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Protein kinase R plays a key role in innate antiviral immune responses of vertebrate animals. Most mammalian poxviruses encode two PKR antagonists, E3 (dsRNA binding) and K3 (eIF2α homolog) proteins. In this study, the role of K3 family proteins from poxviruses with distinct host tropisms in determining the virus host range was examined in a vaccinia E3L deletion mutant virus. It was found that K3 orthologs from the species-specific poxviruses (taterapox virus, sheeppox virus, myxoma virus, swinepox virus and yaba monkey tumor virus) restored the virus replication competency in cells derived from their natural hosts or related animal species. Further, it was found that the residues located in the helix insert region of the protein, K45 of vaccinia K3 and Y47 of the sheep poxvirus ortholog 011, are critical for the virus host species specificity. These observations demonstrate that poxvirus K3 proteins are major determinants of the virus host specificity. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) was shown to activate protein kinase R (PKR), which inhibits expression of interferon (IFN) and IFN-stimulated genes by controlling the translation of newly transcribed mRNAs. However, it is unknown exactly how HCV activates PKR. To address the molecular mechanism(s) of PKR activation mediated by HCV infection, we examined the effects of viral proteins on PKR activation. Here, we show that expression of HCV NS5B strongly induced PKR and eIF2α phosphorylation, and attenuated MHC class I expression. In contrast, expression of Japanese encephalitis virus RNA-dependent RNA polymerase did not induce phosphorylation of PKR. Co-immunoprecipitation analyses showed that HCV NS5B interacted with PKR. Furthermore, expression of NS5B with polymerase activity-deficient mutation failed to phosphorylate PKR, suggesting that RNA polymerase activity is required for PKR activation. These results suggest that HCV activates PKR by association with NS5B, resulting in translational suppression of MHC class I to establish chronic infection. [ABSTRACT FROM AUTHOR]

Ambystoma tigrinum virus (ATV) (family Iridoviridae , genus Ranavirus ) was isolated from diseased tiger salamanders ( Ambystoma tigrinum stebbinsi ) from the San Rafael Valley in southern Arizona, USA in 1996. Genomic sequencing of ATV, as well as other members of the genus, identified an open reading frame that has homology to the eukaryotic translation initiation factor, eIF2α (ATV eIF2α homologue, vIF2αH). Therefore, we asked if the ATV vIF2αH could also inhibit PKR. To test this hypothesis, the ATV vIF2αH was cloned into vaccinia virus (VACV) in place of the well-characterized VACV PKR inhibitor, E3L. Recombinant VACV expressing ATV vIF2αH partially rescued deletion of the VACV E3L gene. Rescue coincided with rapid degradation of PKR in infected cells. These data suggest that the salamander virus, ATV, contains a novel gene that may counteract host defenses, and this gene product may be involved in the presentation of disease caused by this environmentally important pathogen. [ABSTRACT FROM AUTHOR]

The interferon-induced double-strand RNA activated protein kinase (PKR) plays important roles in host defense against viral infection. Here we demonstrate the significant antiviral role of PKR against foot-and-mouth disease virus (FMDV) and report that FMDV infection inhibits PKR expression and activation in porcine kidney (PK-15) cells. The viral nonstructural protein 3 C proteinase (3C pro ) is identified to be responsible for this inhibition. However, it is independent of the well-known proteinase activity of 3C pro or 3C pro -induced shutoff of host protein synthesis. We show that 3C pro induces PKR degradation by lysosomal pathway and no interaction is determined between 3C pro and PKR. Together, our results indicate that PKR acts an important antiviral factor during FMDV infection, and FMDV has evolved a strategy to overcome PKR-mediated antiviral role by downregulation of PKR protein. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) lacking TRS 1 and IRS 1 (HCMV[Δ I /Δ T ]) cannot replicate in cell culture. Although both proteins can block the protein kinase R (PKR) pathway, they have multiple other activities and binding partners. It remains unknown which functions are essential for HCMV replication. To investigate this issue, we first identified a TRS1 mutant that is unable to bind to PKR. Like HCMV[Δ I /Δ T ], a recombinant HCMV containing this mutant (HCMV[TRS1-Mut 1]) did not replicate in wild-type cells. However, HCMV[Δ I /Δ T ] did replicate in cells in which PKR expression was reduced by RNA interference. Moreover, HCMV[Δ I /Δ T ] and HCMV[TRS1-Mut 1] replicated to similar levels as virus containing wild-type TRS1 in cell lines in which PKR expression was knocked out by CRISPR/Cas9-mediated genome editing. These results demonstrate that the sole essential function of TRS1 is to antagonize PKR and that its other activities do not substantially enhance HCMV replication, at least in cultured human fibroblasts. [ABSTRACT FROM AUTHOR]

We have previously shown that the replication of avian reovirus (ARV) in chicken embryo fibroblasts (CEF) is more resistant to the antiviral action of interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study we examined the capacity of these three viruses to induce the expression of IFN when infecting avian cells. Efficient expression of both type-α and type-β IFNs, as well as of the double-stranded RNA (dsRNA)-activated protein kinase (PKR), takes place in ARV-infected CEF, but not in cells infected with VSV or VV. PKR expression is not directly induced by ARV infection, but by the IFN secreted by ARV-infected cells. IFN induction in ARV-infected cells requires viral uncoating, but not viral gene expression, a situation similar to that reported for apoptosis induction by ARV-infected cells. However, our results demonstrate that IFN induction by ARV-infected CEF occurs by a caspase-independent mechanism. [ABSTRACT FROM AUTHOR]

Human adenovirus type 12 (HAdV-12) displays a relatively low virulence and slow replication in cultured human cells, which is manifested by premature death of HAdV-12-infected cells. Whereas HAdV-2 induction of IFN-β expression was transient, HAdV-12-infected cells maintained high levels of IFN-β expression, protein kinase R (PKR) activation and eIF-2α phosphorylation throughout the infectious cycle. The importance of the IFN-inducible PKR kinase in restriction of HAdV-12 was supported by the enhanced growth of the virus following PKR knockdown in HeLa cells. Ectopic expression of HAdV-2 VA RNAI increased HAdV-12 hexon protein expression, suggesting that insufficient VA RNA expression contributes to the restricted growth of HAdV-12. Although some adenovirus species are known to persist in human lymphoid tissues, HAdV12 has so far not been found. Thus, it is possible that the inability of HAdV12 to evade the INF response may have implications for the virus to establish long-lasting or persistent infections. [ABSTRACT FROM AUTHOR]

Nitazoxanide (NTZ) inhibits influenza, Japanese encephalitis, hepatitis B and hepatitis C virus replication but effects on the replication of other members of the Flaviviridae family has yet to be defined. The pestivirus bovine viral diarrhoea virus (BVDV) is a surrogate model for HCV infection and NTZ induced PKR and eIF2α phosphorylation in both uninfected and BVDV-infected cells. This led to the observation that NTZ depletes ATP-sensitive intracellular Ca2+ stores. In addition to PKR and eIF2α phosphorylation, consequences of NTZ-mediated Ca2+ mobilisation included induction of chronic sub-lethal ER stress as well as perturbation of viral protein N-linked glycosylation and trafficking. To adapt to NTZ-mediated ER stress, NTZ treated cells upregulated translation of Ca2+-binding proteins, including the ER chaperone Bip and the cytosolic pro-survival and anti-viral protein TCTP. Depletion of intracellular Ca2+ stores is the primary consequence of NTZ treatment and is likely to underpin all antiviral mechanisms attributed to the thiazolide. [ABSTRACT FROM AUTHOR]

Abstract: Protein kinase R (PKR), a regulator of translation in mammalian cells, possesses two ds-RNA binding domains responsible for kinase activation. Protein kinase Z (PKZ), a PKR-like kinase present in fish, possesses two Z-DNA binding domains. A complementation strategy with cells stably deficient in PKR was used to compare the functions of PKR and PKZ. We found reporter expression was inhibited by wildtype (WT) PKR but not by either catalytic (K296R) or RNA-binding (K64E) mutants. PKZ, like PKR, more potently inhibited 5′ cap-dependent compared to IRES-dependent reporter expression. However, in contrast to PKR-expressing cells, phosphorylation of initiation factor eIF2α was not detectably increased in PKZ-expressing cells. Furthermore, virus-induced stress granule formation was observed in PKR-deficient cells complemented with WT PKR but not K296R mutant PKR or WT PKZ. These results suggest that PKR and PKZ function by distinguishable mechanisms to modulate host responses including protein synthesis inhibition and stress granule formation. [Copyright &y& Elsevier]

Abstract: Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. [Copyright &y& Elsevier]

Abstract: 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 (Cko). Here we used complementation of human cell lines stably deficient in PKR (PKRkd) to probe the basis of these PKR-mediated responses. We found that PKRkd 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 PKRkd 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 Cko virus-mediated IFN-β induction. Furthermore, inhibition of virus growth was observed with Cko-infected PKRkd cells complemented with PKR but not with PKZ. [Copyright &y& Elsevier]

Abstract: dsRNA-activated protein kinase (PKR) is activated by viral dsRNAs and phosphorylates eIF2a reducing translation of host and viral mRNA. Although infection with a chimeric West Nile virus (WNV) efficiently induced PKR and eIF2a phosphorylation, infections with natural lineage 1 or 2 strains did not. Investigation of the mechanism of suppression showed that among the cellular PKR inhibitor proteins tested, only Nck, known to interact with inactive PKR, colocalized and co-immunoprecipitated with PKR in WNV-infected cells and PKR phosphorylation did not increase in infected Nck1,2−/− cells. Several WNV stem-loop RNAs efficiently activated PKR in vitro but not in infected cells. WNV infection did not interfere with intracellular PKR activation by poly(I:C) and similar virus yields were produced by control and PKR−/− cells. The results indicate that PKR phosphorylation is not actively suppressed in WNV-infected cells but that PKR is not activated by the viral dsRNA in infected cells. [Copyright &y& Elsevier]

Abstract: RNA species produced during virus replication are pathogen-associated molecular patterns (PAMPs) triggering cellular innate immune responses including induction of type I interferon expression and apoptosis. Pattern recognition receptors (PRRs) for these RNAs include the retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation associated gene 5 (MDA5) and the dsRNA dependent protein kinase (PKR). Currently, poxvirus PAMPs and their associated PRRs are not well characterized. We report that RNA species generated in vaccinia infected cells can activate MDA5 or RIG-I dependent interferon-β (IFN-β) gene transcription in a cell type-specific manner. These RNA species also induce the activation of apoptosis in a PKR dependent, but MDA5 and RIG-I independent, manner. Collectively our results demonstrate that RNA species generated during vaccinia virus replication are major PAMPs activating apoptosis and IFN-β gene transcription. Moreover, our results delineate the signaling pathways involved in the recognition of RNA-based poxvirus PAMPs. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: We examined the characteristics of interferon alpha/beta (IFN-α/β) induction after alphavirus or control Sendai virus (SeV) infection of murine fibroblasts (MEFs). As expected, SeV infection of wild-type (wt) MEFs resulted in strong dimerization of IRF3 and the production of high levels of IFN-α/β. In contrast, infection of MEFs with multiple alphaviruses failed to elicit detectable IFN-α/β. In more detailed studies, Sindbis virus (SINV) infection caused dimerization and nuclear migration of IRF3, but minimal IFN-β promoter activity, although surprisingly, the infected cells were competent for IFN production by other stimuli early after infection. A SINV mutant defective in host macromolecular synthesis shutoff induced IFN-α/β in the MEF cultures dependent upon the activities of the TBK1 IRF3 activating kinase and host pattern recognition receptors (PRRs) PKR and MDA5 but not RIG-I. These results suggest that wild-type alphaviruses antagonize IFN induction after IRF3 activation but also may avoid detection by host PRRs early after infection. [Copyright &y& Elsevier]

Abstract: Activated PKR protein regulates downstream anti-viral effects, including inhibition of translation. Thus, many viruses encode proteins to inhibit PKR. Here, we provide evidence that the vaccinia virus K1 protein, a host-range protein, possesses this function. First, the expression of the wild-type K1 protein was necessary to inhibit virus-induced eIF2α phosphorylation, an indirect measure of PKR activation, in RK13 and HeLa cells. Second, virus-induced eIF2α phosphorylation no longer occurred in PKR-deficient HeLa cells, suggesting PKR was responsible for vaccinia virus-induced eIF2α modification. Third, in normal HeLa cells, K1 protein expression also prevented virus-mediated PKR phosphorylation (activation). Residues in the C-terminal portion of the ANK2 region of K1 were identified as necessary for this inhibitory phenotype. Interestingly, mutant viruses that failed to inhibit PKR activation, such as S2C#2, also did not replicate in HeLa cells, suggesting that K1''s inhibition of PKR was required for a productive infection. In support of this theory, when PKR was absent from HeLa cells, there was a modest restoration of viral protein synthesis during S2C#2 infection. However, the increased protein synthesis was insufficient for a productive infection. [Copyright &y& Elsevier]

Abstract: Modified vaccinia virus Ankara (MVA), which is a promising replication-defective vaccine vector, is unusual among the orthopoxviruses in activating NF-κB transcription factors in cells of several types. In human embryonic kidney (HEK 293T) cells, the MVA-induced depletion of IκBα required to activate NF-κB is inhibited by UV-inactivation of the virus, and begins before viral DNA replication. In HEK 293T, CHO, or RK13 cells, expression of the cowpox virus CP77 early gene, or the vaccinia virus K1L early gene suppresses MVA-induced IκBα depletion. In mouse embryonic fibroblasts (MEFs), MVA induction of IκBα depletion is dependent on the expression of mouse or human double-stranded RNA-activated protein kinase (PKR). These results demonstrate that events during the early phase of MVA replication can induce PKR-mediated processes contributing both to the activation of NF-κB signaling, and to processes that may restrict viral replication. This property may contribute to the efficacy of this vaccine virus. [Copyright &y& Elsevier]

Abstract: The small molecule salubrinal has antiviral activity against herpes simplex virus-1 (HSV-1) and inhibits dephosphorylation of eIF2α mediated by the HSV-1 protein ICP34.5. We investigated whether salubrinal''s activities in infected cells depend on ICP34.5. An ICP34.5 deletion mutant was as sensitive as wild type HSV-1 to salubrinal inhibition of plaque formation in Vero cells. However, salubrinal induced formation of syncytia in infected Vero cells, which was enhanced by ICP34.5 mutations. Expression of HSV-1 US11 with immediate early kinetics, which is known to suppress the effects of ICP34.5 mutations, resulted in slight resistance to salubrinal in murine embryonic fibroblasts, and substantial resistance in those cells when ICP34.5 was additionally mutated. ICP34.5 mutations, but not immediate early expression of US11, prevented salubrinal''s ability to increase phosphorylation of eIF2α during HSV-1 infection of Vero cells. Taken together, our data indicate that salubrinal has both ICP34.5-dependent and -independent activities in HSV-1 infected cells. [Copyright &y& Elsevier]

Abstract: The E3L protein of vaccinia virus (VV) is well known for its capacity to evade cellular innate antiviral immunity related to interferon (IFN), for example PKR and RNaseL mediated antiviral activities. However, due to the limited range of cells that support VV E3L deletion mutant replication, the full capacity of E3L inhibiting the innate immune response induced by IFNs remains to be examined. In this report, the inhibition activity of VV E3L against a wide spectrum of human IFNs, including type I IFNs (12 IFN-α subtypes, IFN-β, and IFN-ω), and type II IFN (γ), was comparatively examined using the Copenhagen strain E3L deletion mutant and its revertant control virus in a human hepatoma cell line, Huh7. Deletion of the E3L open reading frame rendered the mutant VV sensitive to all types of IFNs, while the revertant VV was strongly resistant to these treatments. Furthermore, we show that the inhibition of VV E3L deletion mutant by IFN occurs at the stage of intermediate gene translation, while the expression of early genes and transcription of intermediate genes are largely unaffected. Using specific siRNAs to suppress the classical IFN-induced antiviral pathways, we found that PKR is the key factor modulated by E3L, while the RNaseL and MxA pathways play limited roles in this Huh7 cell system. Thus, our data demonstrates that VV E3L can mediate strong inhibition activity against all human type I and type II IFNs, mainly through modulation of the PKR pathway in Huh7 cells. [Copyright &y& Elsevier]

Abstract: Vaccinia virus (VACV) C7L is a host-range gene that regulates cellular tropism of VACV. Distantly related C7L homologues are encoded by nearly all mammalian poxviruses, but whether they are host-range genes functioning similar to VACV C7L has not been determined. Here, we used VACV as a model system to analyze five different C7L homologues from diverse mammalian poxviruses for their abilities to regulate poxvirus cellular tropism. Three C7L homologues (myxoma virus M63R, M64R and cowpox virus 020), when expressed with an epitope tag and from a VACV mutant lacking the host-range genes K1L and C7L (vK1L−C7L−), failed to support productive viral replication in human and murine cells. In nonpermissive cells, these viruses did not synthesize viral late proteins, expressed a reduced level of the early protein E3L, and were defective at suppressing cellular PKR activation. In contrast, two other C7L homologues, myxoma virus (MYXV) M62R and yaba-like disease virus (YLDV) 67R, when expressed with an epitope tag and from vK1L−C7L−, supported normal viral replication in human and murine cells and restored the ability of the virus to suppress PKR activation. Furthermore, M62R rescued the defect of vK1L−C7L− at replicating and disseminating in mice following intranasal inoculation. These results show that MYXV M62R and YLDV 67R function equivalently to C7L at supporting VACV replication in mammalian hosts and suggest that a C7L-like host-range gene is essential for the replication of many mammalian poxviruses in mammalian hosts. [Copyright &y& Elsevier]

Abstract: We have previously reported that the addition of interferon (IFN) to the culture medium of Vero cells (which cannot produce IFN) that were infected with the CPI− strain of parainfluenza virus 5 (PIV5, formally known as SV5), that fails to block IFN signaling, rapidly induces alterations in the relative levels of virus mRNA and protein synthesis. In addition, IFN treatment also caused a rapid redistribution of virus proteins and enhanced the formation of cytoplasmic viral inclusion bodies. The most studied IFN-induced genes with known anti-viral activity are MxA, PKR and the Oligo A synthetase/RNase L system. We therefore examined the effects of these proteins on the replication cycle of PIV5. These studies revealed that while these proteins had some anti-viral activity against PIV5 they were not primarily responsible for the very rapid alteration in virus protein synthesis observed following IFN treatment, nor for the IFN-induced formation of virus inclusion bodies, in CPI− infected cells. [Copyright &y& Elsevier]

Abstract: A major component of the cellular antiviral system is the latent protein kinase PKR, which is activated by binding to either double-stranded RNA (dsRNA) or the cellular PACT protein. Activated PKR phosphorylates the translation initiation factor eIF2, thereby inhibiting viral and cellular protein synthesis and virus replication. To evade the antiviral effects of PKR, many viruses, including influenza A virus, have evolved multiple mechanisms. For influenza A virus, the non-structural (NS1A) protein plays a major role in blocking activation of PKR during virus infection. The mechanism by which the NS1A protein inhibits PKR activation in infected cells has not been established. In the present study, we first carried out a series of in vitro experiments to determine whether the NS1A protein could utilize a common mechanism to inhibit PKR activation by both PACT and dsRNA, despite their different modes of activation. We demonstrated that the direct binding of the NS1A protein to the N-terminal 230 amino acid region of PKR can serve as such a common mechanism and that this binding does not require the RNA-binding activity of the NS1A protein. The lack of requirement for NS1A RNA-binding activity for the inhibition of PKR activation in vivo was established by two approaches. First, we showed that an NS1A protein lacking RNA-binding activity, like the wild-type (wt) protein, blocked PKR activation by PACT in vivo, as well as the downstream effects of PKR activation in cells, namely, eIF2 phosphorylation and apoptosis. In addition, we demonstrated that PKR activation is inhibited in cells infected with a recombinant influenza A virus expressing NS1A mutant protein that cannot bind RNA, as is the case in cells infected with wild-type influenza A virus. [Copyright &y& Elsevier]

The process of eukaryotic translation initiation can be regulated by a highly conserved mechanism involving the phosphorylation of the translation initiation factor eIF2 on the α subunit. This mechanism is recognized as an efficient step in the host antiviral response. Vaccinia virus (VV), like many other viruses, encodes proteins to overcome this inhibitory process. The C-terminus of the vaccinia virus E3L is known to bind to double-stranded RNA (dsRNA) thereby sequestering the activator of this antiviral response. In this report, the N-terminus of E3L was found to be required for the additional regulation of eIF2α phosphorylation. This phosphorylation event did not lead to a global shutdown in protein synthesis. Because the N-terminus of E3L is required for full viral pathogenesis in mice, these results suggest an alternative role of eIF2α phosphorylation in regulating viral replication. [Copyright &y& Elsevier]

Reovirus-induced murine myocarditis provides an excellent model for the human disease. Previously, we showed that reovirus induction of and sensitivity to interferon-β (IFN-β) are important determinants of protection against cardiac damage. IFN-β induces a number of genes with antiviral activities, including the dsRNA-activated protein kinase, PKR. Once bound to viral dsRNA, PKR becomes activated and phosphorylates eukaryotic initiation factor-2α (eIF2α) leading to the cessation of host cell translation. Additionally, activated PKR can exert its antiviral effects by inducing phosphorylation of IκB, leading to the activation of the transcription factor NFκB and subsequent induction of IFN-β. Thus, activated PKR can both induce and be induced by IFN-β. Recently, numerous reports have shown PKR to be dispensable for both induction of IFN as well as protection against disease. However, both PKR''s role in the heart in response to viral infection and its ability to prevent cardiac damage have gone largely unexplored. Here, we demonstrate PKR to be critical for viral induction of IFN-β in primary cardiac myocyte cultures. Additionally, we show that loss of PKR leads to an increase in virulence for both myocarditic and nonmyocarditic reoviruses. Finally, we demonstrate PKR to be critical for protection against reovirus-induced viral myocarditis. [Copyright &y& Elsevier]

A study was undertaken to evaluate the efficacy of an adenoviral construct expressing the murine interferon-β (IFN-β) transgene (Ad:IFN-β) 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-β 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-β 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-β 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. [Copyright &y& Elsevier]

Vaccinia virus encodes two regulators of the cellular antiviral response. The E3L gene is thought to act primarily by sequestering double-stranded RNA, whereas the K3L gene is thought to act as a competitive inhibitor of the double-stranded RNA-dependent protein kinase, PKR. The broad host range associated with vaccinia virus replication appears to be related to the presence of these genes. The E3L gene is required for replication in HeLa cells, but is not required for replication in BHK cells. On the contrary, the K3L gene is required for replication in BHK cells, but is dispensable for replication in HeLa cells. Our results suggest that these cell lines varied in the expression of endogenous activatable PKR and that replication of vaccinia virus in different cell lines led to altered levels of double-stranded RNA synthesis from the virus. Vaccinia virus was able to overcome these cellular variations by regulating PKR activity through the synthesis of either E3L or K3L. The results suggest that vaccinia virus has evolved a broad host range by maintaining both the E3L and the K3L genes. [Copyright &y& Elsevier]

All known orthopoxviruses, including ectromelia virus (ECTV), contain a gene in the E3L family. The protein product of this gene, E3, is a double-stranded RNA-binding protein. It can impact host range and is used by orthopoxviruses to combat cellular defense pathways, such as PKR and RNase L. In this work, we constructed an ECTV mutant with a targeted disruption of the E3L open reading frame (ECTVΔE3L). Infection with this virus resulted in an abortive replication cycle in all cell lines tested. We detected limited transcription of late genes but no significant translation of these mRNAs. Notably, the replication defects of ECTVΔE3L were rescued in human and mouse cells lacking PKR. ECTVΔE3L was nonpathogenic in BALB/c mice, a strain susceptible to lethal mousepox disease. However, infection with ECTVΔE3L induced protective immunity upon subsequent challenge with wild-type virus. In summary, E3L is an essential gene for ECTV., (Copyright © 2018 Elsevier Inc. All rights reserved.)