibition of DNA synthesis appeared to be related to the immunosuppressive property of interferon in the absence or presence of 2-ME. 2-ME did not affect the antiviral properties of either virus-type or immune interferon in nonlymphoid cells. Further, the induction of virustype interferon in spleen cells was neither inhibited nor enhanced by 2-ME, while the induction of immune interferon was enhanced. This enhancement is consistent with 2-ME enhancement of the immunosuppressive effects of immune interferon inducers.There are two possibilities for 2-ME blockade of the immunosuppressive effect of virus-type interferon, while not affecting the antiviral property. Firstly, the immunosuppressive and antiviral properties of virus-type interferon may involve different mechanisms at the subcellular level. Secondly, the selectivity of the blockade by 2-ME could be due to the fact that spleen cells are the target cells in immunosuppression, while L cells are the target cells in inhibition of virus replication. Thus, virus-type interferon may suppress the immune response at the level of the macrophage and 2-ME may reverse this effect by replacing a blocked macrophage function.     

Synthesis of two non-phosphorylated proteins induced in mouse L-cells by homologous interferon

We have shown that two proteins P1 and P2 of M_r 43000 and 40800 are always detected by two-dimensional gel electrophoresis of interferon-treated mouse L-929 cell extract. These two proteins have an isoelectric point of pH 4.6 and pH 4.7 respectively. If Pl is detectable in small amount in the control gels, P2 is completely absent. Actinomycin D added at the same time as interferon, prevents both P1 and P2 synthesis, but enhances their production when added between 4 to 6 h after interferon. Using molecular weight and isoelectric point as criteria, we have tried to compare P1 and P2 to enzymes induced by interferon. With double-labelled two-dimensional gels by |³⁵S| methionine and |γ³²P| ATP, we have shown that neither P1 nor P2 is phosphorylated. This experimental procedure has allowed us to obtain new data on substrates phosphorylared by interferon induced protein kinase.     

NS3 of Bluetongue Virus Interferes with the Induction of Type I Interferon

Upon infection with Bluetongue virus (BTV), an arthropod-borne virus, type I interferon (IFN-I) is produced in vivo and in vitro. IFN-I is essential for the establishment of an antiviral cellular response, and most if not all viruses have elaborated strategies to counteract its action. In this study, we assessed the ability of BTV to interfere with IFN-I synthesis and identified the nonstructural viral protein NS3 as an antagonist of the IFN-I system.