Interferon-induced resistance to the killing by NK cells: a preferential effect of IFN-gamma

HeLa cells show a decrease of susceptibility to the killing by natural killer (NK) cells when treated with IFN-alpha, beta, or gamma. The concentrations at which preparations of IFN-alpha or beta induce the resistance to killing are those which also induce resistance of HeLa cells to infection by vesicular stomatitis virus (VSV). Stimulation of the killing activity of NK cells is also induced at that same range of concentrations of IFN-alpha and beta. In contrast with preparations of IFN-gamma, induction of the resistance to killing occurs at IFN concentrations which have only marginal stimulatory effect on the activity of NK cells and have no antiviral effect against VSV. IFN-gamma, produced with cloned IFN-gamma cDNA, is as effective as lymphocyte-produced IFN in inducing the resistance to natural killing. The potent effect of IFN-gamma on the target cells is, therefore, not due to the function of lymphokines which might contaminate lymphocyte-produced preparations of IFN-gamma, but a genuine property of the IFN itself.     

Regulation of the antiviral and anticellular activities of interferon by exogenous double-stranded RNA

The effects of double-stranded RNA (dsRNA) on interferon (IFN)-induced antiviral and anticellular activities was investigated by introducing poly(I)-poly(C) into mouse L-cells. Coprecipitation of dsRNA with calcium phosphate enabled its efficient penetration into cells in culture. Rate of cellular protein synthesis was inhibited by dsRNA only in cultures pretreated with IFN. Moreover, the anticellular effect of IFN, as measured by the inhibition of cell DNA synthesis, was also enhanced by dsRNA. The kinetics of dsRNA-mediated inhibition of protein synthesis were relatively slow as compared with the inhibitory effect of 2'-5' oligoadenylic acid (2'5'A), which was also introduced into cells by the calcium phosphate coprecipitation technique. To analyze the effects of dsRNA on the antiviral state induced by IFN, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMC), replications were followed by measuring viral-specific RNA synthesis in the cell. Introduction of dsRNA after the infection had no effect on VSV and EMC replication in control cells, and it enhanced, to a small extent, the antiviral state of cells pretreated with IFN. In contrast, introduction of 2'5'A into virus-infected cells inhibited VSV and EMC replications regardless of IFN pretreatment. This work demonstrated that the role of dsRNA in regulating the antiviral and anticellular activities of IFN could be studied by introducing exogenous dsRNA into cells in culture by the calcium phosphate coprecipitation technique.     

Purification and some properties of human erythrocyte calpastatin

By means of a new type of microinjection apparatus, which has a micropipette located in a hole through the optical axis of the condenser lens, we injected interferon (IFN) or 2',5'-oligoadenylate (2-5A) into mouse L cells, and observed their antiviral effects on the multiplication of vesicular stomatitis virus (VSV). After injection, cells were infected with VSV, and labeled with [3H]uridine in the presence of actinomycin D. The proportion of cells infected with VSV which carried radioactive virus-RNA was determined by autoradiography. IFN introduced directly into L cells had no effect on the virus growth. This result supports the idea that IFN molecules exert their effect from outside the cell membrane without penetrating into the cytoplasm. 2-5A, on the other hand, was able to inhibit the growth of VSV effectively when injected into L cells. The antiviral effect was dependent on the dose of 2-5A injected, and moreover the effect was transient, since it disappeared completely after 24-h incubation.     

Resistance to Virus Infection Conferred by the Interferon-Induced Promyelocytic Leukemia Protein

The interferon (IFN)-induced promyelocytic leukemia (PML) protein is specifically associated with nuclear bodies (NBs) whose functions are yet unknown. Two of the NB-associated proteins, PML and Sp100, are induced by IFN. Here we show that overexpression of PML and not Sp100 induces resistance to infections by vesicular stomatitis virus (VSV) (a rhabdovirus) and influenza A virus (an orthomyxovirus) but not by encephalomyocarditis virus (a picornavirus). Inhibition of viral multiplication was dependent on both the level of PML expression and the multiplicity of infection and reached 100-fold. PML was shown to interfere with VSV mRNA and protein synthesis. Compared to the IFN mediator MxA protein, PML had less powerful antiviral activity. While nuclear body localization of PML did not seem to be required for the antiviral effect, deletion of the PML coiled-coil domain completely abolished it. Taken together, these results suggest that PML can contribute to the antiviral state induced in IFN-treated cells.     

Negative regulation of the alpha interferon-induced antiviral response by the Ras/Raf/MEK pathway

Interferon (IFN) is one of the molecules released by virus-infected cells, resulting in the establishment of an antiviral state within infected and neighboring cells. IFN-induced antiviral response may be subject to modulation by the cellular signaling environment of host cells which impact the effectiveness of viral replication. Here, we show that cells with an activated Ras/Raf/MEK signaling cascade allow propagation of viruses in the presence of IFN. Ras-transformed (RasV12) and vector control NIH 3T3 cells were infected with vesicular stomatitis virus (VSV) or an IFN-sensitive vaccinia virus (delE3L) in the presence of alpha interferon. While IFN protected vector control cells from infection by both viruses, RasV12 cells were susceptible to viral infection regardless of the presence of IFN. IFN sensitivity was restored in RasV12 cells upon RNA interference (RNAi) knockdown of Ras. We further investigated which elements downstream of Ras are responsible for counteracting IFN-induced antiviral responses. A Ras effector domain mutant that can only stimulate the Raf kinase family of effectors was able to suppress the IFN response and allow VSV replication. IFN-induced antiviral mechanisms were also restored in RasV12 cells by treatment with a MEK inhibitor (U0126 or PD98059). Moreover, by using RNAi to MEK1 and MEK2, we determined that MEK2, rather than MEK1, is responsible for suppression of the IFN response. In conclusion, our results suggest that activation of the Ras/Raf/MEK pathway downregulates IFN-induced antiviral response.     

Use of Getah virus for antiviral assay of human interferon

Antiviral assay is used routinely for measuring the biological activity of interferon (IFN). However, the challenge viruses used in these assays are considered dangerous to the animal industry and pose a risk of human infection. For example, the vesicular stomatitis virus (VSV) is an important exotic disease agent in domestic animals, and the sindbis virus provokes rash, arthralgia, and fever in humans. Therefore, biosafety needs to be considered when antiviral assays are performed. We chose Getah virus as a candidate challenge virus because it is less hazardous to animals and humans. Crystal violet staining 50% CPE inhibition antiviral assay of human IFN using Getah virus was studied. Antiviral assay using Getah virus and FL cells gave a higher titer of human IFN than did assay using VSV. The titer of human IFN alpha was almost the same as that given by standardized control samples. The titer of human IFN by antiviral assay using Getah virus on the sheet method (IFN reacted the sheeted FL cells) was higher than those of the simultaneous reaction method (IFN reacted the suspending FL cells before sheeted). We therefore consider the sheet method useful for detection of small amounts of IFN. Antiviral assay using Getah virus on MDBK cells gave a lower titer of human IFN alpha than did assay using VSV. However, the adjusting the number of MDBK cells and the titer of Getah virus to get the best condition for CPE appearance, gave similar results in the assays using Getah virus and VSV. We consider that Getah virus is a potentially useful challenge virus for antiviral assay of human IFN.     

Adenovirus early region 1A modulation of interferon antiviral activity.

Human adenovirus type 5 (Ad5) is a DNA virus which replicates as efficiently in human A549 cells treated with human interferon-alpha 2 (IFN) as in untreated cells. Vesicular stomatitis virus (VSV), on the other hand, is a negative-strand RNA virus which is very sensitive to the effects of IFN treatment in A549 cells. The IFN-mediated inhibition of VSV replication was not observed in cells coinfected with Ad5. Abrogation of IFN-mediated antiviral activity was maximal when Ad5 infection preceded VSV infection by at least 36 h, but did not require adenovirus DNA synthesis for manifestation. Coinfection experiments with VSV and deletion variants of adenovirus demonstrated that neither virus-associated RNA synthesis nor expression of adenovirus early regions E1B, E2A, E3, or E4 are required for abrogation of IFN-mediated inhibition of VSV replication. However, expression of early region E1A was essential, suggesting that E1A products can modulate, either directly or indirectly, IFN activity in adenovirus-infected cells.     

ISG20, a new interferon-induced RNase specific for single-stranded RNA,defines an alternative antiviral pathway against RNA genomic viruses

Interferons (IFNs) encode a family of secreted proteins that provide the front-line defense against viral infections. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. We have recently isolated a new human IFN-induced gene that we have termed ISG20, which codes for a 3' to 5' exonuclease with specificity for single-stranded RNA and, to a lesser extent, for DNA. In this report, we demonstrate that ISG20 is involved in the antiviral functions of IFN. In the absence of IFN treatment, ISG20-overexpressing HeLa cells showed resistance to infections by vesicular stomatitis virus (VSV), influenza virus, and encephalomyocarditis virus (three RNA genomic viruses) but not to the DNA genomic adenovirus. ISG20 specifically interfered with VSV mRNA synthesis and protein production while leaving the expression of cellular control genes unaffected. No antiviral effect was observed in cells overexpressing a mutated ISG20 protein defective in exonuclease activity, demonstrating that the antiviral effects were due to the exonuclease activity of ISG20. In addition, the inactive mutant ISG20 protein, which is able to inhibit ISG20 exonuclease activity in vitro, significantly reduced the ability of IFN to block VSV development. Taken together, these data suggested that the antiviral activity of IFN against VSV is partly mediated by ISG20. We thus show that, besides RNase L, ISG20 has an antiviral activity, supporting the idea that it might represent a novel antiviral pathway in the mechanism of IFN action.     

Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaling pathway

Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-alpha/beta greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.     

Regulation of macrophage growth and antiviral activity by interferon-gamma

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.     

Interferon Regulatory Factor-1 Protects from Fatal Neurotropic Infection with Vesicular Stomatitis Virus by Specific Inhibition of Viral Replication in Neurons

The innate immune system protects cells against invading viral pathogens by the auto- and paracrine action of type I interferon (IFN). In addition, the interferon regulatory factor (IRF)-1 can induce alternative intrinsic antiviral responses. Although both, type I IFN and IRF-1 mediate their antiviral action by inducing overlapping subsets of IFN stimulated genes, the functional role of this alternative antiviral action of IRF-1 in context of viral infections in vivo remains unknown. Here, we report that IRF-1 is essential to counteract the neuropathology of vesicular stomatitis virus (VSV). IFN- and IRF-1-dependent antiviral responses act sequentially to create a layered antiviral protection program against VSV infections. Upon intranasal infection, VSV is cleared in the presence or absence of IRF-1 in peripheral organs, but IRF-1^−/− mice continue to propagate the virus in the brain and succumb. Although rapid IFN induction leads to a decline in VSV titers early on, viral replication is re-enforced in the brains of IRF-1^−/− mice. While IFN provides short-term protection, IRF-1 is induced with delayed kinetics and controls viral replication at later stages of infection. IRF-1 has no influence on viral entry but inhibits viral replication in neurons and viral spread through the CNS, which leads to fatal inflammatory responses in the CNS. These data support a temporal, non-redundant antiviral function of type I IFN and IRF-1, the latter playing a crucial role in late time points of VSV infection in the brain.     

Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human leukocyte interferon. I. Effect on early and late stages of the viral multiplication cycle

The kinetics of induction in human amnion U cells of the antiviral activity against vesicular stomatitis virus (VSV) produced by a single molecularly cloned subspecies of human leukocyte interferon (IFN-alpha A) were examined. IFN-alpha A-induced inhibition was found to be biphasic over a period of 24 h with the major extent of VSV inhibition occurring within the first 6 h of IFN treatment. The relationship of this major phase of inhibition to the early and late events of the VSV multiplication cycle was investigated. IFN-alpha A treatment had no detectable effect on the adsorption and penetration of VSV virions or on their uncoating to yield viral nucleocapsids. The polypeptides of adsorbed or uncoated VSV particles were neither preferentially degraded nor detectably altered in IFN-treated cells, as compared to untreated cells. Progeny virions released from IFN-treated cells, although greatly reduced in number, were found to be equally as infectious as those released from untreated cells. Progeny virions from IFN-treated cells also had a normal complement of VSV proteins in the same ratios as were seen in virions from untreated cells; specifically, IFN treatment produced no reduction in the incorporation of G or M protein into assembled virions. These results suggest that conditions of IFN treatment sufficient to reduce the yield of infectious VSV progeny greater than 99% do not detectably affect either the early or the late stages of the VSV multiplication cycle.     

Vesicular stomatitis virus (VSV) therapy of tumors

Vesicular stomatitis virus (VSV) is an essentially nonpathogenic negative-stranded RNA virus, the replication of which is extremely sensitive to the antiviral effects of interferon (IFN). We demonstrate here that VSV selectively induces the cytolysis of numerous transformed human cell lines in vitro, with all the morphological characteristics of apoptotic cell death. Importantly, VSV can also potently inhibit the growth of p53-null C6 glioblastoma tumors in vivo without infecting and replicating in normal tissue. With our previous findings demonstrating that primary cells containing the double-stranded RNA-activated protein kinase PKR and a functional IFN system are not permissive to VSV replication, these results suggest that signaling by IFN may be defective in many malignancies. Thus VSV might be useful in novel therapeutic strategies for targeting neoplastic disease.     

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.     

hsp70-Dependent Antiviral Immunity against Cytopathic Neuronal Infection by Vesicular Stomatitis Virus

The major inducible 70-kDa heat shock protein (hsp70) protects against measles virus (MeV) neurovirulence in the mouse that is caused by a cell-associated noncytolytic neuronal infection. Protection is type I interferon (IFN) dependent, and we have established a novel axis of antiviral immunity in which hsp70 is released from virus-infected neurons to induce IFN-β in macrophages. The present work used vesicular stomatitis virus (VSV) to establish the relevance of hsp70-dependent antiviral immunity to fulminant cytopathic neuronal infections. In vitro, hsp70 that was constitutively expressed in mouse neuronal cells caused a modest increase in VSV replication. Infection induced an early extracellular release of hsp70 from viable cells, and the release was progressive, increasing with virus-induced apoptosis and cell lysis. The impact of this VSV-hsp70 interaction on neurovirulence was established in weanling male hsp70 transgenic and nontransgenic mice. Constitutive expression of hsp70 in neurons of transgenic mice enhanced viral clearance from brain and reduced mortality, and it was correlated with enhanced expression of type I IFN mRNA. Nontransgenic mice were also protected against neurovirulence and expressed increased type I IFN mRNA in brain when hsp70 was expressed by a recombinant VSV (rVSV-hsp70), indicating that hsp70 in the virus-infected cell is sufficient for host protection. In vitro data confirmed extracellular release of hsp70 from cells infected with rVSV-hsp70 and also showed that viral replication is not enhanced when hsp70 is expressed in this manner, suggesting that hsp70-mediated protection in vivo is not dependent on stimulatory effects of hsp70 on virus gene expression.     

Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-beta gene. The resultant virus (VSV-IFNbeta) was successfully propagated in cells not receptive to murine IFN-alpha/beta and expressed high levels of functional heterologous IFN-beta. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNbeta was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-beta, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNbeta grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-beta gene (VSV-hIFNbeta) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNbeta was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNbeta retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.     

A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon

Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.     

The target reaction in the antiviral action of mouse interferon against vesicular stomatitis virus multiplication

Treatment of mouse L929 cells with mouse interferon (IFN) lowered the yield of vesicular stomatitis virus (VSV) in a dose-dependent manner. Accumulation of viral proteins was severely inhibited in IFN-treated cells, whereas cellular protein synthesis was not, indicating that the virus-induced shutoff of cellular protein synthesis was prevented by IFN. In order to identify the major target of IFN action precisely, the effect of IFN treatment on the synthesis of viral RNAs and proteins at various stages during the course of viral replication was examined. Accumulation of viral RNAs late in infection was inhibited, as was the case with viral proteins, but the synthesis of leader RNA and mRNAs early in infection was not significantly inhibited by treatment with a moderate dose of IFN. On the other hand, viral protein synthesis at an early stage of infection was strongly inhibited by IFN. The results indicate that the major target reaction of antiviral action of IFN against VSV multiplication is the translation of viral mRNA.