Protein Kinase R

Purpose: To date, the exact cause of abnormal exercise response in chronic fatigue syndrome (CFS) remains to be revealed, but evidence addressing intracellular immune deregulation in CFS is growing. Therefore, the aim of this cross-sectional study was to examine the interactions between several intracellular immune variables and exercise performance in CFS patients. Methods: After venous blood sampling, subjects (16 CFS patients) performed a maximal exercise stress test on a bicycle ergometer with continuous monitoring of cardiorespiratory variables. The following immune variables were assessed: the ratio of 37 kDa Ribonuclease (RNase) L to the 83 kDa native RNase L (using a radiolabeled ligand/receptor assay), RNase L enzymatic activity (enzymatic assay), protein kinase R activity assay (comparison Western blot), elastase activity (enzymatic-colorimetric assay), the percent of monocytes, and nitric oxide determination (for monocytes and lymphocytes; flow cytometry, live cell assay). Results: Forward stepwise multiple regression analysis revealed 1) that elastase activity was the only factor related to the reduction in oxygen uptake at a respiratory exchange ratio (RER) of 1.0 (regression model: R 2 ϭ 0.53, F (1,14) ϭ 15.5, P Ͻ 0.002; elastase activity P Ͻ 0.002); 2) that the protein kinase R activity was the principle factor related to the reduction in workload at RER ϭ 1.0; and 3) that elastase activity was the principle factor related to the reduction in percent of target heart rate achieved. Conclusion: These data provide evidence for an association between intracellular immune deregulation and exercise performance in patients with CFS. To establish a causal relationship, further study of these interactions using a prospective longitudinal design is required.

Dengue virus (DENV) infection is the most prevalent mosquito-borne viral diseases in the world. Vectormediated transmission of DENV is initiated when a blood-feeding female Aedes mosquito injects saliva, together with the virus, into the skin of its mammalian host. Understanding the role of skin immune cells in the activation of innate immunity to DENV at the early times of infection is a critical issue that remains to be investigated. The purpose of our study was to assess the contribution of human keratinocytes as potential host cells to DENV in the activation of immune responses at the anatomical site of mosquito bite. We show that primary keratinocytes support DENV replication with the production of negativestranded viral RNAs inside the infected cells. In the course of DENV life cycle, we observed the activation of host genes involved in the antiviral immune responses such as intracellular RNA virus sensors Toll-Like Receptor-3, Retinoic Acid Inducible Gene-I, Melanoma Differentiation Associated gene-5 and the RNAdependent protein kinase R. DENV infection of primary keratinocytes also resulted in up-regulation of the expression of the antiviral Ribonuclease L gene, which subsequently led to enhanced production of IFN-b and IFN-c. Depending on stages of viral replication, we observed the activation of host genes encoding the antimicrobial proteins b-defensin and RNase 7 in infected keratinocytes. Our data demonstrate for the first time the permissiveness of human epidermal keratinocytes to DENV infection. Remarkably, DENV replication in keratinocytes contributes to the establishment of antiviral innate immunity that might occur in the early times after the bite of mosquito.

The interferon system provides a powerful and universal intracellular defense mechanism against viruses. Knockout mice defective in IFN signaling quickly succumb to all kinds of viral infections. Likewise, humans with genetic defects in interferon signaling die of viral disease at an early age. Among the known interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins belong to the dynamin superfamily of large GTPases and have direct antiviral activity. They inhibit a wide range of viruses by blocking an early stage of the viral replication cycle. Likewise, the protein kinase R (PKR), and the 2-5 OAS/RNaseL system represent major antiviral pathways and have been extensively studied. Viruses, in turn, have evolved multiple strategies to escape the IFN system. They try to go undetected, suppress IFN synthesis, bind and neutralize secreted IFN molecules, block IFN signaling, or inhibit the action of IFN-induced antiviral proteins. Here, we summarize recent findings about the astonishing interplay of viruses with the IFN response pathway. #

The existence of naturally occurring antisense RNAs has been illustrated, in eukaryotes, by an increasing number of reports. The following review presents the major findings in this field, with a special focus on the regulation of gene expression exerted by endogenous complementary transcripts. A large variety of eukaryotic organisms, contains antisense transcripts. Moreover, the great diversity of genetic loci encoding overlapping sense and antisense RNAs suggests that such transcripts may be involved in numerous biological functions, such as control of development, adaptative response, viral infection. The regulation of gene expression by endogenous antisense RNAs seems of general importance in eukaryotes as already established in prokaryotes: it is likely to be involved in the control of various biological functions and to play a role in the development of pathological situations. Several experimental evidences for coupled, balanced or unbalanced expression of sense and antisense RNAs suggest that antisense transcripts may govern the expression of their sense counterparts. Furthermore, documented examples indicate that this control may be exerted at many levels of gene expression (transcription, maturation, transport, stability and translation). This review also addresses the underlying molecular mechanisms of antisense regulation and presents the current mechanistic hypotheses. (1994)]. In all the prokaryotic examples studied so far, Abbreviations: 3∞UTR, 3∞ untranslated region; ASP, antisense protein; antisense transcripts were found to downregulate the bFGF, basic fibroblast growth factor; BrdU, 5-bromo-2∞-deoxyuridine; expression of sense transcripts. Although there is no dsRNA, double-stranded RNA; Env, envelope; FIV, feline immunodeexperimental evidence so far, mechanisms for positive ficiency virus; HIV, human immunodeficiency virus; HSV, human simregulation are, none the less, quite plausible. plex virus; IBP, Inr-associated binding protein; LAT, latency associated transcript; LCE, lin-4-complementary element; MBP, Moreover, the introduction of artificial complemenmyelin basic protein; mld, myelin-deficient; ORF, open reading tary oligonucleotides and expression of transduced antiframe; PKR, ds activated, eIF-2 a-specific protein kinase; RRE, Revsense RNA or ribozymes have been extensively used to responsive element; RT-PCR, reverse transcription-polymerase chain inhibit gene expression. Although variable degrees of reaction; T3, triiodothyronine; TS, thymidylate synthetase; u/m, unwindase/modificase. success were achieved, these techniques have, indeed,

Type I interferon (IFN) production is a critical component of the innate defence against viral infections 1 . Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR) 2-12 . Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-e, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.

Environmental factor(s), such as viral infection, has been implicated as one of the triggering events leading to neuro-in£ammation in multiple sclerosis. This study underlines the importance of double-stranded RNA (dsRNA), the active component of a viral infection, in inducing the expression of inducible nitric oxide synthase (iNOS) in human astroglia. DsRNA in the form of synthetic polyinosinic-polycytidylic acid (poly IC) induced expression of iNOS and iNOS promoter-driven luciferase activity through activation of nuclear factor (NF)-U UB and CCAAT/enhancer-binding proteinL L (C/EBPL L). In addition, we show that inhibitors of protein kinase R attenuated iNOS by suppressing the activation of NF-U UB but not C/EBPL L. In contrast, knock down of p38 mitogen-activated protein kinase (MAPK) attenuated iNOS by suppressing the activation of C/EBPL L but not NF-U UB. This study delineates a novel role of dsRNA in inducing the expression of iNOS through dsRNAactivated protein kinase (PKR)-mediated activation of NF-U UB and p38-mediated activation of C/EBPL L in human astroglia that may participate in virus-induced neurological abnormalities.

Conflicting results addressing reduced levels of two essential fatty acids in the erythrocyte membrane of patients with chronic fatigue syndrome (CFS) have been reported. The utilised diagnostic criteria for CFS may account for the observed discrepancies among investigators. Decreased levels of essential fatty acids in the erythrocyte membrane of CFS patients fit our current understanding of CFS pathophysiology. Activation of the protein kinase R enzyme leads to nuclear factor-kB activation, which in turn generates increased production of nitric oxide (NO). A wide variety of infectious agents are typically seen in CFS patients, and are able to increase NO production. It is hypothesized that oxidative stress accounts for the observed reduction in the levels of essential fatty acids in the erythrocyte membrane of CFS patients.

Background: Hepatitis C is a major health problem affecting more than 200 million individuals in world including Pakistan. Current treatment regimen consisting of interferon alpha and ribavirin does not always succeed to eliminate virus completely from the patient's body. Results: Interferon induced antiviral protein kinase R (PKR) has a role in the hepatitis C virus (HCV) treatment as dsRNA activated PKR has the capacity to phosphorylate the serine and threonine of E2 protein and dimerization viral RNA. E2 gene of hepatitis C virus (HCV) genotype 1 has an active role in IFN resistance. E2 protein inhibits and terminates the kinase activity of PKR by blocking it in protein synthesis and cell growth. This brings forward a possible relation of E2 and PKR through a mechanism via which HCV evades the antiviral effect of IFN.

The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2␣) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2␣ phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2-and eIF3-dependent steps in the viral RNA translation initiation process.

Adenovirus synthesize proteins that interact with oncogene and tumor suppressor gene products to set the cell for virus replication. Mutant viruses defective in these functions replicate selectively in cancer cells and represent new tools to treat cancer. We report a selectivity strategy based on deletions of adenovirus Virus-Associated (VA) RNAs. In normal cells, these RNAs are necessary for virus replication because they inactivate the RNA-dependent protein kinase protein kinase R, a kinase that otherwise would block protein translation in response to infection. However, downstream effectors of Ras can also inactivate protein kinase R, and therefore, the need for VA RNA genes should be bypassed in cells with an active Ras pathway. We demonstrate here that a VAI RNA mutant presents a Ras-dependent replication and can be used for oncolytic virotherapy of pancreatic tumors.

In this study, we investigated the fine regulation of cell-to-cell movement of Bamboo mosaic virus (BaMV). We report that the coat protein (CP) of BaMV is phosphorylated in planta at position serine 241 (S241), in a process involving Nicotiana benthamiana casein kinase 2α (NbCK2α). BaMV CP and NbCK2α colocalize at the plasmodesmata, suggesting that phosphorylation of BaMV may be involved in its movement. S241 was mutated to examine the effects of temporal and spatial dysregulation of phosphorylation on i) the interactions between CP and viral RNA and ii) the regulation of cell-to-cell movement. Replacement of S241 with alanine did not affect RNA binding affinity but moderately impaired cell-to-cell movement. A negative charge at position 241 reduced the ability of CP to bind RNA and severely interfered with cell-to-cell movement. Deletion of residues 240 to 242 increased the affinity of CP to viral RNA and dramatically impaired cell-to-cell movement. A threonine at position 241 chan...

Vaccinia virus has evolved multiple mechanisms to counteract the interferon-induced antiviral host cell response. Recently, two vaccinia virus gene products were shown to interfere with the activity of the double-stranded RNA-dependent protein kinase (PKR): the K3L gene product and the E3L gene product. We have evaluated the efficiency by which these gene products inhibit PKR and whether they act in a synergistic manner. The effects of the two vaccinia virus gene products were compared in an in vivo system in which translation of a reporter gene (dihydrofolate reductase or eukaryotic translation initiation factor 2 alpha [eIF-2 alpha]) was inhibited because of the localized activation of PKR. In this system, the E3L gene product, and to a lesser extent the K3L gene product, potentiated translation of the reporter gene and inhibited eIF-2 alpha phosphorylation. Analysis in vitro demonstrated that the E3L gene product inhibited PKR approximately 50- to 100-fold more efficiently than t...

The relevance of translational control in the gene expression and oncotropism of the autonomous parvoviruses was investigated with MVMp, the prototype strain of minute virus of mice (MVM), infecting normal and transformed rodent and human cells of different tissue origins. Mouse embryo fibroblasts (MEFs) and NIH 3T3 fibroblasts were resistant to MVMp infection, but 3T3 fibroblasts derived from double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) knockout mice (PKR o/o ) behaved in a manner that was highly permissive to productive MVMp replication. NIH 3T3 resistance correlated with significant phosphorylation of eukaryotic translation initiation factor 2 (eIF2) occurring at early time points after infection. Permissive

Background: Hepatitis C is a major health problem affecting more than 200 million individuals in world including Pakistan. Current treatment regimen consisting of interferon alpha and ribavirin does not always succeed to eliminate virus completely from the patient's body. Results: Interferon induced antiviral protein kinase R (PKR) has a role in the hepatitis C virus (HCV) treatment as dsRNA activated PKR has the capacity to phosphorylate the serine and threonine of E2 protein and dimerization viral RNA. E2 gene of hepatitis C virus (HCV) genotype 1 has an active role in IFN resistance. E2 protein inhibits and terminates the kinase activity of PKR by blocking it in protein synthesis and cell growth. This brings forward a possible relation of E2 and PKR through a mechanism via which HCV evades the antiviral effect of IFN.

We previously established a role for the second messenger ceramide in protein kinase R (PKR)-mediated articular cartilage degradation. Ceramide is known to play a dual role in collagen gene regulation, with the effect of ceramide on collagen promoter activity being dependent on its concentration. Treatment of cells with low doses of sphingomyelinase produces small increases in endogenous ceramide. We investigated whether ceramide influences articular chondrocyte matrix homeostasis and, if so, the role of PKR in this process. Bovine articular chondrocytes were stimulated for 7 days with sphingomyelinase to increase endogenous levels of ceramide. To inhibit PKR, 2-aminopurine was added to duplicate cultures. De novo sulphated glycosaminoglycan and collagen synthesis were measured by adding [35S]-sulphate and [3H]-proline to the media, respectively. Chondrocyte phenotype was investigated using RT-PCR and Western blot analysis. Over 7 days, sphingomyelinase increased the release of newl...

Adenovirus synthesize proteins that interact with oncogene and tumor suppressor gene products to set the cell for virus replication. Mutant viruses defective in these functions replicate selectively in cancer cells and represent new tools to treat cancer. We report a selectivity strategy based on deletions of adenovirus Virus-Associated (VA) RNAs. In normal cells, these RNAs are necessary for virus replication because they inactivate the RNA-dependent protein kinase protein kinase R, a kinase that otherwise would block protein translation in response to infection. However, downstream effectors of Ras can also inactivate protein kinase R, and therefore, the need for VA RNA genes should be bypassed in cells with an active Ras pathway. We demonstrate here that a VAI RNA mutant presents a Ras-dependent replication and can be used for oncolytic virotherapy of pancreatic tumors.

Interferons (IFNs) suppress cell growth by inducing cellular genes. The anti-estrogen tamoxifen (Tam), binds to estrogen receptor and inhibits transcription of estrogen stimulated genes. In cells resistant to IFN-induced growth suppression, IFN/ Tam combination causes cell death. We previously reported that the combination of IFN-L and Tam was a more potent growth suppressor of human tumor xenografts than either agent alone. The IFN/Tam combination acts in a manner similar to the IFN/retinoic acid combination. Using a genetic technique, we have recently identified several genes associated with retinoid-IFN-induced mortality (GRIM). One such gene, GRIM-12, was identical to human thioredoxin reductase (TR). In the present study we have examined whether the IFN/Tam combination also requires GRIM-12 for inducing cell death. We report here that GRIM-12 is necessary for mediating the cell death effects of IFN/Tam, and its expression is induced by IFN/ Tam at a post-transcriptional stage. Repression of GRIM-12 levels either by antisense expression or by dominant negative inhibitors caused resistance to IFN/Tam induced death and promoted cell growth. Overexpression of GRIM-12 increased IFN/Tam induced apoptosis. Thus, these studies have identified a critical role for GRIM-12 (TR) in apoptosis.

The existence of naturally occurring antisense RNAs has been illustrated, in eukaryotes, by an increasing number of reports. The following review presents the major findings in this field, with a special focus on the regulation of gene expression exerted by endogenous complementary transcripts. A large variety of eukaryotic organisms, contains antisense transcripts. Moreover, the great diversity of genetic loci encoding

Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72 bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibioticfree vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination.

Rhinovirus infections cause the majority of acute exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease, with increased pro-inflammatory cytokine production by infected bronchial epithelial cells contributing to disease pathogenesis. Theses diseases are a huge cause of morbidity worldwide, and contribute a major economic burden to healthcare costs. Current steroid based treatments are only partially efficient at controlling virus induced inflammation, which remains an unmet therapeutic goal. Although NF-kappaB has been implicated, the precise mechanisms of rhinovirus induction of pro-inflammatory gene expression in bronchial epithelial cells are unclear. We hypothesised that rhinovirus replication and generation of dsRNA was an important process of pro-inflammatory cytokine induction. Using pharmalogical (2-aminopurine and a new small molecule inhibitor) and genetic inhibition of the dsRNA binding kinase protein kinase R, striking inhibition of dsRNA (polyrIC) and rhinovirus induced CCL5, CXCL8 and IL-6 protein was observed. Using confocal microscopy, rhinovirus induced protein kinase R phosphorylation co-located with NF-kappaB p65 nuclear translocation. Focusing on CXCL8, both rhinovirus infection and dsRNA treatment required IkappaB kinase-beta for induction of CXCL8. Analysis of cis-acting sites in the CXCL8 promoter revealed that both rhinovirus infection and dsRNA treatment upregulated CXCL8 promoter activation via NF-kappaB and NF-IL6 binding sites. Together, the results demonstrate the importance of dsRNA in induction of pro-inflammatory cytokines by rhinoviruses, and suggest that protein kinase R is involved in NF-kappaB mediated gene transcription of pro-inflammatory cytokines via IkappaB kinase-beta. These molecules regulating rhinovirus induction of inflammation represent therapeutic targets.

The exacerbation of symptoms after exercise differentiates Chronic fatigue syndrome (CFS) from several other fatigue-associated disorders. Research data point to an abnormal response to exercise in patients with CFS compared to healthy sedentary controls, and to an increasing amount of evidence pointing to severe intracellular immune deregulations in CFS patients. This manuscript explores the hypothetical interactions between these two separately reported observations. First, it is explained that the deregulation of the 2-5A synthetase/RNase L pathway may be related to a channelopathy, capable of initiating both intracellular hypomagnesaemia in skeletal muscles and transient hypoglycemia. This might explain muscle weakness and the reduction of maximal oxygen uptake, as typically seen in CFS patients. Second, the activation of the protein kinase R enzyme, a characteristic feature in atleast subsets of CFS patients, might account for the observed excessive nitric oxide (NO) production in patients with CFS. Elevated NO is known to induce vasidilation, which may limit CFS patients to increase blood flow during exercise, and may even cause and enhanced postexercise hypotension. Finally, it is explored how several types of infections, frequently identified in CFS patients, fit into these hypothetical pathophysiological interactions.

Dengue virus (DENV) infection is the most prevalent mosquito-borne viral diseases in the world. Vectormediated transmission of DENV is initiated when a blood-feeding female Aedes mosquito injects saliva, together with the virus, into the skin of its mammalian host. Understanding the role of skin immune cells in the activation of innate immunity to DENV at the early times of infection is a critical issue that remains to be investigated. The purpose of our study was to assess the contribution of human keratinocytes as potential host cells to DENV in the activation of immune responses at the anatomical site of mosquito bite. We show that primary keratinocytes support DENV replication with the production of negativestranded viral RNAs inside the infected cells. In the course of DENV life cycle, we observed the activation of host genes involved in the antiviral immune responses such as intracellular RNA virus sensors Toll-Like Receptor-3, Retinoic Acid Inducible Gene-I, Melanoma Differentiation Associated gene-5 and the RNAdependent protein kinase R. DENV infection of primary keratinocytes also resulted in up-regulation of the expression of the antiviral Ribonuclease L gene, which subsequently led to enhanced production of IFN-b and IFN-c. Depending on stages of viral replication, we observed the activation of host genes encoding the antimicrobial proteins b-defensin and RNase 7 in infected keratinocytes. Our data demonstrate for the first time the permissiveness of human epidermal keratinocytes to DENV infection. Remarkably, DENV replication in keratinocytes contributes to the establishment of antiviral innate immunity that might occur in the early times after the bite of mosquito.

The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR.

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. 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. 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. Full-length CORE induced apoptosis in approximately 15-20% of transfected cells with low expression and in approximately 40-50% of those with high expression of viral protein. Interestingly, 40-50% of cells transfected with truncated CORE underwent apoptosis, independently of protein expre...

However, the mechanisms of WNV-induced neuronal damage are not well understood. We investigated WNV neuropathogenesis by using human neuroblastoma cells and primary rat hippocampal neurons. We observed that WNV activates multiple unfolded protein response (UPR) pathways, leading to transcriptional and translational induction of UPR target genes. We evaluated the role of the three major UPR pathways, namely, inositol-requiring enzyme 1-dependent splicing of X box binding protein 1 (XBP1) mRNA, activation of activating transcription factor 6 (ATF6), and protein kinase R-like endoplasmic reticulum (ER) kinase-dependent eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation, in WNVinfected cells. We show that XBP1 is nonessential or can be replaced by other UPR pathways in WNV replication. ATF6 was rapidly degraded by proteasomes, consistent with induction of ER stress by WNV. We further observed a transient phosphorylation of eIF2␣ and induction of the proapoptotic cyclic AMP response element-binding transcription factor homologous protein (CHOP). WNV-infected cells exhibited a number of apoptotic phenotypes, such as (i) induction of growth arrest and DNA damage-inducible gene 34, (ii) activation of caspase-3, and (iii) cleavage of poly(ADP-ribose) polymerase. The expression of WNV nonstructural proteins alone was sufficient to induce CHOP expression. Importantly, WNV grew to significantly higher viral titers in chop ؊/؊ mouse embryonic fibroblasts (MEFs) than in wild-type MEFs, suggesting that CHOP-dependent premature cell death represents a host defense mechanism to limit viral replication that might also be responsible for the widespread neuronal loss observed in WNV-infected neuronal tissue. Cells, cell culture, and virus. SK-N-MC neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin, streptomycin, and glutamine. HEK293T cells were cultured in minimal essential medium containing 10% fetal bovine serum, penicillin, streptomycin, and glutamine. xbp1 Ϫ/Ϫ and xbp1 ϩ/ϩ cells were obtained from Laurie Glimcher and grown as described previously . chop Ϫ/Ϫ cells were obtained from David Ron and cultured as described previously . The WNV strain used in this study has been described before (56). Viral stocks and infection and plaque assays were as described previously . In some experiments, viruses from WNV-infected mosquito cell (C6/36) supernatants were used. WNV replicons were packaged into virus-like particles (VLPs) by using a cell line persistently expressing a Venezuelan equine encephalitis virus replicon encoding the WNV structural proteins (10). To obtain high-titer WNV VLPs, we utilized a WNV replicon (WNR-CNS1-5) containing a full capsid-encoding region, since this replicon has been shown to produce high-titer VLP preparations (10; unpublished data). Rat hippocampal neurons were isolated and cultured in Gary Banker's laboratory as described previously .

stress. This response centers on the phosphorylation state of eukaryotic initiation factor (eIF)-2␣, which is induced by kinases like protein kinase R-like ER kinase (PERK) and GCN2 to suppress translation and is later reversed so translation resumes. GADD34 was recently identified as the factor that activates the type 1 protein serine/threonine phosphatase (PP1), which dephosphorylates eIF-2␣ during cellular stresses. Our study delineates a negative feedback regulatory loop in which the eIF-2␣-controlled inhibition of protein translation leads to GADD34 induction, which promotes translational recovery. We show that activating transcription factor-4 (ATF4), which is paradoxically translated during the eIF-2␣-mediated translational block, is required for the transactivation of the GADD34 promoter in response to ER stress and amino acid deprivation. ATF4 directly binds to and trans-activates a conserved ATF site in the GADD34 promoter during ER stress. Examination of ATF4؊/؊ MEFs revealed an absence of GADD34 induction, prolonged eIF-2␣ phosphorylation, delayed protein synthesis recovery, and diminished translational upregulation of BiP during ER stress. These studies demonstrate the essential role of GADD34 in the resumption of protein synthesis, define the pathway for its induction, and reveal that cytoprotective unfolded protein response targets like BiP are sensitive to the eIF-2␣mediated block in translation.

Metabolic syndrome greatly increases the risk for developing metabolic and cardiovascular disorders and has reached epidemic proportions globally. Despite recent advances in medical science, scientific understandings on the root mechanisms of metabolic syndrome are still not fully understood, and such insufficient knowledge contributes to the relative lack of effective treatments for such diseases. Protein Kinase R (PKR) is a serine threonine kinase activated during various stress conditions. Activation of PKR can increase reactive oxygen product generation, cause oxidative stress and inflammation. In this review we discuss the potential role of PKR in metabolic syndrome, pathways activated by it and the interrelationship between pathways activated, modes of propagation if one of the pathways is inhibited or activated. Specific and effective inhibitors of PKR are being developed and can become potential treatment for metabolic syndrome and prevent many diseases.

Double-stranded RNA binding domains of human protein kinase R (dsRBD-PKR) regulate distinct cellular functions and the fate of an RNA molecule in the cell. This highly homologous domains present in multiple copies in a number of species, exhibit individual and specific functional specificity. Number of NMR and X-ray crystallographic structural studies reveals that such domains take a common ␣-␤-␤-␤-␣ tertiary fold. However, the functional specificities of these domains could be due to the dynamics of the individual amino acid residues, as has been shown earlier in the case of backbone dynamics of 15 N-1 H of dsRNA binding motifs (dsRBMs) of human protein kinase R (PKR) (Nanduri S, Rahman F, Williams BRG, Qin J. EMBO J 2000;19:5567-5574). To further investigate if the differences in dynamics of the two dsRBMs are restricted to only backbone, or if the side-chain motions are also different to the extent of influencing their packing of the two hydrophobic cores, we have investigated the methyl group dynamics using 13 C-methyl relaxation measurements. The results show that the hydrophobic core of dsRBM1 is more tightly packed than dsRBM2, and it seems to undergo less fast scale motions in the subnanosecond regime. Proteins 2006;62:501-508.