VSV replication in neurons is inhibited by type I IFN at multiple stages of infection

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.     

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.     

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.     

Protein kinase PKR catalytic activity is required for the PKR-dependent activation of mitogen-activated protein kinases and amplification of interferon beta induction following virus infection

The protein kinase regulated by RNA (PKR) enhances both activation of mitogen-activated protein kinases and the induction of interferon beta (IFN-β) by measles virus defective in C-protein expression (C^ko). Here we used complementation of human cell lines stably deficient in PKR (PKR^kd) to probe the basis of these PKR-mediated responses. We found that PKR^kd HeLa and amnion U cell lines were defective for virus-mediated activation of IFN induction signaling components compared to PKR-sufficient control cells. Complementation of PKR^kd cells with wildtype PKR, but not with PKR mutants defective in either catalytic activity or dsRNA-binding activity, restored JNK, p38 and ATF-2 phosphorylation and enhanced IFN-β induction following infection. By contrast to mammalian PKR, the Z-DNA binding domain-containing fish homologue of PKR, PKZ, lacked the capacity to enhance C^ko virus-mediated IFN-β induction. Furthermore, inhibition of virus growth was observed with C^ko-infected PKR^kd cells complemented with PKR but not with PKZ.     

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.     

Protein kinase PKR and RNA adenosine deaminase ADAR1: new roles for old players as modulators of the interferon response

Double-stranded RNA (dsRNA) plays a centrally important role in antiviral innate immunity, both for the production of interferon (IFN) and also in the actions of IFN. Among the IFN-inducible gene products are the protein kinase regulated by RNA (PKR) and the adenosine deaminase acting on RNA 1 (ADAR1). PKR is an established key player in the antiviral actions of IFN, through dsRNA-dependent activation and subsequent phosphorylation of protein synthesis initiation factor eIF2α thereby altering the translational pattern in cells. In addition, PKR plays an important role as a positive effector that amplifies the production of IFN. ADAR1 catalyzes the deamination of adenosine (A) in RNA with double-stranded (ds) character, leading to the destabilization of RNA duplex structures and genetic recoding. By contrast to the antiviral and proapoptotic functions associated with PKR, the actions of ADAR1 in some instances are proviral and cell protective as ADAR1 functions as a suppressor of dsRNA-mediated antiviral responses including activation of PKR and interferon regulatory factor 3.     

Hepatitis C virus: The role of molecular mimicry in response to interferon treatment

Chronic hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. In order for HCV to persist, the virus must escape immune recognition or inhibit the host immune response. The NS5A protein contains the interferon sensitivity‐determining region (ISDR) and is able to repress dsRNA‐dependent protein kinase (PKR) thus influencing the response to interferon (IFN) therapy. Patients who respond to IFN therapy have stronger antibody reactivity against the NS5A compared to IFN non‐responders. Therefore, given the possible role for the ISDR in IFN resistance and differential antibody reactivity, it is possible that variation in ISDR may be involved in viral immune escape and development of persistent HCV infection employing aspects of host mimicry. In this study, pre‐treatment samples obtained from HCV infected patients were used to investigate the effect of different NS5A ISDR variants on the IFN antiviral response and their involvement in immune evasion. The NS5A was identified as a homologue of the variable region of immunoglobulins (Ig). The IFN resistant genotypes had higher levels of similarity to Ig compared to IFN sensitive genotypes. Expression of NS5A‐6003 (HCV genotype 1b) and NS5A‐6074 (HCV genotype 2a) was able to rescue vesicular stomatitis virus (VSV) from IFN inhibition and restore luciferase activity. A correlation between Ig‐like NS5A structure and also antibody response with the outcome of IFN treatment was observed. J. Med. Virol. 84:1571–1585, 2012. © 2012 Wiley Periodicals, Inc.     

Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection

The double-stranded (ds) RNA-dependent protein kinase PKR is considered to play an important role in interferon's (IFN's) response to viral infection. Here, we demonstrate that mice lacking PKR are predisposed to lethal intranasal infection by the usually innocuous vesicular stomatitis virus, and also display increased susceptibility to influenza virus infection. Our data indicate that in normal cells, PKR primarily prevents virus replication by inhibiting the translation of viral mRNAs through phosphorylation of eIF2alpha, while concomitantly assisting in the production of autocrine IFN and the establishment of an antiviral state. These results show that PKR is an essential component of innate immunity that acts early in host defense prior to the onset of IFN counteraction and the acquired immune response.     

Multiple levels of PKR inhibition during HIV-1 replication

Recent therapeutic approaches against HIV-1 include IFN in combination therapy for patients with coinfections or as an alternative strategy against the virus. These treatment options require a better understanding of the weak efficacy of the IFN-stimulated genes, such as the protein kinase RNA-activated (PKR), which results in viral progression. Activated PKR has a strong antiviral activity on HIV-1 expression and production in cell culture. However, PKR is not activated upon HIV-1 infection when the virus reaches high levels of replication, due to viral and cellular controls. PKR is activated by low levels of the HIV-1 trans-activation response (TAR) RNA element, but is inhibited by high levels of this double-stranded RNA. The viral Tat protein also counteracts PKR activation by several mechanisms. In addition, HIV-1 replicates only in cells that have a high level of the TAR RNA binding protein (TRBP), a strong inhibitor of PKR activation. Furthermore, increased levels of adenosine deaminase acting on RNA (ADAR1) are observed when HIV-1 replicates at high levels and the protein binds to PKR and inhibits its activation. Finally, the PKR activator (PACT) also binds to PKR during HIV-1 replication with no subsequent kinase activation. The combination of all the inhibiting pathways that prevent PKR phosphorylation contributes to a high HIV-1 production in permissive cells. Enhancing PKR activation by counteracting its inhibitory partners could establish an increased innate immune antiviral pathway against HIV-1 and could enhance the efficacy of the IFN treatment.     

Identification by electron microscopy of the maturation steps in vaccinia virus morphogenesis inhibited by the interferon-induced enzymes, protein kinase (PKR), 2-5A synthetase, and nitric oxide synthase (iNOS).

Interferons (IFN) play a major role as a first-line host defense mechanism against viral infections. As treatment of animal cells with IFN induces a large number of genes, it has been difficult to assign the role of these genes in the antiviral action of IFN. Vaccinia virus (VV) is an ideally suited system to study IFN action because all steps in viral morphogenesis can be followed easily by electron microscopy (EM) of ultrathin sections from infected cells. To define the role of IFN-induced genes in viral morphogenesis, we have independently expressed from VV recombinants in primary chicken embryo fibroblast (CEF) cells each of the three IFN-induced genes encoding protein kinase (PKR), 2-5A synthetase, and inducible nitric oxide synthase (iNOS). By EM analysis, we have identified the steps in VV morphogenesis that are affected by each of the IFN-induced enzymes in comparison with untreated and IFN-treated cells. We found that in cells pretreated with IFN and infected with VV, immature virus (IV) is formed, but further stages of maturation are blocked. In cells infected with a VV recombinant expressing PKR (VV-PKR), there is severe inhibition on virus factories, and only few IV are formed. In cells infected with a VV recombinant expressing 2-5A synthetase (VV-2-5A), VV assembly is inhibited at or after IV formation. In cells infected with a VV recombinant expressing iNOS (VV-iNOS), all stages in VV morphogenesis are observed but with aberrant forms. In addition to the effects on viral assembly, in cells infected with either VV-PKR, VV-2-5AS, or VV-iNOS, there is nucleus condensation characteristic of apoptosis. Our findings have identified the steps in VV morphogenesis inhibited by PKR, 2-5A, and iNOS, provided a distinction between these effects, and highlighted a functional redundancy of the IFN system to block viral infection and to induce apoptosis.     

B‐cell‐activating factor expressions in salivary epithelial cells after dsRNA virus infection depends on RNA‐activated protein kinase activation

B‐cell‐activating factor (BAFF) plays a key role in promoting activation of autoimmune B cells. This cytokine may be expressed in and secreted by salivary gland epithelial cells (SGEC) after stimulation with type I IFN or viral or synthetic dsRNA. Because this BAFF expression depends only in part on endosomal TLR and type I IFN, we investigated whether other dsRNA sensors could be implicated in BAFF expression. Using human SGEC, we confirmed the partial dependence of BAFF expression on TLR‐3 by replicating the partial inhibition of BAFF expression observed upon endosomal inhibition using TLR‐3 or Toll/IL‐1R domain‐containing protein inducing IFN‐β silencing mRNA, but not with TLR‐7 silencing mRNA. Melanoma differentiation‐associated gene 5 silencing mRNA had no effect on BAFF expression, but retinoic acid‐inducible gene I silencing mRNA had a slight effect observed following infection with dsRNA reovirus‐1. Inhibition of RNA‐activated protein kinase (PKR) by 2‐aminopurine completely abolished both BAFF mRNA and protein production after reovirus‐1 infection and poly(I:C) stimulation through NF‐κB and p38 MAPK pathways, with the latter implicated only after poly(I:C) stimulation. Thus, PKR is the dsRNA sensor implicated in BAFF induction in SGEC after dsRNA stimulation. In autoimmune diseases, PKR may be an interesting target for preventing BAFF following the induction of innate immunity.     

Hepatitis C virus IRES-dependent translation is insensitive to an eIF2alpha-independent mechanism of inhibition by interferon in hepatocyte cell lines

Interferon (IFN) in combination with ribavirin is the main treatment for hepatitis C virus (HCV) infection. The sensitivity or resistance of the virus to IFN has been linked to certain types of the interferon sensitivity determining region (ISDR) and PKR-eIF2alpha phosphorylation homology domain (PePHD) sequences in the NS5A and E2 regions of the viral genome, respectively. In search of the other potential mechanisms of HCV resistance to IFN, we tested the effect of IFN-alpha on translational activity of the HCV IRES in various cell types. Using bicistronic dual luciferase reporter RNAs in direct RNA transfection studies, we found that the cap-dependent translation was dramatically inhibited by IFN (5- to 16-fold), whereas HCV IRES translation was inhibited only marginally in two hepatoma cell lines, Huh7 and HepG2 cells. No difference in IFN sensitivity was observed among IRESs of genotypes 1a, 1b, and 2a. Translation under the control of encephalomyocarditis virus (EMCV) IRES was inhibited by IFN to the same extent as cap-dependent translation. In cells of nonhepatic origin (HeLa and Raji), however, HCV IRES-, EMCV IRES-, and cap-dependent translation were dramatically inhibited to similar levels. The PKR expression level was enhanced by IFN in all cells, but eIF2alpha phosphorylation level was not changed, probably due to the absence of double-stranded RNA species. There was also no evidence of RNase L activation. Therefore, inhibition of translation by IFN under these conditions was probably mediated by novel IFN-induced inhibitory pathways, independent of eIF2alpha phosphorylation, while HCV IRES was not subject to this inhibition in hepatoma cell lines. Thus, HCV IRES-driven translation was resistant to IFN-induced, eIF2alpha-independent inhibition in human hepatoma cells that are frequently used in studies on HCV replication. This may present a new potential mechanism of viral resistance to IFN treatment during the early steps of virus infection.     

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.     

Prolonged infection of L cells with vesicular stomatitis virus. Defective interfering forms and temperature-sensitive mutants as factors in the infection.

When L_y cells were treated with 100 units per milliliter of mouse interferon and then infected with vesicular stomatitis virus (VSV) at a multiplicity of 10–60 PFU/cell, a prolonged infection of cultures ensued, lasting in one case longer than 60 days. After four passages in L_y cells at high multiplicities of VSV from a prolonged infection, there was no inhibition of virus growth, and virus from the prolonged infection had the same distribution of intracellular and extracellular RNA forms as was found in wild-type virus passed four times in L_y cells. Also, the intracellular and extracellular RNA forms of virus taken directly from a prolonged infection of L_y cells were indistinguishable from those of the wild-type virus. A morphological examination of VSV from a prolonged infection did not show a significant number of small defective viral forms after four passages in L_y cells at a high multiplicity of infection. There was, however, evidence for the emergence of temperature-sensitive mutants of VSV during the course of the prolonged infection. Changing the incubation temperature of the cultures from 37° to 32° resulted in an increase in virus production and virus-induced cytopathology. The virus produced during prolonged infections grew to higher titers at 32° than at 37° and its plaque size at 39° progressively decreased with the length of time that the infection persisted. Furthermore, interferon production also seemed to have a role in the persistence of infections such as the treatment of cultures with rabbit anti-mouse interferon globulin and resulted in a significant rise in virus production and viral cytopathology in the cell cultures.     

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.     

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.     

PKR protection against intranasal vesicular stomatitis virus infection is mouse strain dependent

The interferon-induced antiviral state is mediated by interferon-stimulated genes that are upregulated in concert after stimulation by type I interferons. Because so many viruses encode strategies to inactivate the interferon-inducible double-stranded RNA (dsRNA)-dependent protein kinase PKR, this protein is likely to be a major player in antiviral defense. Here we demonstrate the increased susceptibility of PKR-/- animals to vesicular stomatitis virus (VSV) by the intranasal route, but also demonstrate that the protective effects of PKR are mouse strain dependent. We have found the difference between wild-type-BALB/c and 129SvEv animals to be on the order of 5 logs, with high levels of virus present in the lungs of BALB/c but not 129SvEv animals. To evaluate the sensitivity of PKR-/- mice to VSV clearly, the PKR mutation was bred onto the resistant 129SvEv background. The increased sensitivity of PKR-/- mice, compared to PKR+/+ strain-matched controls, is on the order of 10-fold as measured by median lethal dose (LD50). PKR-/- 129 mice support VSV replication in the lung unlike controls. While this result clearly demonstrates an important role for PKR in protection against VSV infection of the lung, it also underlines the importance of other host factors in containing a viral infection.