Eliane F. Meurs

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.

Cutting Edge: Influenza A Virus Activates TLR3-Dependent Inflammatory and RIG-I-Dependent Antiviral Responses in Human Lung Epithelial Cells

Influenza A virus (IAV) triggers a contagious acute respiratory disease that causes considerable mortality annually. Recently, we established a role for the pattern-recognition TLR3 in the response of lung epithelial cells to IAV-derived dsRNA. However, additional nucleic acid-recognition proteins have lately been implicated as key viral sensors, including the RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene (MDA)-5. In this study, we investigated the respective role of TLR3 vs RIG-I/MDA-5 signaling in human respiratory epithelial cells infected by IAV using BEAS-2B cells transfected with vectors encoding either a dominant-negative form of TLR3 or of mitochondrial antiviral signaling protein (MAVS; a signaling intermediate of RIG-I and MDA-5), or with plasmids overexpressing functional RIG-I or MDA-5. We demonstrate that the sensing of IAV by TLR3 primarily regulates a proinflammatory response, whereas RIG-I (but not MDA-5) mediates both a type I IFN-dependent antiviral signaling and a proinflammatory response.

Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.

The human p68 kinase is an interferon‐regulated enzyme that inhibits protein synthesis when activated by double‐stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N‐terminus, shown to bind double‐stranded RNA in vitro and expression of a mutant form of the alpha‐subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha‐subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.

Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKepsilon molecular complex from the mitochondrial outer membrane by hepatitis C virus NS3-4A proteolytic cleavage

ABSTRACT Intracellular RNA virus infection is detected by the cytoplasmic RNA helicase RIG-I that plays an essential role in signaling to the host antiviral response. Recently, the adapter molecule that links RIG-I sensing of incoming viral RNA to downstream signaling and gene activation events was characterized by four different groups; MAVS/IPS-1-1/VISA/Cardif contains an amino-terminal CARD domain and a carboxyl-terminal mitochondrial transmembrane sequence that localizes to the mitochondrial membrane. Furthermore, the hepatitis C virus NS3-4A protease complex specifically targets MAVS/IPS-1/VISA/Cardif for cleavage as part of its immune evasion strategy. With a novel search program written in python, we also identified an uncharacterized protein, KIAA1271 (K1271), containing a single CARD-like domain at the N terminus and a Leu-Val-rich C terminus that is identical to that of MAVS/IPS-1/VISA/Cardif. Using a combination of biochemical analysis, subcellular fractionation, and confocal microscopy, we now demonstrate that NS3-4A cleavage of MAVS/IPS-1/VISA/Cardif/K1271 results in its dissociation from the mitochondrial membrane and disrupts signaling to the antiviral immune response. Furthermore, virus-induced IKKε kinase, but not TBK1, colocalized strongly with MAVS at the mitochondrial membrane, and the localization of both molecules was disrupted by NS3-4A expression. Mutation of the critical cysteine 508 to alanine was sufficient to maintain mitochondrial localization of MAVS/IPS-1/VISA/Cardif and IKKε in the presence of NS3-4A. These observations provide an outline of the mechanism by which hepatitis C virus evades the interferon antiviral response.

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

ABSTRACT 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.

Inhibition of RIG-I-Dependent Signaling to the Interferon Pathway during Hepatitis C Virus Expression and Restoration of Signaling by IKKε

ABSTRACT Interferon (IFN) is one important effector of the innate immune response, induced by different viral or bacterial components through Toll-like receptor (TLR)-dependent and -independent mechanisms. As part of its pathogenic strategy, hepatitis C virus (HCV) interferes with the innate immune response and induction of IFN-β via the HCV NS3/4A protease activity which inhibits phosphorylation of IRF-3, a key transcriptional regulator of the IFN response. In the present study, we demonstrate that inhibition by the protease occurs upstream of the noncanonical IKK-related kinases IKKε and TBK-1, which phosphorylate IRF-3, through partial inhibition of the TLR adapter protein TRIF/TICAM1-dependent pathway. Use of TRIF−/− mouse embryo fibroblasts however revealed the presence of a TRIF-independent pathway involved in IFN induction that was also inhibited by NS3/4A. Importantly, we show that NS3/4A can strongly inhibit the ability of the recently described RIG-I protein to activate IFN, suggesting that RIG-I is a key factor in the TRIF-independent, NS3/4A-sensitive pathway. Expression of IFN signaling components including IKKε, TBK-1, TRIF, and wild type or constitutively active forms of RIG-I in the HCV replicon cells resulted in IFN-β promoter transactivation, with IKKε displaying the highest efficiency. Subsequently, overexpression of IKKε resulted in 80% inhibition of both the positive and negative replicative strands of the HCV replicon. The partial restoration of the capacity of the host cell to transcribe IFN-β indicates that IKKε expression is able to bypass the HCV-mediated inhibition and restore the innate antiviral response.

Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.

Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain II. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors.

PKR Stimulates NF-κB Irrespective of Its Kinase Function by Interacting with the IκB Kinase Complex

ABSTRACT The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-κB. NF-κB is retained in the cytoplasm through binding to its inhibitor IκBα. The critical step in NF-κB activation is the phosphorylation of IκBα by the IκB kinase (IKK) complex. This activity releases NF-κB from IκBα and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-κB in a reporter assay and have shown for the first time that two catalytically inactive PKR mutants, PKR/KR296 and a deletion mutant (PKR/Del42) which lacks the potential eIF2α-binding domain, can also activate NF-κB. This result indicated that NF-κB activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2α relationship. Transfection of either wild-type PKR or catalytically inactive PKR in PKR0/0 mouse embryo fibroblasts resulted in the activation of the IKK complex. By using a glutathioneS-transferase pull-down assay, we showed that PKR interacts with the IKKβ subunit of the IKK complex. This interaction apparently does not require the integrity of the IKK complex, as it was found to occur with extracts from cells deficient in the NF-κB essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-κB signaling pathway without using its kinase activity.

Serum-Derived Hepatitis C Virus Infection of Primary Human Hepatocytes Is Tetraspanin CD81 Dependent

ABSTRACT Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.

Expression of Hepatitis C Virus Proteins Interferes with the Antiviral Action of Interferon Independently of PKR-Mediated Control of Protein Synthesis

ABSTRACT Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an IFN-induced kinase involved in the antiviral action of IFN, at the level of control of protein synthesis through the phosphorylation of the initiation factor eIF2α (M. Gale, Jr., C. M. Blakely, B. Kwieciszewski, S. L. Tan, M. Dossett, N. M. Tang, M. J. Korth, S. J. Polyak, D. R. Gretch, and M. G. Katze, Mol. Cell. Biol. 18:5208–5218, 1998). Accordingly, cell lines inducibly expressing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:1262–1271, 1999). In the present study we analyzed whether the resistance of UHCV-11 cells to IFN could also be attributed to inhibition of PKR. Confocal laser scanning microscopy showed no colocalization of PKR, which is diffuse throughout the cytoplasm, and the induced HCV proteins, which localize around the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by direct analysis of the in vivo phosphorylation of eIF2α after treatment of cells with poly(I)-poly(C). We found that neither PKR activity nor eIF2α phosphorylation was affected by coexpression of the HCV proteins. In conclusion, expression of HCV proteins in their biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.