Characterization of rainbow trout and zebrafish eukaryotic initiation factor 2alpha and its response to endoplasmic reticulum stress and IPNV infection

The cDNAs of rainbow trout and zebrafish eIF2alpha have been isolated and found to encode proteins of similar molecular weight and isoelectric point to the alpha-subunit of the human translational initiation factor, eIF2. The rainbow trout (36.0kDa) and zebrafish (36.2kDa) eIF2alphas share 93 and 91% identity to the human protein, respectively, and are recognized by antibodies raised to the human form. In mammals, the phosphorylation of the alpha-subunit of eIF2 plays a key role in the regulation of protein synthesis in response to a range of cellular stresses. Regions corresponding to the human phosphorylation and kinase-docking sites are identical in the proteins of both fish species, as are residues that interact with the eIF2 recycling factor, eIF2B. Moreover, both recombinant rainbow trout and zebrafish eIF2alphas can be phosphorylated in vitro by the mammalian heme-sensitive eIF2alpha-kinase, HRI/HCR, as well as the interferon-inducible, dsRNA sensitive kinase, PKR. Phosphorylation of rainbow trout and zebrafish eIF2alpha can also occur in vivo. RTG-2 and ZFL cells subjected to endoplasmic reticulum (ER) stress by treatment with the Ca(2+)-ionophore A23187 showed increased levels of eIF2alpha phosphorylation, suggesting similarity between the ER stress response in fish and other higher eukaryotes. Furthermore, RTG-2 cells responded to treatment with poly(I).poly(C) or to infection by infectious pancreatic necrosis virus, IPNV, by increasing eIF2alpha phosphorylation. These data imply that RTG-2 cells express the interferon-induced eIF2alpha-kinase, PKR and suggests that the interferon/eIF2alpha/PKR response to virus infection may be a conserved vertebrate characteristic. Overall these data are consistent with the premise that fish are able to regulate protein synthesis in response to cellular stresses through phosphorylation of eIF2alpha.     

Pseudosubstrate inhibition of protein kinase PKR by swine pox virus C8L gene product

The interferon-induced protein kinase PKR is activated upon binding double-stranded RNA and phosphorylates the translation initiation factor eIF2alpha on Ser-51 to inhibit protein synthesis in virally infected cells. Swinepox virus C8L and vaccinia virus K3L gene products structurally resemble the amino-terminal third of eIF2alpha. We demonstrate that the C8L protein, like the K3L protein, can reverse the toxic effects caused by high level expression of human PKR in yeast cells. In addition, expression of either the K3L or C8L gene product was found to reverse the inhibition of reporter gene translation caused by PKR expression in mammalian cells. The inhibitory function of the K3L and C8L gene products in these assays was found to be critically dependent on residues near the carboxyl-termini of the proteins including a sequence motif shared among eIF2alpha and the C8L and K3L gene products. Thus, despite significant sequence differences both the C8L and K3L proteins function as pseudosubstrate inhibitors of PKR.     

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

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

Hepatitis C virus IRES-dependent translation is insensitive to an eIF2alpha-independent mechanism of inhibition by interferon in hepatocyte cell lines

Interferon (IFN) in combination with ribavirin is the main treatment for hepatitis C virus (HCV) infection. The sensitivity or resistance of the virus to IFN has been linked to certain types of the interferon sensitivity determining region (ISDR) and PKR-eIF2alpha phosphorylation homology domain (PePHD) sequences in the NS5A and E2 regions of the viral genome, respectively. In search of the other potential mechanisms of HCV resistance to IFN, we tested the effect of IFN-alpha on translational activity of the HCV IRES in various cell types. Using bicistronic dual luciferase reporter RNAs in direct RNA transfection studies, we found that the cap-dependent translation was dramatically inhibited by IFN (5- to 16-fold), whereas HCV IRES translation was inhibited only marginally in two hepatoma cell lines, Huh7 and HepG2 cells. No difference in IFN sensitivity was observed among IRESs of genotypes 1a, 1b, and 2a. Translation under the control of encephalomyocarditis virus (EMCV) IRES was inhibited by IFN to the same extent as cap-dependent translation. In cells of nonhepatic origin (HeLa and Raji), however, HCV IRES-, EMCV IRES-, and cap-dependent translation were dramatically inhibited to similar levels. The PKR expression level was enhanced by IFN in all cells, but eIF2alpha phosphorylation level was not changed, probably due to the absence of double-stranded RNA species. There was also no evidence of RNase L activation. Therefore, inhibition of translation by IFN under these conditions was probably mediated by novel IFN-induced inhibitory pathways, independent of eIF2alpha phosphorylation, while HCV IRES was not subject to this inhibition in hepatoma cell lines. Thus, HCV IRES-driven translation was resistant to IFN-induced, eIF2alpha-independent inhibition in human hepatoma cells that are frequently used in studies on HCV replication. This may present a new potential mechanism of viral resistance to IFN treatment during the early steps of virus infection.     

The PKR activator PACT is induced by Aβ: involvement in Alzheimer's disease

The neuropathological hallmarks of Alzheimer's disease (AD) include senile plaques made of Aβ peptide, neurofibrillary tangles containing hyperphosphorylated tau protein and neuronal loss. The pro-apoptotic kinase PKR can be activated by Aβ and can phosphorylate tau protein via GSK3β kinase activation. The activated form of PKR (pPKR) accumulates in affected neurons and could participate in neuronal degeneration in AD. The mechanism of abnormal PKR activation in AD is not elucidated but could be linked to the PKR activator PACT. PACT stainings, and levels were assessed in the brains of AD patients and in APP/PS1 knock-in transgenic mice and in cell cultures exposed to stresses. We showed that PACT and pPKR colocalizations are enhanced in AD brains. Their levels are increased and correlated in AD and APP/PS1 knock-in mice brains. In human neuroblastoma cells exposed to Aβ, tunicamycin or H2O2, PACT and pPKR concentrations are increased. PACT then PKR inhibitions indicate that PACT is upstream of PKR activation. Our findings demonstrate that PACT levels are enhanced in AD brains and could partly be caused by the action of Aβ. In addition, PACT participates in PKR activation. The PACT-PKR pathway represents a potential link between Aβ accumulation, PKR activation and tau phosphorylation.     

The effect of the vaccinia K1 protein on the PKR-eIF2α pathway in RK13 and HeLa cells

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

Biochemical analyses of multiple fractions of PKR purified from Escherichia coli.

PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.     

The oncogenic potential of hepatitis C virus NS5A sequence variants is associated with PKR regulation.

The NS5A protein of hepatitis C virus (HCV) confers cell growth regulation and has been implicated in viral oncogenesis. Here, we investigated whether highly divergent NS5A proteins obtained from HCV-infected patients presented an oncogenic potential when expressed in mammalian cells. In general, NS5A expression was associated with increased rates of cell growth and culture proliferation. Immortalized primary hepatocyte and immortalized fibroblast cell lines expressing a subset of these sequences exhibited a significant increase in protein synthetic rate, culture saturation density, and a transformed cellular phenotype, as shown by anchorage-independent cell growth and colony formation in soft agar assays. Oncogenic transformation correlated with inhibition of protein kinase R (PKR) activity and concomitant reduction of eukaryotic initiation factor 2alpha (elF2alpha) phosphorylation levels that caused stimulation of mRNA translation. The extent of sequence variation throughout NS5A or within the previously characterized PKR-binding domain was not a predictive indicator of this cellular phenotype, suggesting that sequences outside this region contribute to PKR regulation. Our data indicate that NS5A oncogenic potential is conditional through viral sequence variation. These results provide further evidence to define the PKR pathway as a mediator of cell growth control and suggest that viral regulation of PKR may contribute to hepatocyte growth deregulation during chronic HCV infection.     

West Nile virus infection does not induce PKR activation in rodent cells

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.     

Modulation of PKR activity in cells infected by bovine viral diarrhea virus

Bovine viral diarrhea virus is an important animal pathogen. The cytopathic and noncytopathic biotypes of the virus are associated with distinct pathologic entities. A striking difference between the two biotypes is viral RNA accumulation in infected cells. Viral dsRNA is thought to activate protein kinase PKR; an important mediator of innate immunity. In this study, we investigated PKR activation and its consequences in BVDV-infected cells. Infection with cp BVDV was found to induce PKR activation, eIF2alpha phosphorylation, translation inhibition and NF-kappaB activation. In contrast, PKR activity and eIF2alpha phosphorylation were not induced during infection with the ncp BVDV. In addition, cells infected with ncp BVDV showed no PKR phosphorylation in response to infection with the unrelated poliovirus whereas uninfected ncp BVDV cells when infected with poliovirus showed high levels of phosphorylated PKR. Cells infected with ncp BVDV failed to respond to synthetic dsRNA (poly I:C) treatment with NF-kappaB activation. However, the NF-kappaB response to bacterial lipopolysaccarides (LPS) was normal in these cells, suggesting a specific suppression of antiviral response signaling in ncp BVDV infected cells. These results indicate that ncp BVDV has evolved specific mechanisms to prevent activation of PKR and its antiviral effectors, most likely to facilitate the establishment and maintenance of persistent infection.     

Polycystin-2 down-regulates cell proliferation via promoting PERK-dependent phosphorylation of eIF2alpha

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of renal, hepatic and pancreatic cysts and by non-cystic manifestations such as abnormal vasculature and embryo left-right asymmetry development. Polycystin-2 (PC2), in which mutations account for 10-15% of ADPKD, was previously shown to down-regulate cell proliferation, but the underlying mechanism was not elucidated. Here, we demonstrate that PC2, but not pathogenic mutants E837X and R872X, represses cell proliferation through promoting the phosphorylation of eukaryotic translation initiation factor eIF2alpha by pancreatic ER-resident eIF2alpha kinase (PERK). ER stress is known to enhance eIF2alpha phosphorylation through up-regulating PERK kinase activity (assessed by phosphorylated PERK). During ER stress, PC2 knockdown also repressed eIF2alpha phosphorylation but did not alter PERK phosphorylation, indicating that PC2 facilitates the eIF2alpha phosphorylation by PERK. PC2 was found to be in the same complex as PERK and eIF2alpha. Together, we demonstrate that PC2 negatively controls cell growth by promoting PERK-mediated eIF2alpha phosphorylation, presumably through physical interaction, which may underlie a pathogenesis mechanism of ADPKD and indicates that PC2 is an important regulator of the translation machinery.     

Neutralizing innate host defenses to control viral translation in HSV-1 infected cells

Lytic replication of many viruses activates an innate host response designed to prevent the completion of the viral lifecycle, thus impeding the spread of the infection. One branch of the host's complex reaction functions to incapacitate the cellular translational machinery on which the synthesis of viral polypeptides completely depends. This is achieved through the activation of specific protein kinases that phosphorylate eIF2 on its alpha subunit and inactivate this critical translation initiation factor. However, as continued synthesis of viral proteins is required to assemble the viral progeny necessary to transmit the infection to neighboring cells, viruses have developed a variety of strategies to counter this cellular response. Genetic and biochemical studies with herpes simplex virus type 1 (HSV-1) have revealed that the virus produces at least two discrete products at different times during its replicative program that act to prevent the accumulation of phosphorylated eIF2alpha. The gamma(1)34.5 gene product is expressed first, encoding a regulatory subunit that binds the cellular protein phosphatase 1alpha and regenerates pools of active eIF2 by removing the inhibitory phosphate from the alpha subunit. The second function, encoded by the product of the Us11 gene, specifies a double-stranded RNA-binding protein that prevents activation of PKR, a cellular eIF2alpha kinase. Together, both proteins cooperate to overcome the antiviral response of the host and properly regulate translation in HSV-1-infected cells.     

PKR is not required for interferon-gamma inhibition of VSV replication in neurons

In this report, the contribution of PKR to the IFN-gamma mediated inhibition of VSV replication in neurons was examined. IFN-gamma treatment of NB41A3 murine neuroblastoma cells resulted in the reduced expression of VSV protein during infection. PKR was found to be modestly upregulated in NB41A3 cells following IFN-gamma treatment. The phosphorylation state of PKR and its downstream target, eIF2alpha, were unaffected by either IFN-gamma or VSV infection. Inhibition of PKR through the use of 2-aminopurine or the expression of the Influenza A NS1 gene had no effect on the ability of IFN-gamma to inhibit the replication of VSV in vitro. These data indicate that endogenously expressed PKR is not required for the IFN-gamma mediated inhibition of VSV replication in NB41A3 neuroblastoma cells.     

Phosphorylation of translation initiation factor eIF2alpha in the brain during pilocarpine-induced status epilepticus in mice

In this work, we show extensive phosphorylation of the alpha subunit of translation initiation factor 2 (eIF2alpha) occurring in the brain of mice subjected to 30 min of status epilepticus induced by pilocarpine. eIF2alpha(P) immunoreactivity was detected in the hippocampal pyramidal layer CA1 and CA3, cortex layer V, thalamus and amygdala. After 2 h of recovery, there was a marked decrease in total brain eIF2alpha(P), with the cortex layer V showing the most pronounced loss of anti-eIF2alpha(P) labeling, whereas the CA1 subregion had a significant increase in eIF2alpha(P). These results indicate that inhibition of protein synthesis in experimental models of epilepsy might be due to low levels of eIF2-GTP caused by the phosphorylation of eIF2alpha, and suggest that translational control may contribute to cell fate in the affected areas.     

PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

dsRNA-dependent protein kinase R (PKR) is a ubiquitously expressed enzyme well known for its roles in immune response. Upon binding to viral dsRNA, PKR undergoes autophosphorylation, and the phosphorylated PKR (pPKR) regulates translation and multiple signaling pathways in infected cells. Here, we found that PKR is activated in uninfected cells, specifically during mitosis, by binding to dsRNAs formed by inverted Alu repeats (IRAlus). While PKR and IRAlu-containing RNAs are segregated in the cytosol and nucleus of interphase cells, respectively, they interact during mitosis when nuclear structure is disrupted. Once phosphorylated, PKR suppresses global translation by phosphorylating the α subunit of eukaryotic initiation factor 2 (eIF2α). In addition, pPKR acts as an upstream kinase for c-Jun N-terminal kinase and regulates the levels of multiple mitotic factors such as CYCLINS A and B and POLO-LIKE KINASE 1 and phosphorylation of HISTONE H3. Disruption of PKR activation via RNAi or expression of a transdominant-negative mutant leads to misregulation of the mitotic factors, delay in mitotic progression, and defects in cytokinesis. Our study unveils a novel function of PKR and endogenous dsRNAs as signaling molecules during the mitosis of uninfected cells.