Protein Kinase R

Viral infection activates host defense mechanisms, including the production of type I interferon (IFN) and the apoptosis of infected cells. We investigated whether these two antiviral responses were differentially regulated in infected cells. We showed that the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) apoptosis signal-regulating kinase 1 (ASK1) was activated in cells by the synthetic double-stranded RNA analog polyinosinic:polycytidylic acid [poly(I:C)] and by RNA viruses, and that ASK1 played an essential role in both the induction of the gene encoding IFN-β (IFNB) and apoptotic cell death. In contrast, we found that the MAPKKK ASK2, a modulator of ASK1 signaling, was essential for ASK1-dependent apoptosis, but not for inducing IFNB expression. Furthermore, genetic deletion of either ASK1 or ASK2 in mice promoted the replication of influenza A virus in the lung. These results indicated that ASK1 and ASK2 are components of the antiviral defense mechanism and su...

Satratoxin G (SG) is a macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum, a mold suggested to play an etiologic role in damp building-related illnesses. Acute intranasal exposure of mice to SG specifically induces apoptosis in olfactory sensory neurons of the nose. The PC-12 rat pheochromocytoma cell model was used to elucidate potential mechanisms of SGinduced neuronal cell death. Agarose gel electrophoresis revealed that exposure to SG at 10 ng/ml or higher for 48-h induced DNA fragmentation characteristic of apoptosis in PC-12 cells. SGinduced apoptosis was confirmed by microscopic morphology, hypodiploid fluorescence and annexin V-fluorescein isothiocyanate (FITC) uptake. Messenger RNA expression of the proapoptotic genes p53, double-stranded RNA-activated protein kinase (PKR), BAX, and caspase-activated DNAse was significantly elevated from 6 to 48 h after SG treatment. SG also induced apoptosis and proapoptotic gene expression in neural growth factor-differentiated PC-12 cells. Although SG-induced caspase-3 activation, caspase inhibition did not impair apoptosis. Moreover, SG induced nuclear translocation of apoptosis-inducing factor (AIF), a known contributor to caspase-independent neuronal cell death. SG-induced apoptosis was not affected by inhibitors of oxidative stress or mitogen-activated protein kinases but was suppressed by the PKR inhibitor C16 and by PKR siRNA transfection. PKR inhibition also blocked SG-induced apoptotic gene expression and AIF translocation but not caspase-3 activation. Taken together, SG-induced apoptosis in PC-12 neuronal cells is mediated by PKR via a caspase-independent pathway possibly involving AIF translocation.

Tumour necrosis factor-K K (TNF) has a variety of cellular effects including apoptotic and necrotic cytotoxicity. TNF activates a range of kinases, but their role in cytotoxic mechanisms is unclear. HeLa cells expressing elevated type II 75 kDa TNF receptor (TNFR2) protein, analysed by flow cytometry and Western analysis, showed altered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK; but not MAPK) protein content and activation. There was greater JNK activation, but reduced p38MAPK activation in dying cells compared to those still to enter TNFinduced apoptosis. Moreover, cells displaying more rapid apoptosis possess higher levels of type I 55 kDa TNFR1 receptor isoform, but less TNFR2. These findings reveal differential kinase activation in TNF-induced apoptotic death.

The NS1 gene of influenza A virus encodes a multi-functional protein that plays an important role in counteracting cellular antiviral mechanisms such as the interferon (IFN), protein kinase R and retinoic acid-inducible gene product I pathways. In addition, NS1 has recently been shown to have RNA interference (RNAi) or RNA silencing suppression (RSS) activity. This study analysed the IFN antagonistic activity of NS1 and the RSS activity for several influenza subtypes: H1N1, H3N2, H5N1 and H7N7. It was shown that the various NS1 proteins were capable of inhibiting the activation of an IFN-responsive promoter. However, differential RSS activity was measured among the NS1 variants. The NS1 protein of strain A/WSN/33 (H1N1) was most potent in suppressing short hairpin RNA-mediated gene silencing. In contrast, NS1 proteins of the highly pathogenic H5N1 strains A/VN/1194/04 and A/HK/156/97 were most potent in complementing the RSS function of the human immunodeficiency virus type 1 Tat pr...

Background and Hypothesis: During viral infection, viral double stranded RNAs (dsRNAs) are bound by and activate pattern recognition receptors (PRRs). Activated PRRs signal several downstream cellular events, including activating transcription of interferon (IFN). IFN-β can bind to cellular receptors and increase transcription of PRRs, interferon stimulated genes (ISGs), activate Protein Kinase R (PKR) and trigger apoptosis. The ability of IFN to trigger apoptosis makes it a potential chemotherapeutic. However, IFN therapy has had mixed efficacy in inducing tumor cell apoptosis.  One mechanism for this failure may be intrinsic mechanisms that prevent dsRNA from binding to and activating PRRs. ADAR1 binds to dsRNA and catalyzes deamination of adenosine to inosine, a process known as RNA editing. Normally, ADAR1 inhibits cellular dsRNAs from initiating the IFN response. Recent studies have shown ADAR1 is overexpressed in several cancers and contributes to oncogenic phenotypes, suggest...

Abstract: We investigated whether the activity of double-stranded RNA activated kinase (p68/PKR) is necessary for induction of IDO (indolamine 2, 3-dioxygenase) gene by IFN-g. For this purpose, we planned to abrogate the function of cellular wt-PKR gene. Clones of ...

Oncolytic viruses are multifunctional cancer agents with huge clinical potential, and recently the first Herpes Simplex Virus (HSV) oncolytic virus has been approved as a licensed cancer treatment. Increasingly, it is becoming apparent that no one cancer treatment is likely to be a ‘golden bullet’ – a treatment that, on its own is enough to cure all cancers. The answer seems to lie in combination therapies; by combining more than one type of treatment the chances of success, in terms of patient survival, increase. The aim of the project was to investigate the potential of HSV1716 in combination with other anti-cancer agents. As there is a vast array of current and potential cancer therapies, a high throughput screen using a range of cancer cell lines spanning a number of indications currently of clinical interest to Virttu Biologics was set up. This exploratory screen revealed a number of interesting results – synergies between HSV1716 and other drugs were seen across a number of di...

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.

Autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn's disease (CD) 1 . Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity causes Paneth cell dysfunction 2,3 . As Atg16l1 HM mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells (IECs), whose human orthologue harbors rare inflammatory bowel disease (IBD) risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis 4 . Unresolved ER stress is a common feature of IBD epithelium 4,5 , and several genetic risk factors of CD affect Paneth cells . Here we show that impairment in either UPR (Xbp1 ΔIEC ) or autophagy function (Atg16l1 ΔIEC or Atg7 ΔIEC ) in IECs results in each other's compensatory engagement, and severe spontaneous CD-like transmural ileitis if both mechanisms are compromised. Xbp1 ΔIEC mice exhibit autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased IEC death, inositol requiring enzyme 1α (IRE1α)-regulated NFκB activation and tumor necrosis factor signaling which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity and augmentation of autophagy in IECs ameliorates ER stress-induced intestinal inflammation and eases NFκB overactivation and IEC death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal CD as a specific disorder of Paneth cells.

A GGGGCC repeat expansion in C9orf72 is the most common genetic cause of ALS and FTD (C9ALS/FTD). The presence of dipeptide repeat (DPR) proteins, generated by translation of the expanded repeat, is a major pathogenic feature of C9ALS/FTD pathology, but their most relevant effects in a physiological context are not known. Here, we generated C9orf72 DPR knock-in mouse models characterised by physiological expression of 400 codon-optimised polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice exhibit cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction, showing that they recapitulate key features of C9FTD/ALS. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. This signature of increased ECM proteins was also present in C9ALS patient iPSC-motor neurons indicating it is a conserved feature of C9ALS/FTD. TGF-β1 was one of the top predicted regulators of this ECM signature and polyGR expression in human iPSC-neurons was sufficient to induce TGF-β1 followed by COL6A1, indicating TGF-β1 is one driver of the ECM signature. Knockdown of the TGF-β1 or COL6A1 orthologue in Drosophila dramatically and specifically exacerbated neurodegeneration in polyGR flies, showing that TGF-β1 and COL6A1 protect against polyGR toxicity. Altogether, our physiological C9orf72 DPR knock-in mice have revealed a neuroprotective and conserved ECM signature in C9FTD/ALS.

A GGGGCC repeat expansion inC9orf72is the most common genetic cause of ALS and FTD (C9ALS/FTD). The presence of dipeptide repeat (DPR) proteins, generated by translation of the expanded repeat, is a major pathogenic feature of C9ALS/FTD pathology, but their most relevant effects in a physiological context are not known. Here, we generatedC9orf72DPR knock-in mouse models characterised by physiological expression of 400 codon-optimised polyGR or polyPR repeats, and heterozygousC9orf72reduction. (GR)400 and (PR)400 knock-in mice exhibit cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction, showing that they recapitulate key features of C9FTD/ALS. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. This signature of increased ECM proteins was also present in C9ALS patient iPSC-motor neurons indicating it is a co...

The most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion in intron 1 of the C9orf72 gene. This repeat expansion undergoes bidirectional transcription to produce sense and antisense repeat RNA species. Both sense and antisense-derived repeat RNAs undergo repeat-associated non-AUG translation in all reading frames to generate five distinct dipeptide repeat proteins (DPRs). Importantly, toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs. This suggests targeting both sense and antisense repeat RNA may provide the most effective therapeutic strategy. The RNA-targeting CRISPR-Cas13 systems offer a promising avenue for simultaneous targeting of multiple RNA transcripts, as they mature their own guide arrays, thus allowing targeting of more than one RNA species from a single construct. We show that CRISPR-Cas13d originating from Ruminococcus flavefaciens (CasRx) can successfully reduce C9orf72 sense and antisense repeat transcripts and DPRs to background levels in HEK cells overexpressing C9orf72 repeats. CRISPR-CasRx also markedly reduced the endogenous sense and antisense repeat RNAs and DPRs in three independent C9orf72 patient-derived iPSCneuron lines, without detectable off-target effects. To determine whether CRISPR-CasRx is effective in vivo, we treated two distinct C9orf72 repeat mouse models using AAV delivery and observed a significant reduction in both sense and antisense repeat-containing transcripts. Taken together this work highlights the potential for RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

ABSTRACTOligoadenylate synthetase 3 (OAS3) and Ribonuclease L (RNase L) are components of a mammalian RNA decay pathway that is critical for combating viral infection. Nevertheless, the subcellular localization of OAS3 and RNase L during the antiviral response has remained elusive. Herein, we show that upon activation in response to double-stranded RNA or viral infection, OAS3 and RNase L integrate into higher-order cytoplasmic assemblies distinct from stress granules (SGs), RNase L-induced bodies (RLBs), or processing bodies (P-bodies). We identified these assemblies as double-stranded RNA-induced foci (dRIF), which also contain activated protein kinase R (PKR). We observed incorporation of RNase L, OAS3, and PKR in dRIFs during Zika virus and dengue virus infection. Our data support that condensation of dsRNA, OAS3 and RNase L at dRIFs promotes the homodimerization and oligomerization of RNase L required for its rapid activation, demonstrating a fundamental role for biological con...

The impact of pseudorabies virus (PRV) infection on the synthesis of host cell proteins was studied. By metabolic labeling of protein synthesis with [ 35 S]methionine, it was observed that the translation of cellular proteins was inhibited globally in the late phase of infection and viral proteins became the dominating products. Furthermore, immunoblot analysis showed that the total protein levels of two genes involved in translational regulation, namely the dsRNA-dependent protein kinase (PKR) and extracellular signal-regulated kinase 2 (ERK2), decreased during late time of infection. Using [ 32 P]orthophosphate labeling, it was observed that PRV infection also caused a decrease in the phosphorylation of intracellular PKR. Finally, using 2-aminopurine (2-AP, an inhibitor of serine/threonine protein kinase) or adenine (an isomer of 2-AP) to treat PRV-infected cells, we found that the inhibition of host protein synthesis by PRV was partially prevented by these two drugs, suggesting that 2-AP and adenine may share a same target and pathway to manifest the effect.

The effect of double-stranded RNA on the dephosphorylation of purified, 32P-labeled eIF-2 was examined in cell-free extracts prepared from interferon-treated mouse L929 cells. Dephosphorylation of the a subunit of eIF-2 occurred at comparable rates in the presence and in the absence of reovirus dsRNA. By contrast, the ß subunit of eIF-2 was not dephosphorylated to any significant extent in either the presence or the absence of dsRNA. These results indicate that the enhanced phosphorylation of eIF-2a observed in IFN-treated systems in the presence of double-stranded RNA (dsRNA) is indeed caused by an activation of a protein kinase rather than to an inhibition of a phosphoprotein phos- phatase by the dsRNA.

J. Neurochem. (2010) 115, 1421–1433.Type I interferons (IFNs) are known to cause neuropsychiatric side effects, which have been proposed to be mediated by either peripheral actions or activation of glial cells. In the present study, we have investigated whether these cytokines could act directly on neuronal cells and regulate signaling pathways involved in cell death. In human SH‐SY5Y neuroblastoma cells, type I IFNs rapidly stimulated tyrosine phosphorylation of Janus kinase and signal transducer and activator of transcription (STAT) through type I IFN receptor. Prolonged exposure to IFN‐β induced apoptotic cell death accompanied by cytochrome C release, cleavage of caspases 9, 7, 3 and poly‐(ADP ribose) polymerase and DNA fragmentation. Janus kinase inhibition reduced IFN‐β‐stimulated TyK2 and STAT1 phosphorylation, STAT1 transcriptional activity, induction of double‐stranded RNA‐activated protein kinase (PKR) and caspase cleavage. PKR induction was associated with enhanced PKR ac...

Protein kinase C-(PKC) is a Ca 2؉ -independent PKC isoform that is selectively expressed in T lymphocytes (and muscle), and is thought to play an important role in T cell receptor-induced activation. To gain a better understanding of the function and regulation of PKC, we have employed the yeast two-hybrid system to identify PKC-interacting proteins. We report the isolation and characterization of a cDNA encoding a novel 335-amino acid (37.5-kDa) PKC-interacting protein termed PICOT (for PKC-interacting cousin of thioredoxin). PICOT is expressed in various tissues, including in T cells, where it colocalizes with PKC. PICOT displays an N-terminal thioredoxin homology domain, which is required for the interaction with PKC. Comparison of the unique C-terminal region of PICOT with expressed sequence tag data bases revealed two tandem repeats of a novel domain that is highly conserved from plants to mammals. Transient overexpression of full-length PICOT (but not its N-or C-terminal fragments) in T cells inhibited the activation of c-Jun N-terminal kinase (but not extracellular signal-regulated kinase), and the transcription factors AP-1 or NF-B. These findings suggest that PICOT and its evolutionary conserved homologues may interact with PKC-related kinases in multiple organisms and, second, that it plays a role in regulating the function of the thioredoxin system. Members of the protein kinase C (PKC) 1 family of intracellular serine/threonine kinases play critical roles in the regulation of cellular differentiation and proliferation in many cell types and in response to diverse stimuli, including hormones, neurotransmitters, and growth factors (1-3). The PKC family

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Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW-264.7 cells stably expressing a dominant-negative (DN) construct of PKR (DN-PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double-stranded RNA poly(I:C), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, as well as by the expression of DN-PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF-B modulation. PKR activation induced by dsRNA also resulted in IL-10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL-10 modulation.

Hypoxia within the tumor microenvironment promotes angiogenesis, metabolic reprogramming, and tumor progression. In addition to activating hypoxia-inducible factor-1α (HIF-1α), cells also respond to hypoxia by globally inhibiting protein synthesis via serine 51 phosphorylation of translation eukaryotic initiation factor 2α (eIF2α). In this study, we investigated potential roles for stress-activated eIF2α kinases in regulation of HIF-1α. Our investigations revealed that the double-stranded RNA–dependent protein kinase R (PKR) plays a significant role in suppressing HIF-1α expression, acting specifically at the level of transcription. HIF-1α transcriptional repression by PKR was sufficient to impair the hypoxia-induced accumulation of HIF-1α and transcriptional induction of HIF-1α–dependent target genes. Inhibition of HIF-1A transcription by PKR was independent of eIF2α phosphorylation but dependent on inhibition of the signal transducer and activator of transcription 3 (Stat3). Furth...

Background-Brain cell death is a major pathological consequence of alcohol neurotoxicity. However, the molecular cascades in alcohol-induced brain tissue injury are unclear. Western blot and double immunofluorescence, we examined the expression of interferon (IFN)-induced protein kinase R (PKR), phosphorylated-PKR (p-PKR), and IFN gamma (IFNγ) in the prefrontal cortex (PFC) of postmortem brains from subjects with alcohol use disorders (AUD). Results-The protein levels of PKR, p-PKR, and IFNγ were significantly increased in subjects with AUD compared with control subjects without AUD, and a younger age of onset of AUD was significantly correlated with higher protein levels of p-PKR. In addition, elevated PKR-and p-PKR-IR were observed in both neurons and astrocytes in the PFC of subjects with AUD compared to subjects without AUD. Conclusions-The activation of the IFNγ-PKR pathway in PFC of humans is associated with chronic excessive ethanol use with an age of onset dependent manner, and activation of this pathway may play a pivotal role in AUD-related brain tissue injury. This study provides insight into neurodegenerative key factors related to AUD and identifies potential targets for the treatment of alcohol-induced neurotoxicity.

Brain cell death is a major pathological consequence of alcohol neurotoxicity. However, the molecular cascades in alcohol-induced brain tissue injury are unclear. Using Western blot and double immunofluorescence, we examined the expression of interferon (IFN)-induced protein kinase R (PKR), phosphorylated-PKR (p-PKR), and IFN gamma (IFNγ) in the prefrontal cortex (PFC) of postmortem brains from subjects with alcohol use disorders (AUD). The protein levels of PKR, p-PKR, and IFNγ were significantly increased in subjects with AUD compared with control subjects without AUD, and a younger age of onset of AUD was significantly correlated with higher protein levels of p-PKR. In addition, elevated PKR- and p-PKR-IR were observed in both neurons and astrocytes in the PFC of subjects with AUD compared to subjects without AUD. The activation of the IFNγ-PKR pathway in PFC of humans is associated with chronic excessive ethanol use with an age of onset dependent manner, and activation of this p...

We previously have reported that the mitogen-activated protein kinase (MAPK) pathway is stimulated by adhesion of human chondrocytes to anti-␤ 1 -integrin antibodies or collagen type II in vitro. These mechanisms most likely prevent chondrocyte dedifferentiation to fibroblast-like cells and chondrocyte death. To investigate whether this pathway plays an essential role for the differentiation, phenotype, and survival of chondrocytes, we blocked mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) (MEK), a kinase upstream of the kinase Erk by using U0126. Exposure of chondrocytes to U0126 caused activation of caspase-3 in a dose-dependent manner. Western blot analysis with an antibody specific for dually phosphorylated Erk shows that collagen type II induced phosphorylation of Erk1/2 was specifically blocked by U0126 in a dose-dependent manner. Immunohistochemical analysis showed that treated chondrocytes were caspase-3 positive. In treated chondrocytes, the cleavage of 116-kDa poly(ADP-ribose)polymerase resulted in the 85-kDa apoptosis-related cleavage fragment and was associated with caspase-3 activity. Analysis by electron microscopy showed typical morphological signs of apoptosis, such as crescent-shaped clumps of heterochromatin, and a degraded pericellular matrix. Thus, these results indicate that the MEK/Erk signal transduction pathway is involved in the maintenance of chondrocytes differentiation and survival. These data stimulate further investigations on the role of mitogen-activated protein kinase pathways in human chondrocytes.

Toll-like receptors (TLRs), a family of mammalian receptors, are able to recognize nucleic acids. TLR3 recognizes double-stranded (ds)RNA, a product of the replication of certain viruses. Polyinosinic-polycytidylic acid, referred to as poly(I:C), an analog of viral dsRNA, interacts with TLR3 thereby eliciting immunoinflammatory responses characteristic of viral infection or down-regulating the expression of chemokine receptor CXCR4. It is known that dsRNA also directly activates interferon (IFN)-induced enzymes, such as the RNA-dependent protein kinase (PKR). In the present study, the mRNA expression of TLR3, CXCR4, IFNγ and PKR was investigated in a culture of peripheral blood mononuclear cells (PBMCs) stimulated with poly(I:C) and endogenous RNA from human PBMCs. No cytotoxic effect on the cells or on the proliferation of CD3 + , CD4 + and CD8 + cells was observed. TLR3 expression in the PBMCs in the presence of poly(I:C) was up-regulated 9.5-fold, and TLR3 expression in the PBMCs treated with endogenous RNA was down-regulated 1.8-fold (p=0.002). The same trend was observed for IFNγ where in the presence of poly(I:C) an 8.7-fold increase was noted and in the presence of endogenous RNA a 3.1-fold decrease was observed. In the culture activated with poly(I:C), mRNA expression of CXCR4 increased 8.0-fold and expression of PKR increased 33.0-fold. Expression of these genes decreased in the culture treated with endogenous RNA when compared to the culture without stimulus. Thus, high expression of mRNA for TLR3, IFNγ, CXCR4 and PKR was observed in the presence of poly(I:C) and low expression was observed in the cells cultured with endogenous RNA. In conclusion, TLR3 may play major physiological roles that are not in the context of viral infection. It is possible that RNA released from cells could contain enough double-stranded structures to regulate cell activation. The involvement of endogenous RNA in endogenous gene expression and its implications in the regulation thereof, are still being studied, and will have significant implications in the future.

Toll-like receptors (TLRs), a family of mammalian receptors, are able to recognize nucleic acids. TLR3 recognizes double-stranded (ds)RNA, a product of the replication of certain viruses. Polyinosinic-polycytidylic acid, referred to as poly(I:C), an analog of viral dsRNA, interacts with TLR3 thereby eliciting immunoinflammatory responses characteristic of viral infection or down-regulating the expression of chemokine receptor CXCR4. It is known that dsRNA also directly activates interferon (IFN)-induced enzymes, such as the RNA-dependent protein kinase (PKR). In the present study, the mRNA expression of TLR3, CXCR4, IFNγ and PKR was investigated in a culture of peripheral blood mononuclear cells (PBMCs) stimulated with poly(I:C) and endogenous RNA from human PBMCs. No cytotoxic effect on the cells or on the proliferation of CD3(+), CD4(+) and CD8(+) cells was observed. TLR3 expression in the PBMCs in the presence of poly(I:C) was up-regulated 9.5-fold, and TLR3 expression in the PBM...

Cell division cycle 2 (Cdc2) protein is an essential subunit of M-phase kinase (MPK), which has a key role in G2/M transition. Even though the control of MPK activity has been well established with regard to the phosphorylation of Cdc2 at Thr 14 and/or Tyr 15 and Thr 161, little is known about the proteolytic control of Cdc2. In this study, we observed that Cdc2 was downregulated under genotoxic stresses and that double-stranded RNA-activated protein kinase (PKR) was involved in the process. The PKR-mediated Tyr4 phosphorylation triggered Cdc2 ubiquitination. Phospho-mimic mutations at the Tyr 4 residue (Y4D or Y4E) caused significant ubiquitination of Cdc2 even in the absence of PKR. Our findings demonstrate that (i) PKR, Ser/Thr kinase, phosphorylates its new substrate Cdc2 at the Tyr 4 residue, (ii) PKR-mediated Tyr 4phosphorylation facilitates Cdc2 ubiquitination and proteosomal degradation, (iii) unphosphorylated Tyr 4 prevents Cdc2 ubiquitination, and (iv) downstream from p53, PKR has a crucial role in G2 arrest and triggers Cdc2 downregulation under genotoxic conditions.

There is a growing evidence describing a decline in adaptive homeostasis in agingrelated diseases affecting the central nervous system (CNS), many of which are characterized by the appearance of non-native protein aggregates. One signaling pathway that allows cell adaptation is the integrated stress response (ISR), which senses stress stimuli through four kinases. ISR activation promotes translational arrest through the phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α) and the induction of a gene expression program to restore cellular homeostasis. However, depending on the stimulus, ISR can also induce cell death. One of the ISR sensors is the double-stranded RNA-dependent protein kinase [protein kinase R (PKR)], initially described as a viral infection sensor, and now a growing evidence supports a role for PKR on CNS physiology. PKR has been largely involved in the Alzheimer's disease (AD) pathological process. Here, we reviewed the antecedents supporting the role of PKR on the efficiency of synaptic transmission and cognition. Then, we review PKR's contribution to AD and discuss the possible participation of PKR as a player in the neurodegenerative process involved in aging-related pathologies affecting the CNS.

Autophagy is a highly conserved cellular catabolic pathway for degradation and recycling of intracellular components in response to nutrient starvation or environmental stress. Endoplasmic reticulum (ER) homeostasis can be disturbed by physiological and pathological influences, resulting in accumulation of misfolded and unfolded proteins in the ER lumen, a condition referred to as ER stress. Imiquimod (IMQ), a Toll-like receptor (TLR) 7 ligand, possesses anti-tumor and anti-viral activities in vitro and in vivo. IMQ has been reported to promote the apoptosis of THP-1-derived macrophages through an ER stress-dependent pathway. However, the role of ER stress in IMQ-induced autophagy is unknown. In this study, we investigated the relationship between ER stress and IMQ-induced autophagy. The expression of LC3, P62, p-PERK, Grp78, p-elF2α and IRE1α proteins were determined by immunoblotting. The relationship between ER stress and IMQ-induced autophagy were analyzed by ER stress inhibitor...

Activation of the double‐stranded RNA‐dependent protein kinase (PKR) has been implicated in the pathogenesis of several neurodegenerative diseases. We find that a compound widely used as a pharmacological inhibitor of this enzyme, referred to as PKR inhibitor (PKRi), {8‐(imidazol‐4‐ylmethylene)‐6H‐azolidino[5,4‐g]benzothiazol‐7‐one}, protects against the death of cultured cerebellar granule and cortical neurons. PKRi also prevents striatal neurodegeneration and improves behavioral outcomes in a chemically induced mouse model of Huntington’s disease. Surprisingly, PKRi fails to block the phosphorylation of eIF2α, a downstream target of PKR, and does not reduce the autophosphorylation of PKR enzyme immunoprecipitated from neurons. Furthermore, neurons lacking PKR are fully protected from apoptosis by PKRi, demonstrating that neuroprotection by this compound is not mediated by PKR inhibition. Using in vitro kinase assays we investigated whether PKRi affects any other protein kinase. Th...

Porcine reproductive and respiratory syndrome virus (PRRSV) infects alveolar macrophages (AM), causing dysregulated alpha interferon (IFN-␣) and tumor necrosis factor alpha (TNF-␣) production through a mechanism(s) yet to be resolved. Here, we show that AM infected with PRRSV secreted a reduced quantity of IFN-␣ following exposure of the cell to synthetic double-stranded RNA (dsRNA). This reduction did not correlate with reduced IFNA1 gene transcription. Rather, it coincided with two events that occurred late during infection and that were indicative of translational attenuation, specifically, the activation of eukaryotic translation initiation factor 2␣ (eIF2␣) and the appearance of stress granules. Notably, the typical rapid production of TNF-␣ by AM exposed to lipopolysaccharide (LPS) was suppressed or enhanced by PRRSV, depending on when the LPS exposure occurred after virus infection. If exposure was delayed until 6 h postinfection (hpi) so that the development of the cytokine response coincided with the time in which phosphorylation of eIF2␣ by the stress sensor PERK (protein kinase RNA [PKR]-like ER kinase) occurred, inhibition of TNF-␣ production was observed. However, if LPS exposure occurred at 2 hpi, prior to a detectable onset of eIF2␣ phosphorylation, a synergistic response was observed due to the earlier NF-B activation via the stress sensor IRE1␣ (inositol-requiring kinase 1␣). These results suggest that the asynchronous actions of two branches of the unfolded protein response (UPR), namely, IRE1␣, and PERK, activated by ER stress resulting from the virus infection, are associated with enhancement or suppression of TNF-␣ production, respectively. IMPORTANCE The activation of AM is controlled by the microenvironment to deter excessive proinflammatory cytokine responses to microbes that could impair lung function. However, viral pneumonias frequently become complicated by secondary bacterial infections, triggering severe inflammation, lung dysfunction, and death. Although dysregulated cytokine production is considered an integral component of the exacerbated inflammatory response in viral-bacterial coinfections, the mechanism responsible for this event is unknown. Here, we show that PRRSV replication in porcine AM triggers activation of the IRE1␣ branch of the UPR, which causes a synergistic TNF-␣ response to LPS exposure. Thus, the severe pneumonias typically observed in pigs afflicted with PRRSV-bacterial coinfections could result from dysregulated, overly robust TNF-␣ production in response to opportunistic pathogens that is not commensurate with the typical restrained reaction by uninfected AM. This notion could help in the design of therapies to mitigate the severity of viral and bacterial coinfections.

This study investigated whether treatment of articular cartilage chondrocytes with a combination of oncostatin M (OSM) and interleukin-1 (IL-1) could induce a degradative phenotype that was mediated through the protein kinase R (PKR) signalling pathway. High-density monolayer cultures of full depth, bovine chondrocytes were treated with a combination of OSM and IL-1 (OSM+IL-1) for 7 days. To inhibit the activation of PKR, a pharmacological inhibitor of PKR was added to duplicate cultures. Pro-and active matrix metalloproteinase-9 (MMP9) and MMP9 mRNA were signifi cantly upregulated by OSM+IL-1 through a PKR dependent mechanism. ADAMTS4 and ADAMTS5 mRNA were also upregulated by OSM+IL-1. The upregulation of ADAMTS4 and ADAMTS5 were, in part, mediated through PKR. OSM+IL-1 resulted in a loss of type II collagen, which could not be rescued by PKR inhibition. OSM+IL-1 reduced the expression of COL2A1 (type II collagen), COL9A1 (type IX collagen), COL11A1 (type XI collagen), and ACAN (aggrecan) mRNAs. Expression of type II and XI collagen and aggrecan was reduced further when PKR was inhibited. OSM+IL-1 resulted in an 11-fold increase in TNFα mRNA which was, in part, mediated through the PKR pathway. This study demonstrates, for the fi rst time, that a number of catabolic and pro-infl ammatory effects known to be important in human arthritis and induced by OSM and IL-1, are mediated by the PKR signalling pathway.

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-B and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

Background/Aims: HCV-CORE protein has been implicated in the regulation of apoptosis of infected cells acting as full-length or C-terminus deleted forms and resulting in both proapoptotic and antiapoptotic effects in different experimental conditions. Methods: We have fused full-length and C-terminus deleted CORE with GFP to assess intracellular localization in transiently transfected cell lines and primary hepatocytes. Apoptosis of cells expressing different levels of chimeric proteins was quantified by cytometry. Results: Full-length CORE localized mainly in the cytoplasm, but nuclear staining was also observed, being more evident in primary human hepatocytes. Nuclear staining only was observed in cells expressing truncated CORE. Fulllength CORE induced apoptosis in , 15 -20% of transfected cells with low expression and in , 40 -50% of those with high expression of viral protein. Interestingly, 40 -50% of cells transfected with truncated CORE underwent apoptosis, independently of protein expression levels. CORE-induced apoptosis was significantly reduced in the presence of a protein kinase R (PKR) inhibiting peptide and truncated CORE was able to enhance translocation of PKR into nucleoli where CORE/PKR colocalization was observed. Conclusions: These results suggest that nuclear forms of HCV-CORE are generated in vivo in primary hepatocytes and induce PKR-dependent apoptosis, a mechanism that might have a relevant role during natural infection.

Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the α4 gene, induces apoptosis in all cell lines tested. In some cell lines d120-induced apoptosis, manifested by the release of cytochrome c , activation of caspase 3, and fragmentation of cellular DNA, is blocked by the overexpression of Bcl-2. In these cells viral protein kinase U S 3 delivered in trans blocks apoptosis induced by the mutant virus at a premitochondrial stage. We report that the U S 3 protein kinase targets the pro-apoptotic BAD member of the Bcl-2 family. Specifically, the U S 3 protein kinase mediates a posttranslational modification of BAD and blocks its cleavage, which is reported to activate apoptosis. Thus, U S 3 protein kinase is the sole viral protein required to block activation of caspase 3, prevent cleavage of poly(ADP-ribose) polymerase, and block fragmentation of cellular DNA induced by BAD.

The double-stranded RNA-dependent protein kinase (PKR) is induced in mouse and human cells on treatment with interferon. In this study, we have cloned and determined the nucleotide sequences of chicken PKR cDNA in various chicken breeds. Chicken PKR was a 550-amino-acid protein as deduced from the cDNA open reading frame (ORF) , and there were specific domains (two double-stranded RNA binding domains (DRBDs) and numerous kinase subdomains) characterized in RNA binding proteins and kinase families. Furthermore, it was suggested that chicken PKR was polymorphic. Transfected cell clones expressing chicken PKR mRNA were demonstrated to confer antiviral responses to vesicular stomatitis virus, except for Koshamo type-3 (KS-3). KS-3 PKR, which has an amino acid substitution at position 507 (Arg to Gln), showed amphibious antiviral responses. This specific amino acid substitution was considered to determine the antiviral function of chicken PKR in addition to essential domains as DRBDs and kinase subdomains.

6-Hydroxydopamine, 1-methyl-4-phenyl-pyridinium (MPP ϩ), and rotenone cause the death of dopaminergic neurons in vitro and in vivo and are widely used to model Parkinson's disease. To identify regulated genes in such models, we performed serial analysis of gene expression on neuronal PC12 cells exposed to 6-hydroxydopamine. This revealed a striking increase in transcripts associated with the unfolded protein response. Immunoblotting confirmed phosphorylation of the key endoplasmic reticulum stress kinases IRE1␣ and PERK (PKR-like ER kinase) and induction of their downstream targets. There was a similar response to MPP ϩ and rotenone, but not to other apoptotic initiators. As evidence that endoplasmic reticulum stress contributes to neuronal death, sympathetic neurons from PERK null mice in which the capacity to respond to endoplasmic reticulum stress is compromised were more sensitive to 6-hydroxydopamine. Our findings, coupled with evidence from familial forms of Parkinson's disease, raise the possibility of widespread involvement of endoplasmic reticulum stress and the unfolded protein response in the pathophysiology of this disease.

BAFF expression, but through a PKR-independent mechanism for these 3 cell types and a type 1 IFN-dependent mechanism in monocytes and SGECs. Thus, BAFF induction by viral infection is a general phenomenon, but the types of viruses and mechanisms of the induction depend on the cell type.

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ϳ9,000 host proteins immobilized in a microarray, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA and protein levels of MAPKAPK3 were elevated in both HCV subgenomic replicon cells and cell culture-derived HCV (HCVcc)-infected cells. Silencing of MAPKAPK3 expression resulted in decreases in both protein and HCV infectivity levels but not in the intracellular HCV RNA level. We showed that MAPKAPK3 increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation.

Measles virus (MV) lacking expression of C protein (C KO) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C KO mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product. Quantitative PCR (qPCR) analyses revealed reduced viral RNA synthesis and a steepened transcription gradient in C KO virus-infected cells compared to those in parental virus-infected cells. The observed alterations were further reflected in lower viral protein expression levels and reduced C KO virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between C KO mutant and parental viruses. After one subsequent passage of the C KO virus, defective interfering RNA (DI-RNA) with a duplex structure was obtained that was not seen with the parental virus. We conclude that in the absence of C protein, the amount of PKR activator RNA, including DI-RNA, is increased, thereby triggering innate immune responses leading to impaired MV growth.

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.

Detection of foreign RNA by the innate immune system can trigger the induction of type I interferon (IFN) and apoptosis. Important antiviral defense pathways that result in type I IFN production following the recognition of foreign doublestranded RNA (dsRNA) include the RIG-I family helicases and IPS-1 adaptor cytosolic pathway and the Toll-like receptor 3 and TIR domain-containing adaptor-inducing IFN-␤ (TRIF) adaptor membrane-associated pathway, both of which activate IFN regulatory factor 3 (IRF3). In addition to triggering an innate immune response, dsRNAs are widely used to mediate geneselective silencing in mammalian cells by the RNA interference pathway. We investigated the ability of short interfering RNAs, including T7 phage polymerase-synthesized RNA (PRNA), which like some viral RNAs contains a 5-triphosphate, to selectively silence gene expression and to cause induction of IFN-␤ and apoptosis. We found that PRNA-mediated gene silencing and associated nonspecific pro-apoptotic and IFN-inducing effects were dependent on the cell line and RNA length. Double-stranded PRNAs 50 nucleotides long as well as polyinosinic-polycytidylic acid activated the RNA-dependent protein kinase (PKR) and induced significant levels of IFN-␤ and apoptosis, whereas shorter PRNAs and chemically synthesized dsRNAs did not. Effector caspase activation and apoptosis following RNA transfection was enhanced by pretreatment with IFN, and removal of the 5-phosphate from PRNAs decreased induction of both IFN-␤ and apoptosis. PKR, in addition to IPS-1 and IRF3 but not TRIF, was required for maximal type I IFN-␤ induction and the induction of apoptosis by both transfected PRNAs and polyinosinic-polycytidylic acid. The mammalian innate immune system recognizes and responds to a diverse array of pathogen-associated molecular patterns, one of which is foreign RNA (1-6). Characterization of viral RNAs that activate the innate immune response identified the retinoic acid-inducible protein (RIG-I) 2-like receptor

The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNAinduced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-␣)induced apoptosis was comparable between the HeLa lines. NF-B was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-␣. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus, was unaffected by the PKR knockdown. Surprisingly, the yield of mutant adenovirus that fails to encode VAI RNA was not enhanced in PKR-deficient cells, indicating the importance of host factors in addition to PKR in conferring the VAI RNA phenotype.

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 (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 in RNA with double-stranded character, leading to the destablization 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.

Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2␣ and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as ␤-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and ␤-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and ␤-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and ␤-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR.

ADAR1, an interferon (IFN)-inducible double-stranded (ds) RNA-specific adenosine deaminase, downregulates host innate responses, including activation of the dsRNA-dependent protein kinase (PKR) and induction of IFN-␤ mRNA. Conversely, PKR amplifies IFN-␤ induction by measles virus (MV) and inhibits virus protein synthesis. Formation of stress granules (SGs), cytoplasmic aggregates of stalled translation complexes and RNA-binding proteins, is a host response to virus infection mediated by translation initiation factor eIF2␣ phosphorylation. We examined the roles of PKR and ADAR1 in SG formation using HeLa cells stably deficient in either PKR (PKR kd) or ADAR1 (ADAR1 kd) compared to control (CON kd) cells. Infection with either wildtype (WT) MV or an isogenic mutant lacking C protein expression (C ko) comparably induced formation of SG in ADAR1 kd cells, whereas only the C ko mutant was an efficient inducer in control cells. Both ADAR1 and PKR colocalized with SG following infection. MV-induced; SG formation was PKR dependent but impaired by ADAR1. Complementation of ADAR1 kd cells by expression of either p150 WT isoform or the p150 Z␣ (Y177A) Z-DNA-binding mutant of ADAR1 restored suppression of host responses, including SG formation and PKR activation. In contrast, neither the p110 WT isoform nor the p150 catalytic (H910A, E912A) mutant of ADAR1 complemented the ADAR1 kd phenotype. These results further establish ADAR1 as a suppressor of host innate responses, including activation of PKR and the subsequent SG response.