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.     

Hepatitis C virus expression and interferon antiviral action is dependent on PKR expression

Interferon (IFN)-inducible double-stranded RNA-activated protein kinase (PKR) is thought to play a key antiviral role against hepatitis C virus (HCV). However, demonstrating the importance of PKR expression on HCV protein synthesis in the presence or absence of IFN has proven difficult in vivo. In the present experiment, full-length HCV constructs were transiently transfected into two cell lines stably expressing T7 RNA polymerase. HCV expression was monitored under conditions of upregulated or downregulated PKR expression. In addition, IFN was monitored during downregulation of PKR. HCV expression effectively increased PKR expression, as well as that of its regulated proteins. PKR was obviously knocked down by PKR-specific siRNA, which resulted in significantly increased HCV core protein levels. Conversely, over-expression of PKR significantly suppressed HCV core levels in both cell lines. Furthermore, IFN induced high levels of PKR, whereas downregulation of PKR reversed IFN's antiviral effects and increased HCV core levels. Based on these results, it appears that HCV protein expression is directly dependent on PKR expression. PKR is antiviral toward HCV and responsible for IFN's effect against HCV.     

Inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine

Summary.  Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a group of RNA viruses that establish persistent infections. A proposed strategy for evading immunity during persistent PRRSV infection is by preventing the induction of IFN activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. IFN-γ mRNA expression was observed in the lymph nodes and lungs of pigs infected with wild-type PRRSV strain SDSU-23983. Pretreatment of MARC-145 cells with IFN-γ inhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-γ on virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells. Virus replication was partially restored by the addition of 2-aminopurine (2-AP), an inhibitor of dsRNA inducible protein kinase (PKR). The addition of 2-AP also restored the viral RNA content per cell to near normal levels, suggesting that inhibition of viral RNA synthesis was through PKR. The principal difference between P6 and P136 isolates was the recovery of P136 replication with lower concentrations of 2-AP. Immunostaining with anti-PKR antibody showed a redistribution of PKR from the cytoplasm into nucleoli of infected cells. Received March 7, 2000 Accepted August 16, 2000     

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.     

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.     

Activation of protein kinase R is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication

We performed experiments to determine the effect of PKR activation on respiratory syncytial virus (RSV) replication. We first determined that RSV infection activates PKR which induces the phosphorylation of eIF2α, resulting in the formation of host stress granules. We used RNA interference to decrease endogenous PKR levels. RSV replication was not altered in cells deficient for PKR expression. However, RSV-mediated stress granule formation was significantly reduced in PKR-knockdown cells. As an alternative method to block PKR activation, we used treatment with the kinase inhibitor 2-aminopurine (2-AP). We observed that 2-AP treatment significantly reduced viral replication. We also treated PKR-knockdown cells with 2-AP and inoculated with RSV. Under these conditions, 2-AP treatment diminished viral replication in the absence of PKR expression. These results suggest that PKR activation has a minimal effect on RSV replication and that the antiviral effect of 2-AP during RSV infection likely occurs via a PKR-independent mechanism.     

Translational resistance of late alphavirus mRNA to eIF2alpha phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR

The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian kinases that phosphorylates the translation initiation factor 2alpha in response to virus infection. This kinase is induced by interferon and activated by double-stranded RNA (dsRNA). Phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) blocks translation initiation of both cellular and viral mRNA, inhibiting virus replication. To counteract this effect, most viruses express inhibitors that prevent PKR activation in infected cells. Here we report that PKR is highly activated following infection with alphaviruses Sindbis (SV) and Semliki Forest virus (SFV), leading to the almost complete phosphorylation of eIF2alpha. Notably, subgenomic SV 26S mRNA is translated efficiently in the presence of phosphorylated eIF2alpha. This modification of eIF2 does not restrict viral replication; SV 26S mRNA initiates translation with canonical methionine in the presence of high levels of phosphorylated eIF2alpha. Genetic and biochemical data showed a highly stable RNA hairpin loop located downstream of the AUG initiator codon that is necessary to provide translational resistance to eIF2alpha phosphorylation. This structure can stall the ribosomes on the correct site to initiate translation of SV 26S mRNA, thus bypassing the requirement for a functional eIF2. Our findings show the existence of an alternative way to locate the ribosomes on the initiation codon of mRNA that is exploited by a family of viruses to counteract the antiviral effect of PKR.     

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.     

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.     

Abortive HIV-1 RNA induces pro-IL-1β maturation via protein kinase PKR and inflammasome activation in humans

The proinflammatory cytokine IL-1β mediates high levels of immune activation observed during acute and chronic human immunodeficiency virus 1 (HIV-1) infection. Little is known about the mechanisms that drive IL-1β activation during HIV-1 infection. Here, we have identified a crucial role for abortive HIV-1 RNAs in inducing IL-1β in humans. Abortive HIV-1 RNAs were sensed by protein kinase RNA-activated (PKR), which triggered activation of the canonical NLRP3 inflammasome and caspase-1, leading to pro-IL-1β processing and secretion. PKR activated the inflammasome via ROS generation and MAP kinases ERK1/2, JNK, and p38. Inhibition of PKR during HIV-1 infection blocked IL-1β production. As abortive HIV-1 RNAs are produced during productive infection and latency, our data strongly suggest that targeting PKR signaling might attenuate immune activation during acute and chronic HIV-1 infection.     

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.     

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.     

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.     

HSV gene functions: what have we learned that could be generally applicable to its near and distant cousins?

Herpes simplex virus 1 (HSV-1) encodes at least 84 polypeptides to perform two functions: to enable viral replication and to create the environment in which the entry of the virus into host cells, synthesis of virion components, assembly and egress are optimized. Whereas the former are indispensable for viral replication, the latter, numbering 47, can be deleted without a major effect on viral replication in cells in culture. Of particular interest are gene products whose function is either to modify cellular proteins (set 1) or to block entirely their function (set 2). An example of set 1 is the infected cell protein No. 0 (ICP0), a promiscuous transactivator of genes introduced into cells by infection or transfection. In its nuclear phase this protein binds to cyclin D3, extends its life by many hours, and sequesters it in nuclear structures known as ND10. In its cytoplasmic phase, ICP0 binds the translation elongation factor EF-1 delta. Another viral protein, the UL13 protein kinase, hyper-phosphorylates EF-1 delta. ICP0 and the protein kinase stimulate protein synthesis and cause the cell to induce the synthesis of pre-S phase cellular proteins the virus needs for its replication. The gamma 134.5 protein, a prototype of set 2, also has multiple functions. One, mapped at its carboxyl terminus, blocks the effects of double-stranded RNA-dependent protein kinase R (PKR) that is activated by all wild-type and mutant viruses examined to date. PKR phosphorylates eIF-2 alpha and shuts off protein synthesis. gamma 134.5 protein binds protein phosphatase 1 and redirects it to dephosphorylate eIF-2 alpha. Although PKR is activated in wild-type-infected cells, protein synthesis is unaffected. HSV-1 encodes in addition at least two proteins, ORF O and ORF P that are repressed during productive infection. The ORF P protein localizes in spliceosomes and blocks the synthesis of viral proteins derived from spliced mRNA. The ORF O protein binds ICP4, the major regulatory protein, and prevents it from binding to DNA. The role of ORF O and ORF P proteins in the establishment of latency is uncertain. A significant discovery that has emerged from these studies is that viral proteins can perform several functions that may be totally unrelated.     

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.     

Mechanisms of RNA interference in the HIV-1-host cell interplay

The RNA interference pathway is a mechanism to regulate gene expression that acts in mammalian antiviral immunity as a complement of interferon- and cytokine-based innate immunity. RNA interference has been proposed as an ancient mechanism against viruses since several components of this system show an effect against viral replication. In fact, protein effectors of this pathway, as well as synthesized microRNA, act against HIV-1, exerting a partial control over HIV-1 latency and replication. Conversely, HIV-1 may counteract this antiviral cell response through two major lines of attack: first, its main regulator Tat suppresses the cellular RNA interference pathway; and second, the virus synthesizes viral microRNA that alter specific cellular functions to enhance HIV-1 replication. As a result of this complex interaction, the microRNA profile in an HIV-1-infected cell is deeply modified. One key application of all this knowledge is the development of pharmacological treatment since microRNA expression can be manipulated and artificial small interference RNA can be delivered into the infected cell to inhibit viral replication. This strategy, combined with current antiretroviral therapy, could be valuable in controlling HIV-1 replication in infected cells.     

A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation

The Tat protein of human immunodeficiency virus type 1 (HIV-1) trans-activates viral gene expression and is obligatory for virus replication. Tat function is mediated through a sequence termed TAR that comprises part of the 5'-noncoding region of all HIV-1 mRNAs. This region forms a stable stem-loop structure in vitro. Recent evidence indicates that Tat binds directly to the TAR RNA sequence, and this binding is independent of the nucleotide sequence in the loop but dependent on the integrity of the upper stem. We used the electrophoretic mobility-shift assay to identify the sequence and structure specificity of this interaction and its correlation with Tat trans-activation. We show that a 3-nucleotide bulge structure (positions +23 to +25) in TAR RNA is important for both Tat interaction with TAR RNA and Tat-mediated trans-activation of gene expression. Single base substitutions at position +23 that impair Tat-mediated trans-activation in vivo also reduce binding of Tat to TAR in vitro, suggesting that the first uridine residue in the bulge is the critical base for both functions. In contrast, mutations in the loop (positions +31 to +34) and the stem (positions +9 to +12 and +49 to +52), which reduce Tat-mediated trans-activation, had no effect on Tat binding. We also show that a Tat peptide that includes the basic region required for nucleolar localization binds to TAR RNA with the same specificity as the full-length protein. We conclude that Tat binding to TAR is necessary but not sufficient by itself to account for trans-activation.