PKR is not required for interferon-gamma inhibition of VSV replication in neurons

In this report, the contribution of PKR to the IFN-gamma mediated inhibition of VSV replication in neurons was examined. IFN-gamma treatment of NB41A3 murine neuroblastoma cells resulted in the reduced expression of VSV protein during infection. PKR was found to be modestly upregulated in NB41A3 cells following IFN-gamma treatment. The phosphorylation state of PKR and its downstream target, eIF2alpha, were unaffected by either IFN-gamma or VSV infection. Inhibition of PKR through the use of 2-aminopurine or the expression of the Influenza A NS1 gene had no effect on the ability of IFN-gamma to inhibit the replication of VSV in vitro. These data indicate that endogenously expressed PKR is not required for the IFN-gamma mediated inhibition of VSV replication in NB41A3 neuroblastoma cells.     

Disruption of IFN-gamma- mediated antiviral activity in neurons: the role of cannabinoids

Interferon-gamma (IFN-gamma) has potent antiviral activity in neurons which is affected by the production of nitric oxide (NO). This study examines the interactions between cannabinoid receptor-1 (CB(1)), IFNgamma-induced pathways, and inhibition of vesicular stomatitis virus (VSV) replication in neuronal cells. CB(1) is abundantly expressed in neurons of the CNS and the NB41A3 neuroblastoma cell line. CB(1) activation of NB41A3 cells by the synthetic cannabinoid, WIN55,212-2, is associated with an inhibition of Ca(2+) mobilization, leading to diminished nitric oxide synthase (NOS)-1 activity and the production of NO, in vitro. This ultimately results in antagonism of IFN-gamma-mediated antiviral activity and enhanced viral replication. Therefore, activation of cells expressing CB(1) by endogenous (or exogenous) ligands may contribute to decreased inflammation and to increased viral replication in neurons and disease in the CNS.     

Mechanisms of cytokine-mediated inhibition of viral replication.

In this report, the role of nitric oxide synthase (NOS) and IL-12 administration in inhibition of vesicular stomatitis virus (VSV) from infected neuroblastoma cells was examined. We previously have shown that cytokine treatment of cells results in the induction of NOS-1, and this is associated with a 2 log inhibition of VSV production. We performed these studies to examine the mechanism by which viral replication is suppressed. Neuroblastoma cells (NB41A3) were treated with either IL-12 or medium and subsequently infected with VSV. Viral protein and mRNA were isolated from these cells, and their levels were measured by Western or Northern blots, respectively. mRNA levels were decreased modestly, but viral proteins were decreased substantially in cells pretreated with IL-12, suggesting that the inhibitory effect of NO is working at the translational level. Cytokine treatment of cells was not associated with oxidative stress. The viral proteins also were nitrosylated. These data suggest that the mechanism of NO inhibition of viral replication occurs through translational interference and posttranslational modifications of viral components.     

Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L: HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes.

Control of viral replication by interferon (IFN) is thought to be principally mediated by the 2',5'-oligoadenylate synthetase (OAS)/RNAse L, double-stranded dependent protein kinase (PKR), and myxovirus resistance protein (Mx) pathways. In this study, we monitored the constitutive and IFN-induced antiviral activity in mouse embryo fibroblasts lines derived from mice with targeted disruption of either PKR or PKR/RNAse L genes. At high multiplicity of infection (moi = 10), the absence of PKR had no effect on replication of vesicular stomatitis virus (VSV) but moderately enhanced encephalomyocarditis virus (EMCV) growth and greatly increased replication of herpes simplex virus-1 (HSV-1). Replication of EMCV, HSV-1, and VSV was modestly higher in PKR-/- RNAse L-/- fibroblasts when compared with control cells. Although the antiviral action of IFN-alpha was unaffected by the absence of PKR, IFN action was significantly impaired in the double knockout cells but was dependent on the stage of the virus cycle. At early stages, it appeared that anti-EMCV and anti-HSV-1 action of IFN-alpha was significantly compromised, although weak residual antiviral activity was seen. The action of IFN-alpha against VSV was specifically compromised at a late stage of virus replication. The results showed that PKR is an important mediator in constitutive resistance against HSV-1 and that RNAse L is also necessary for the full antiviral activity of IFN against a variety of viruses. These results supported the existence of novel pathways aimed toward specific stages of the virus life cycle.     

Absence of PKR attenuates the anti-HSV-1 activity of an adenoviral vector expressing murine IFN-beta.

A study was undertaken to evaluate the efficacy of an adenoviral vector containing the murine interferon-beta (IFN-beta) transgene (Ad:IFN-beta) against herpes simplex virus type 1 (HSV-1) infection in two transduced cell lines. The transduction of the adenoviral vector efficiency, ranging from 2% to 100%, was dependent on the multiplicity of infection (moi) (0.4-50 plaque-forming units [pfu]/cell). Supernatants from cells transduced with the Ad:IFN-beta but not the adenoviral null vector (Ad:Null) contained biologically active IFN-beta (6.6-106 U/ml depending on the moi). Cells transduced with the Ad:IFN-beta displayed up to 25-fold reduction in viral titers compared with cells transduced with the Ad:Null or nontransduced cell controls. The suppression in viral titer correlated with a reduction in viral gene (alpha, beta, and gamma) and protein expression. The expression of IFN beta-responsive genes, including protein kinase R (PKR) and 2',5'-oligoadenylate synthetase (OAS), were significantly elevated in the Ad:IFN-beta-transduced cells by 12-fold and 25-fold, respectively. However, after infection with HSV-1, a transient but significant drop in PKR but not OAS gene expression was observed 10 h postinfection. The absence of PKR but not RNase L significantly attenuated the antiviral efficacy of the transgene. Collectively, these results illustrate the feasibility of employing a viral vector to deliver a potent antiviral gene to targeted cells without any obvious detriment to the vector itself and support an important role for PKR as a mediator of the anti-HSV-1 activity of type I IFN.     

Nitric oxide radical suppresses replication of wild-type dengue 2 viruses in vitro

Nitric oxide is well accepted as one of the defenses for inhibiting viral dissemination. Macrophages and cells in the macrophage lineage are professional nitric oxide producers which sub-serve as target for dengue virus. The interaction between nitric oxide and dengue virus in such target cell is unknown. In this report, the impact of nitric oxide on infectious dengue virus serotype 2 production and RNA replication was investigated in vitro. Primary isolates of dengue virus serotype 2 from dengue patients were replicated in mouse neuroblastoma cells in the presence of an exogenous nitric oxide donor, s-nitroso-N-acethylpennicillamine, SNAP, at the concentration of 50 or 75 or 100 microM. Nitric oxide inhibited viral replication in a dose and a multiplicity of infection dependent manner. Nitric oxide from 50 and 75 microM SNAP delayed and suppressed replication of dengue virus isolates while higher concentration of nitric oxide, 100 microM SNAP, completely inhibited production of infectious particles up to 36 hr study. Twenty-four out of forty tested isolates, 60%, were susceptible to 50 microM SNAP inhibitory effect. The mechanism of inhibition was investigated at the level of RNA synthesis and was found that RNA production was suppressed which correlated to production of the infectious particles. Down-regulation of the RNA synthesis resulted in reduction of protein synthesis which was detected by lower level of NS1 protein synthesis using immunoblotting. In conclusion, nitric oxide from exogenous nitric oxide donor down regulated replication of dengue virus serotype 2 isolates from dengue patients. The suppression was clearly shown at the level of viral RNA and protein synthesis resulting in reduction of viral progenies production. This phenomenon implies that nitric oxide may serve as a defense which diminishes viral load in patients.     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

RNA adenosine deaminase ADAR1 deficiency leads to increased activation of protein kinase PKR and reduced vesicular stomatitis virus growth following interferon treatment

Two size forms of ADAR1 adenosine deaminase are known, one constitutively expressed (p110) and the other interferon (IFN)-induced (p150). To test the role of ADAR1 in viral infection, HeLa cells with ADAR1 stably knocked down and 293 cells overexpressing ADAR1 were utilized. Overexpression of p150 ADAR1 had no significant effect on the yield of vesicular stomatitis virus. Likewise, reduction of p110 and p150 ADAR1 proteins to less than ∼10 to 15% of parental levels (ADAR1-deficient) had no significant effect on VSV growth in the absence of IFN treatment. However, inhibition of virus growth following IFN treatment was ∼1 log₁₀ further reduced compared to ADAR1-sufficient cells. The level of phosphorylated protein kinase PKR was increased in ADAR1-deficient cells compared to ADAR1-sufficient cells following IFN treatment, regardless of viral infection. These results suggest that ADAR1 suppresses activation of PKR and inhibition of VSV growth in response to IFN treatment.     

Evidence That Hepatitis C Virus Resistance to Interferon Is Mediated through Repression of the PKR Protein Kinase by the Nonstructural 5A Protein

Hepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide. While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment. Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV. To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors. Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR. Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Finally, the inhibition of the PKR protein kinase by NS5A is the first described function for this HCV protein.     

The murine double-stranded RNA-dependent protein kinase PKR and the murine 2',5'-oligoadenylate synthetase-dependent RNase L are required for IFN-beta-mediated resistance against herpes simplex virus type 1 in primary trigeminal ganglion culture

A study was undertaken to evaluate the efficacy of an adenoviral construct expressing the murine interferon-beta (IFN-beta) transgene (Ad:IFN-beta) against herpes simplex virus type 1 (HSV-1) infection in a primary trigeminal ganglion (TG) cell culture. The transduction efficiency ranged from 0.2 to 11.0% depending on the multiplicity of infection (m.o.i.) of the adenoviral vector (0.5-50.0). Moreover, neurons were the main target of the adenoviral transduction. TG cultures transduced with Ad:IFN-beta displayed up to a 19-fold reduction in viral titers compared with cells transduced with an Ad:Null or nontransduced TG culture controls. Transduction with Ad:IFN-beta up-regulated two critical antiviral genes, double-stranded RNA-dependent protein kinase R (PKR) and 2',5'-oligoadenylate synthetase (OAS). The absence of PKR or RNase L (downstream effector molecule of OAS) attenuated Ad:IFN-beta efficacy against HSV-1 replication, implicating a critical role for PKR and OAS/RNase systems in the establishment of IFN-induced resistance against HSV-1 in TG cells.     

Involvement of receptor-bound protein methyltransferase PRMT1 in antiviral and antiproliferative effects of type I interferons.

Protein arginine N-methyltransferase (PRMT1) is one of the proteins that bind to the intracytoplasmatic domain of the IFNAR-1 chain of the type I interferon (IFN) receptor system. The attachment is specific and is not seen with PRMT2, another member of this protein family. Antisense PRMT1 cDNA constructs expressed under the early cytomegalovirus (CMV) promoter were transfected into HeLa cells, and stable transformants were selected. Antibodies to PRMT1 were used to identify clones with reduced PRMT1 expression. In such clones, IFN-beta inhibited three to five times less the replication of vesicular stomatitis virus (VSV) than in the original HeLa cells. The antiproliferative effect of IFN-beta was also reduced up to fivefold in the clones with low PRMT1 expression. No difference was seen when IFN-gamma was used alone to inhibit cell growth. The protein methylating enzyme, bound to IFNAR-1, appears to regulate positively the biologic activity of type I IFN.     

Interferon inhibits dengue virus infection by preventing translation of viral RNA through a PKR-independent mechanism.

Previously, we demonstrated that pretreatment of cells with interferon (IFN) alpha + gamma or beta + gamma inhibited dengue virus (DV) replication. In this study, experiments were performed to better define the mechanism by which IFN blocks the accumulation of dengue virus (DV) RNA. Pretreatment of human hepatoma cells with IFN beta + gamma did not significantly alter virus attachment, viral entry, or nucleocapsid penetration into the cytoplasm. The inhibitory effect of IFN was retained even when naked DV RNA was transfected directly into cells, confirming that steps associated with viral entry were not the primary target of IFN action. Biosynthetic labeling experiments revealed that IFN abolished the translation of infectious viral RNA that occurred prior to RNA replication. Subcellular fractionation experiments demonstrated that IFN did not significantly alter the ability of viral RNA to attach to ribosomes. The antiviral effect of IFN appeared independent of the IFN-induced, double-stranded RNA-activated protein kinase (PKR) and RNase L, as genetically deficient PKR- RNase L- cells that were infected by DV retained sensitivity to inhibition by IFN. We conclude that IFN prevents DV infection by inhibiting translation of the infectious viral RNA through a novel, PKR-independent mechanism.     

A bioassay for interferon type I based on inhibition of Sendai virus growth

The expression of type I interferons (IFNs) in eukaryotic cells represents a first line of defense against viral infection. Cells pretreated by IFNs do not support viral replication and are protected from virus-induced cell destruction. A challenge of IFN-pretreated cells with vesicular stomatitis virus (VSV) is frequently used to quantitate this cytokine because, on the one hand, the replication of VSV is highly sensitive to IFNs and, on the other hand, in unprotected cells this virus induces a rapid cytopathic effect that can readily be quantified. However, as VSV may infect humans and is known to cause severe disease in a variety of animal species, this virus must be considered a biohazard. In this paper, we describe a bioassay for bovine IFN using Sendai virus, a paramyxovirus that grows readily in MDBK cells yet is released from these cells in a non-infectious form. The sensitivity and dynamic range of this assay are similar to those of the popular VSV-based IFN assay. We demonstrate that the Sendai-virus-based IFN assay permits rapid quantitation of recombinant bovine type I IFN, and also of native type I IFNs which are present in the supernatants of monocyte-derived macrophages infected with various pathogens. In view of the possible artifacts induced by viruses in samples to be assayed for IFN activity, we evaluated several methods of virus inactivation. Treatment with beta-propiolactone led to virus inactivation without affecting the bioactivity of IFNs as detected in the Sendai-virus-based assay.     

Expression of HCV structural proteins impairs IFN-mediated antiviral response

Hepatitis C virus (HCV), especially the genotype 1, is naturally resistant to the antiviral effects of interferon-alpha (IFN-alpha). Expression of the whole HCV genome and the NS5A protein has been suggested to interfere with the antiviral activity of IFN-alpha. Here we have analyzed the effect of individual or various combinations of HCV proteins on IFN-alpha-mediated antiviral effect against vesicular stomatitis virus (VSV). When the structural proteins (core-E1-E2) of HCV genotype 1 were expressed in human osteosarcoma cells in a tetracycline-regulated manner, partial VSV resistance to IFN-alpha was established. This was seen as an enhancement of both viral protein synthesis and production of infectious virus. Priming of core-E1-E2-expressing cells with low doses of IFN-gamma (10 IU/ml) partially restored the antiviral activity of IFN-alpha. The core (high-level expression) and NS4B protein expression also showed some rescue of VSV replication. In this model cell system NS3A-NS4A complex and NS5A showed no inhibition of IFN-alpha-induced antiviral activity. Our results indicate that the expression of structural proteins of HCV may impair the antiviral activity of IFNs.     

Rescue of SV 40 from interferon-treated heterokaryons

Summary Simian virus 40 (SV 40)-transformed nonpermissive cells express only the early products of SV 40. Heterokaryons formed by fusion of these transformed cells with uninfected permissive cells support the activation of the resident viral genome leading to subsequent viral DNA replication, late protein synthesis and release of progeny virus. Pretreatment of heterokaryon cultures with either mouse or monkey interferon (IFN) before fusion with polyethylene glycol (PEG) produced a dose-dependent inhibition in the appearance of free viral DNA as well as production of infectious virus. The decreased yield of SV 40 in these cultures was similar to the inhibition which was observed in mouse or monkey cells incubated with homologous IFN prior to exogenous infection with SV 40. when IFN was added to the cultures at progressively later times after fusion with PEG, there was less inhibition of virus production. Although there was a comparable decrease in the production of virus by pretreatment with either mouse or monkey IFN, monkey IFN exerted the inhibition for a longer period of time when added after heterokaryon formation. These results demonstrate that IFN treatment applied even after initiation of SV 40 replication can still inhibit virus multiplication.With 1 Figure