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1.  RIG-I and MDA-5 Detection of Viral RNA-dependent RNA Polymerase Activity Restricts Positive-Strand RNA Virus Replication 
PLoS Pathogens  2013;9(9):e1003610.
Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 5′-triphosphate (5′-ppp) RNA and mediate IFN production. Cytosolic 5′-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-β independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 5′-ppp dsRNA and induces IFN-β through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-β induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-β production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.
Author Summary
Type I interferons (IFN) are critical for mounting effective antiviral responses by the host cells. For RNA viruses, it is believed that IFN is triggered exclusively by viral double-stranded RNA (dsRNA) or RNA containing a 5′-triphosphate (5′-ppp) that is produced during viral genome replication or transcription driven by viral replicases. Here, we provide strong evidence suggesting that the viral replicase also generates 5′-ppp dsRNA using cellular RNA templates, which trigger IFN. This finding indicates that viral replicase is capable of activating the host innate immune response, deviating from the paradigm that viral nucleic acid replication or transcription must be initiated in the host cell to trigger IFN production. Using Semliki Forest virus (SFV) as a model, we show that the magnitude of innate immune response activation by the viral replicase plays a decisive role in establishing viral infection. We demonstrate that in contrast to the wild-type SFV replicase, a non-pathogenic mutant replicase triggers increased IFN production, which leads to a shutdown of virus replication. Consequently, excessive IFN induction by the viral replicase can be dangerous for an RNA virus. Thus, we delineate a novel mechanism by which an RNA virus triggers the host cell immune response leading to RNA virus replication shutdown.
PMCID: PMC3764220  PMID: 24039580
2.  Upregulation of CHOP/GADD153 during Coronavirus Infectious Bronchitis Virus Infection Modulates Apoptosis by Restricting Activation of the Extracellular Signal-Regulated Kinase Pathway 
Journal of Virology  2013;87(14):8124-8134.
Induction of the unfolded protein response (UPR) is an adaptive cellular response to endoplasmic reticulum (ER) stress that allows a cell to reestablish ER homeostasis. However, under severe and persistent ER stress, prolonged UPR may activate unique pathways that lead to cell death. In this study, we investigated the activation of the protein kinase R-like ER kinase (PERK) pathway of UPR in cells infected with the coronavirus infectious bronchitis virus (IBV) and its relationship with IBV-induced apoptosis. The results showed moderate induction of PERK phosphorylation in IBV-infected cells. Meanwhile, activating transcription factor 4 (ATF4) was upregulated at the protein level in the infected cells, resulting in the induction in trans of the transcription factor ATF3 and the proapoptotic growth arrest and DNA damage-inducible protein GADD153. Knockdown of PERK by small interfering RNA (siRNA) suppressed the activation of GADD153 and the IBV-induced apoptosis. Interestingly, knockdown of protein kinase R (PKR) by siRNA and inhibition of the PKR kinase activity by 2-aminopurine (2-AP) also reduced the IBV-induced upregulation of GADD153 and apoptosis induction. In GADD153-knockdown cells, IBV-induced apoptosis was suppressed and virus replication inhibited, revealing a key role of GADD153 in IBV-induced cell death and virus replication. Analysis of the pathways downstream of GADD153 revealed much more activation of the extracellular signal-related kinase (ERK) pathway in GADD153-knockdown cells during IBV infection, indicating that GADD153 may modulate apoptosis through suppression of the pathway. This study provides solid evidence that induction of GADD153 by PERK and PKR plays an important regulatory role in the apoptotic process triggered by IBV infection.
PMCID: PMC3700216  PMID: 23678184
3.  DHX36 Enhances RIG-I Signaling by Facilitating PKR-Mediated Antiviral Stress Granule Formation 
PLoS Pathogens  2014;10(3):e1004012.
RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).
Author Summary
Cellular responses to environmental stress are critical for maintaining homeostasis in living organisms. In one type of response, eukaryotic cells exhibit rapid formation of aggregates with RNA and multiple RNA-binding proteins in the cytoplasm termed stress granules (SGs). Over the past decade, SGs have been suggested to be important compartments and play essential roles in cellular stress responses. We have previously reported that virus infection induced SG-like aggregates and are crucial for antiviral response, therefore termed them as antiviral (av) SGs. In this report, we describe a novel function of DExD/H box RNA helicase 36 (DHX36), as a critical activator of double-stranded RNA (dsRNA)-dependent protein kinase (PKR), which directly trigger avSG formation in virus-infected cells. Our results reveal a novel link between DHX36 and avSG which functions as a platform to facilitate sensing of viral invasion and triggering antiviral responses.
PMCID: PMC3961341  PMID: 24651521
4.  Rotavirus Infection Induces the Unfolded Protein Response of the Cell and Controls It through the Nonstructural Protein NSP3▿ 
Journal of Virology  2011;85(23):12594-12604.
The unfolded protein response (UPR) is a cellular mechanism that is triggered in order to cope with the stress caused by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). This response is initiated by the endoribonuclease inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and PKR-like ER kinase, which increase the expression of the genes involved in the folding and degradation processes and decrease the protein input into the ER by inhibiting translation. It has been shown that viruses both induce and manipulate the UPR in order to protect the host cells from an ER stress-mediated death, thus permitting the translation of viral proteins and the efficient replication of the virus. To understand the cellular events that occur during the rotavirus replication cycle, we examined the activation of the three UPR arms following infection, using luciferase reporters driven by promoters of the ER stress-responsive genes and real-time reverse transcription-PCR to determine the levels of the stress-induced mRNAs. Our findings indicated that during rotavirus infection two of the three arms of the UPR (IRE1 and ATF6) become activated; however, these pathways are interrupted at the translational level by the general inhibition of protein synthesis caused by NSP3. This response seems to be triggered by more than one viral protein synthesized during the replication of the virus, but not by the viral double-stranded RNA (dsRNA), since cells transfected with psoralen-inactivated virions, or with naked viral dsRNA, did not induce UPR.
PMCID: PMC3209385  PMID: 21937647
5.  Coronavirus Infection Modulates the Unfolded Protein Response and Mediates Sustained Translational Repression▿  
Journal of Virology  2008;82(9):4492-4501.
During coronavirus replication, viral proteins induce the formation of endoplasmic reticulum (ER)-derived double-membrane vesicles for RNA synthesis, and viral structural proteins assemble virions at the ER-Golgi intermediate compartment. We hypothesized that the association and intense utilization of the ER during viral replication would induce the cellular unfolded protein response (UPR), a signal transduction cascade that acts to modulate translation, membrane biosynthesis, and the levels of ER chaperones. Here, we report that infection by the murine coronavirus mouse hepatitis virus (MHV) triggers the proximal UPR transducers, as revealed by monitoring the IRE1-mediated splicing of XBP-1 mRNA and the cleavage of ATF6α. However, we detected minimal downstream induction of UPR target genes, including ERdj4, ER degradation-enhancing α-mannosidase-like protein, and p58IPK, or expression of UPR reporter constructs. Translation initiation factor eIF2α is highly phosphorylated during MHV infection, and translation of cellular mRNAs is attenuated. Furthermore, we found that the critical homeostasis regulator GADD34, which recruits protein phosphatase 1 to dephosphorylate eIF2α during the recovery phase of the UPR, is not expressed during MHV infection. These results suggest that MHV modifies the UPR by impeding the induction of UPR-responsive genes, thereby favoring a sustained shutdown of the synthesis of host cell proteins while the translation of viral proteins escalates. The role of this modified response and its potential relevance to viral mechanisms for the evasion of innate defense signaling pathways during coronavirus replication are discussed.
PMCID: PMC2293058  PMID: 18305036
6.  Role of the Host Cell's Unfolded Protein Response in Arenavirus Infection▿  
Journal of Virology  2010;85(4):1662-1670.
Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.
PMCID: PMC3028902  PMID: 21106748
7.  Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR 
PLoS Pathogens  2009;5(6):e1000473.
Activation of the latent kinase PKR is a potent innate defense reaction of vertebrate cells towards viral infections, which is triggered by recognition of viral double-stranded (ds) RNA and results in a translational shutdown. A major gap in our understanding of PKR's antiviral properties concerns the nature of the kinase activating molecules expressed by influenza and other viruses with a negative strand RNA genome, as these pathogens produce little or no detectable amounts of dsRNA. Here we systematically investigated PKR activation by influenza B virus and its impact on viral pathogenicity. Biochemical analysis revealed that PKR is activated by viral ribonucleoprotein (vRNP) complexes known to contain single-stranded RNA with a 5′-triphosphate group. Cell biological examination of recombinant viruses showed that the nucleo-cytoplasmic transport of vRNP late in infection is a strong trigger for PKR activation. In addition, our analysis provides a mechanistic explanation for the previously observed suppression of PKR activation by the influenza B virus NS1 protein, which we show here to rely on complex formation between PKR and NS1's dsRNA binding domain. The high significance of this interaction for pathogenicity was revealed by the finding that attenuated influenza viruses expressing dsRNA binding-deficient NS1 proteins were rescued for high replication and virulence in PKR-deficient cells and mice, respectively. Collectively, our study provides new insights into an important antiviral defense mechanism of vertebrates and leads us to suggest a new model of PKR activation by cytosolic vRNP complexes, a model that may also be applicable to other negative strand RNA viruses.
Author Summary
Upon viral infection of vertebrate cells, a vigorous innate defense response is initiated via the recognition of viral double-stranded (ds) RNA by the protein kinase PKR, resulting in the cessation of protein synthesis and subsequent blockage of viral propagation. The activation of PKR's potent antiviral response against influenza and other viruses with a negative strand RNA genome has presented a conundrum, however, as previous attempts failed to detect dsRNA in cells infected with these viruses. Here, we identify genomic RNA within the ribonucleoprotein (RNP) of influenza viruses as a non-canonical activator of the latent kinase PKR. Cell biological examinations revealed that the transfer of viral RNP from the nucleus to the cytoplasm provides a strong stimulus for PKR activation. Moreover, we provide insight into mechanisms of pathogenesis by showing PKR and the NS1 protein of influenza B virus forms a complex in infected cells, which inhibits PKR activation. This interaction seems to be crucial for viral pathogenicity, as a strong attenuation of NS1 mutant viruses was largely rescued in PKR-deficient mice and cells. Taken together, these findings suggest a new model for the induction and inhibition of PKR by influenza virus that may also apply to viruses with a similar genome structure.
PMCID: PMC2688073  PMID: 19521506
8.  Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2α phosphorylation 
Virology Journal  2013;10:36.
Chikungunya (CHIKV) and Sindbis (SINV) are arboviruses belonging to the alphavirus genus within the Togaviridae family. They cause frequent epidemics of febrile illness and long-term arthralgic sequelae that affect millions of people each year. Both viruses replicate prodigiously in infected patients and in vitro in mammalian cells, suggesting some level of control over the host cellular translational machinery that senses and appropriately directs the cell’s fate through the unfolded protein response (UPR). The mammalian UPR involves BIP (or GRP78), the master sensor in the endoplasmic reticulum (ER) together with the three downstream effector branches: inositol-requiring ser/thr protein kinase/endonuclease (IRE-1), PKR-like ER resident kinase (PERK) and activating transcription factor 6 (ATF-6). Through careful analysis of CHIKV and SINV infections in cell culture we found that the former selectively activates ATF-6 and IRE-1 branches of UPR and suppresses the PERK pathway. By separately expressing each of the CHIKV proteins as GFP-fusion proteins, we found that non-structural protein 4 (nsP4), which is a RNA-dependent-RNA polymerase, suppresses the serine-51 phosphorylation of eukaryotic translation initiation factor, alpha subunit (eIF2α), which in turn regulates the PERK pathway. This study provides insight into a mechanism by which CHIKV replication responds to overcome the host UPR machinery.
PMCID: PMC3605262  PMID: 23356742
9.  Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway 
The Biochemical journal  2009;417(3):695-703.
The UPR (unfolded protein response) pathway is comprised of three signalling cascades mediated by the ER (endoplasmic reticulum) stress sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (asparagine synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPβ (CCAAT/enhancer-binding protein β) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared to triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box binding protein 1), ATF6α or ATF6β expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2α (phosphorylated eukaryotic initiation factor 2α)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, the ASNS NSRE-1 and NSRE-2 elements, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian UPR responsive sequences.
PMCID: PMC3595601  PMID: 18840095
activating transcription factor 3 (ATF3); activating transcription factor 4 (ATF4); asparagine synthetase (ASNS); CCAAT/enhancer-binding protein (C/EBP); endoplasmic reticulum stress; nutrient sensing; unfolded protein response (UPR)
10.  Activation of p38 Mitogen-Activated Protein Kinase and c-Jun NH2-Terminal Kinase by Double-Stranded RNA and Encephalomyocarditis Virus: Involvement of RNase L, Protein Kinase R, and Alternative Pathways 
Molecular and Cellular Biology  2000;20(2):617-627.
Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH2-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i.e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.
PMCID: PMC85147  PMID: 10611240
11.  An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA, Mediating Entry and Leading to Subsequent Type I IFN Responses 
PLoS Pathogens  2010;6(3):e1000829.
Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.
Author Summary
Nearly all viruses produce dsRNA during their replication cycle. This molecule is not normally found in a healthy host cell and thus functions as a flag, alerting the host to a viral infection. Cells can die by lysis during virus infections, and the intracellular dsRNA is then released into the extracellular space. This dsRNA is stable in the extracellular milieu, and is able to function as a signaling molecule, detected by neighboring cells. This has been observed experimentally, as extracellular dsRNA has been used for years to trigger host antiviral responses. It has also been suggested that extracellular dsRNA plays a role in causing pathological symptoms in virus infected patients. Our data suggests that class A scavenger receptors (SR-As) function as cell surface receptors for dsRNA. SR-As bind extracellular, viral dsRNA and mediate its entry into the cell, where it delivers the dsRNA to other known intracellular dsRNA sensors, activating intracellular antiviral responses. These findings shed new light on how the host detects and responds to virus infection.
PMCID: PMC2847946  PMID: 20360967
12.  Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus-Infected Cells▿  
Journal of Virology  2009;83(23):12462-12472.
A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production. One of the common viral targets is the α subunit of eukaryotic initiation factor 2 (eIF-2α). In this report, we show that phosphorylation of eIF-2α was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus (IBV). To understand whether this suppression is through inhibition of protein kinase R (PKR), the double-stranded-RNA-dependent kinase that is one of the main kinases responsible for phosphorylation of eIF-2α, cells infected with IBV were analyzed by Western blotting. The results showed that the level of phosphorylated PKR was greatly reduced in IBV-infected cells. Overexpression of IBV structural and nonstructural proteins (nsp) demonstrated that nsp2 is a weak PKR antagonist. Furthermore, GADD34, a component of the protein phosphatase 1 (PP1) complex, which dephosphorylates eIF-2α, was significantly induced in IBV-infected cells. Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34, eIF-2α, and PKR, as well as their mutant constructs in virus-infected cells, showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication. Taken together, these results confirm that IBV has developed a combination of two mechanisms, i.e., blocking PKR activation and inducing GADD34 expression, to maintain de novo protein synthesis in IBV-infected cells and, meanwhile, to enhance viral replication.
PMCID: PMC2786722  PMID: 19776135
13.  Enhanced selenium effect on growth arrest by BiP/GRP78 knockdown in p53-null human prostate cancer cells 
Oncogene  2006;25(4):546-554.
Redox modification of thiol/disulfide interchange in proteins by selenium could lead to protein unfolding. When this occurs in the endoplasmic reticulum (ER), a process known as unfolded protein response (UPR) is orchestrated for survival through activation of PERK–eIF2α(PERK: double-stranded RNA-activated protein kinase-like ER kinase; eIF2α: eucaryotic initiation factor 2α), ATFα(ATFα: activating transcription factor 6) and inositol requiring 1 (IRE1)-x-box-binding protein 1 (XBP1) signalings. All three UPR transducer pathways were upregulated very rapidly when PC-3 cells were exposed to selenium. These changes were accompanied by increased expression of UPR target genes, including immunoglobulin heavy chain-binding protein/glucose-regulated protein, 78 kDa and CCAAT/enhancer binding protein-homologous protein/growth arrest- and DNA damage- inducible gene (CHOP/GADD153). Induction of BiP/GRP78, an ER-resident chaperone, is part of the damage control mechanism, while CHOP/GADD153 is a transcription factor associated with growth arrest and apoptosis in the event of prolonged ER stress. Knocking down BiP/GRP78 induction by small interference RNA produced a differential response of the three transducers to selenium, suggesting that the signaling intensity of each transducer could be fine-tuned depending on BiP/GRP78 availability. In the presence of selenium, CHOP/GADD153 expression was raised even higher by BiP/GRP78 knockdown. Under this condition, the selenium effect on wild-type p53-activated fragment p21 (p21WAF), cyclin-dependent kinase (CDK)1 and CDK2 was also magnified in a manner consistent with enhanced cell growth arrest. Additional experiments with CHOP/GADD153 siRNA knockdown strongly suggested that CHOP/GADD153 may play a positive role in upregulating the expression of p21WAF in a p53-independent manner (PC-3 cells are p53 null). Collectively, the above findings support the idea that UPR could be an important mechanism in mediating the anticancer activity of selenium.
PMCID: PMC2424019  PMID: 16205645
selenium; unfolded protein response; BiP/GRP78 knockdown; ER stress signalings
14.  Activation of PKR by Bunyamwera Virus Is Independent of the Viral Interferon Antagonist NSs 
Journal of Virology  2003;77(9):5507-5511.
Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant virus that does not express NSs (BUNdelNSs virus). Furthermore, the sensitivity of viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaviruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.
PMCID: PMC153953  PMID: 12692253
15.  Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR by Vaccinia Virus E3: Role of Complex Formation and the E3 N-Terminal Domain 
Molecular and Cellular Biology  1998;18(12):7304-7316.
The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2α (eIF2α). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2α, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and λ repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.
PMCID: PMC109312  PMID: 9819417
16.  Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells. 
Journal of Virology  1997;71(3):1992-2003.
The vaccinia virus E3L gene codes for double-stranded RNA (dsRNA) binding proteins which can prevent activation of the dsRNA-dependent, interferon-induced protein kinase PKR. Activated PKR has been shown to induce apoptosis in HeLa cells. HeLa cells infected with vaccinia virus with the E3L gene deleted have also been shown to undergo apoptosis, whereas HeLa cells infected with wild-type vaccinia virus do not. In this report, using virus recombinants expressing mutant E3L products or alternative dsRNA binding proteins, we show that suppression of induction of apoptosis correlates with functional binding of proteins to dsRNA. Infection of HeLa cells with ts23, which leads to synthesis of increased dsRNA at restrictive temperature, induced apoptosis at restrictive but not permissive temperatures. Treatment of cells with cytosine arabinoside, which blocks the late buildup of dsRNA in vaccinia virus-infected cells, prevented induction of apoptosis by vaccinia virus with E3L deleted. Cells transfected with dsRNA in the absence of virus infection also underwent apoptosis. These results suggest that dsRNA is a trigger that can initiate a suicide response in virus-infected and perhaps uninfected cells.
PMCID: PMC191284  PMID: 9032331
17.  Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response 
Journal of Virology  2005;79(11):6890-6899.
Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2α kinase PERK; however, the amount of phosphorylated eIF2α was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2α phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing α-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.
PMCID: PMC1112127  PMID: 15890928
18.  Measles Virus C Protein Impairs Production of Defective Copyback Double-Stranded Viral RNA and Activation of Protein Kinase R 
Journal of Virology  2014;88(1):456-468.
Measles virus (MV) lacking expression of C protein (CKO) 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 CKO 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 CKO 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 CKO virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between CKO mutant and parental viruses. After one subsequent passage of the CKO 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.
PMCID: PMC3911759  PMID: 24155404
19.  West Nile Virus-Induced Interferon Production Is Mediated by the Double-Stranded RNA-Dependent Protein Kinase PKR▿ †  
Journal of Virology  2007;81(20):11148-11158.
Cells carry a variety of molecules, referred to as pathogen recognition receptors (PRRs), which are able to sense invading pathogens. Interaction of PRRs with viral compounds instigates a signaling pathway(s), resulting in the activation of genes, including those for type I interferon (IFN), which are critical for an effective antiviral response. Here we demonstrate that the double-stranded RNA (dsRNA)-dependent protein kinase PKR, which has been shown to function as a PRR in cells treated with the dsRNA mimetic poly(I:C), serves as a PRR in West Nile virus (WNV)-infected cells. Evidence for PKR's role as a PRR was obtained from both human and murine cells. Using mouse embryonic fibroblasts (MEFs), we demonstrated that PKR gene knockout, posttranscriptional gene silencing of PKR mRNA using small interfering RNA (siRNA), and chemical inhibition of PKR function all interfered with IFN synthesis following WNV infection. In three different human cell lines, siRNA knockdown and chemical inhibition of PKR blocked WNV-induced IFN synthesis. Using the same approaches, we demonstrated that PKR was not necessary for Sendai virus-induced IFN synthesis, suggesting that PKR is particularly important for recognition of WNV infection. Taken together, our data suggest that PKR could serve as a PRR for recognition of WNV infection.
PMCID: PMC2045557  PMID: 17686861
20.  Characterization of RNA Determinants Recognized by the Arginine- and Proline-Rich Region of Us11, a Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation 
Journal of Virology  2002;76(23):11971-11981.
The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I · C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I · C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I · C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2′ hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA.
PMCID: PMC136894  PMID: 12414939
21.  Hepatitis C Virus Reveals a Novel Early Control in Acute Immune Response 
PLoS Pathogens  2011;7(10):e1002289.
Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.
Author Summary
Hepatitis C Virus (HCV) is a poor interferon (IFN) inducer, despite recognition of its RNA by the cytosolic RNA helicase RIG-I. This is due in part through cleavage of MAVS, a downstream adapter of RIG-I, by the HCV NS3/4A protease and through activation of the eIF2α-kinase PKR to control IFN translation. Here, we show that HCV also inhibits RIG-I activation through the ubiquitin-like protein ISG15 and that HCV triggers rapid induction of 49 genes, including ISG15, through a novel signaling pathway that precedes RIG-I and involves PKR as an adapter to recruit MAVS. Hence, we propose to divide the acute response to HCV infection into one early (PKR) and one late (RIG-I) phase, with the former controlling the latter. Furthermore, these data emphazise the need to check compounds designed as immune adjuvants for activation of the early acute phase before using them to sustain innate immunity.
PMCID: PMC3192838  PMID: 22022264
22.  Protein Kinase Regulated by dsRNA Downregulates the Interferon Production in Dengue Virus- and dsRNA-Stimulated Human Lung Epithelial Cells 
PLoS ONE  2013;8(1):e55108.
Dengue virus (DENV) is found in the tropical and subtropical regions and affects millions of people annually. Currently, no specific vaccine or antiviral treatment against dengue virus is available. Innate immunity has been shown to be important for host resistance to DENV infection. Although protein kinase regulated by double-stranded RNA (PKR) has been found to promote the innate signaling in response to infection by several viruses, its role in the innate response to DENV infection is still unclear. Our study aimed to investigate the role of PKR in DENV-induced innate immune responses.
Methodology/Principal Findings
By RNAi, silencing of PKR significantly enhanced the expression of interferon (IFN)-β in DENV infected human lung epithelial A549 cells. Western blot and immunofluorescence microscopy data showed that PKR knockdown upregulated the activation of innate signaling cascades including p38 and JNK mitogen-activated protein kinases (MAPKs), interferon regulatory factor-3 and NF-κB, following DENV2 infection. Likewise, a negative regulatory effect of PKR on the IFN production was also observed in poly(IC) challenged cells. Moreover, the PKR knockdown-mediated IFN induction was attenuated by RIG-I or IPS-1 silencing. Finally, overexpression of a catalytically inactive PKR mutant (K296R), but not of a mutant lacking dsRNA binding activity (K64E) or the double mutant (K64EK296R), reversed the IFN induction mediated by PKR knockdown, suggesting that the dsRNA binding activity is required for PKR to downregulate IFN production.
PKR acts as a negative regulator of IFN induction triggered by DENVs and poly(IC), and this regulation relies on its dsRNA binding activity. These findings reveal a novel regulatory role for PKR in innate immunity, suggesting that PKR might be a promising target for anti-DENV treatments.
PMCID: PMC3555826  PMID: 23372823
23.  Inhibition of Cellular 2′-5′ Oligoadenylate Synthetase by the Herpes Simplex Virus Type 1 Us11 Protein▿  
Journal of Virology  2007;81(7):3455-3464.
Among the many host genes induced by virus infection and interferon, the eIF2α protein kinase PKR and the 2′-5′ oligoadenylate synthetase (OAS) are both activated by double-stranded RNA (dsRNA) produced in virus-infected cells. Furthermore, each is a critical component that independently acts to inhibit virus replication and thereby contributes to the establishment of an antiviral state. As part of their tactics to foil host defense mechanisms, some viruses prevent the induction of interferon-responsive genes at the level of transcription. Other viruses, such as herpes simplex virus type 1 (HSV-1), can additionally replicate in interferon-treated cells and must also evade the actions of host defense proteins such as PKR and OAS that have been previously synthesized and merely await detection of an activating signal. Whereas HSV-1 gene products γ134.5 and Us11 are required for viral replication in interferon-treated cells and both act in a temporally coordinated manner during infection to counteract PKR, HSV-1 functions that target OAS have not been described. Here, we demonstrate that HSV-1 infection inhibits 2′-5′ oligoadenylate synthesis in interferon-stimulated primary human cells. The OAS-inhibiting activity is generated late in the virus' productive life cycle and requires the Us11 gene product. Moreover, we establish that the Us11 protein is sufficient to block OAS activation in extracts from uninfected, interferon-treated cells. Inhibition of OAS specifically requires the Us11 dsRNA-binding domain, suggesting a mechanism that, in part, relies on sequestering available dsRNA produced during infection. Thus, in addition to PKR and its protein activator, PACT, the HSV-1 Us11 gene product is able to counteract the activity of OAS, a third cellular protein critical for host defense.
PMCID: PMC1866071  PMID: 17229694
24.  Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease? 
PLoS ONE  2011;6(10):e25589.
Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously.
Methodology/Principal Findings
Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn's disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa.
Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.
PMCID: PMC3196494  PMID: 22028783
25.  Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors 
PLoS ONE  2013;8(1):e53845.
The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.
PMCID: PMC3540029  PMID: 23320106

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