Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) genotype 1 is thought to interact with several cellular proteins, including the double-stranded RNA-dependent protein kinase (PKR) induced by interferon (IFN). The PKR-binding domain (PKR-BD; aa 2209-2274), including the IFN sensitivity-determining region (aa 2209-2248) and other regions, could be linked to IFN resistance. Thus, the entire NS5A sequence of 27 isolates of HCV genotype 3a was investigated in relation to the clinical response to IFN. The NS5A 3a protein presented a low variability with some specific variable regions. Differential analysis between IFN-resistant and -sensitive isolates identified 5 regions in NS5A, 2 of them inside the PKR-BD and another around the variable 3 region. However, using the yeast growth suppression assay, no interaction was found between 5 resistant NS5A 3a proteins and PKR. Some amino acid changes of the NS5A protein of genotype 3a seemed to relate to IFN resistance independently of the PKR pathway.
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PMID:Variability of the nonstructural 5A protein of hepatitis C virus type 3a isolates and relation to interferon sensitivity. 1186 13

The virus-host interactions that influence hepatitis C virus (HCV) replication are largely unknown but are thought to involve those that disrupt components of the innate intracellular antiviral response. Here we examined cellular antiviral pathways that are triggered during HCV RNA replication. We report that (i) RNA replication of HCV subgenomic replicons stimulated double-stranded RNA (dsRNA) signaling pathways within cultured human hepatoma cells, and (ii) viral RNA replication efficiency corresponded with an ability to block a key cellular antiviral effector pathway that is triggered by dsRNA and includes IFN regulatory factor-1 (IRF-1) and protein kinase R (PKR). The block to dsRNA signaling was mapped to the viral nonstructural 5A (NS5A) protein, which colocalized with PKR and suppressed the dsRNA activation of PKR during HCV RNA replication. NS5A alone was sufficient to block both the activation of IRF-1 and the induction of an IRF-1-dependent cellular promoter by dsRNA. Mutations that clustered in or adjacent to the PKR-binding domain of NS5A relieved the blockade to this IRF-1 regulatory pathway, resulting in induction of IRF-1-dependent antiviral effector genes and the concomitant reduction in HCV RNA replication efficiency. Our results provide further evidence to support a role for PKR in dsRNA signaling processes that activate IRF-1 during virus infection and suggest that NS5A may influence HCV persistence by blocking IRF-1 activation and disrupting a host antiviral pathway that plays a role in suppressing virus replication.
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PMID:Regulation of PKR and IRF-1 during hepatitis C virus RNA replication. 1190 69

The interferon (IFN) sensitivity-determining region (ISDR) of the hepatitis C virus (HCV) NS5A protein is controversially implicated in determining IFN-alpha-sustained viral response for HCV genotype 1-infected patients. Because the NS5A protein interferes with protein kinase antiviral activity, this study attempted to determine whether ISDR amino acid mutation number would correlate better with IFN effectiveness to inhibit virus production and elicit early virus clearance. Early viral kinetic data from 22 genotype 1-infected patients treated with high-dose IFN was compared with ISDR mutation number. IFN effectiveness and first-phase viral log decline correlated directly with ISDR mutation number (P=.02). Mean mutation number was higher among rapid responders (3.7+/-1.0; n=6) than among nonresponders (1.3+/-1.0; n=16) (P=.001). Also, second-phase viral log decline and calculated hepatocyte death rate correlated directly with ISDR mutation number (P=.01). This supports a dual effector role for NS5A, which regulates virus production and immune clearance early in therapy.
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PMID:Relationship of hepatitis C genotype 1 NS5A sequence mutations to early phase viral kinetics and interferon effectiveness. 1192 Mar 10

As popular strategies used by numerous viruses, interception of interferon (IFN) signaling and inhibition of IFN-induced antiviral functions allow viruses to evade the host immune response and set up successful infections. Hepatitis C virus (HCV), the leading cause of chronic liver disease worldwide and a major public health hazard, causes persistent infection in the majority of infected individuals. IFN-based therapies, currently the only ones available for HCV infection, have been unable to eliminate viral infection in the majority of patients, and many studies suggest that HCV possesses mechanisms to antagonize the IFN-induced antiviral response. Multiple viral, host, and IFN-associated factors have been implicated in the interplay between HCV and IFN. Two viral proteins, NS5A and E2, became the focus of much attention and extensive study because of their abilities to inhibit IFN-induced, double-stranded RNA-activated protein kinase (PKR), a major mediator of the IFN-induced biologic response, and to perturb the IFN signaling pathway. In this review, we discuss the significance of the interferon sensitivity determining region (ISDR) within NS5A, which has been the subject of intense debates. In addition, we discuss the potential mechanisms by which NS5A interferes with IFN signaling and the current working models. Further understanding of the molecular mechanisms underlying the interaction between HCV and IFN will likely facilitate improvement of current IFN-based therapies and development of novel treatments for the HCV pandemic. Future HCV research will benefit from both the development of efficient, convenient model systems for viral propagation, and the utilization of high throughput, genomic-scale approaches.
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PMID:To interfere and to anti-interfere: the interplay between hepatitis C virus and interferon. 1195 50

The hepatitis C virus (HCV) NS5A gene product is a phosphorylated 56- to 58-kD nonstructural protein that displays a multitude of activities related to enhancement of viral pathogenesis. Although associated with other viral encoded proteins as part of the viral replicase complex positioned on the cytoplasmic side of the endoplasmic reticulum, a role for NS5A in viral replication has not been defined. Post-translational modifications of NS5A include phosphorylation and potential proteolytic processing to smaller molecular weight forms able to translocate to the nucleus. Both the identification of a putative interferon (IFN) sensitivity-determining region within NS5A, as well as the direct interaction with and inhibition of the IFN-induced double-stranded RNA-dependent protein kinase (PKR) by NS5A remain controversial. Truncated versions of NS5A can act as transcriptional activators, while other recently characterized interactions of NS5A with cellular proteins indicate its pleiotropic role in HCV-host interactions. NS5A itself has no direct effect on IFN-alpha signaling or activation, but other abundant interactions with members of the cellular signaling apparatus, transcription activation machinery and cell cycle-regulatory kinases have been described (e.g. growth factor receptor-bound protein 2, p53, p21/waf and cyclins). Many of these interactions block the apoptotic cellular response to persistent HCV infection. More recently, another altogether different mechanism attenuating the IFN-alpha response was reported based on induction of interleukin (IL)-8. IL-8, in model systems, potentiates viral replication and mutes the nonspecific intracellular IFN antiviral response. Evidence supporting a complex multimechanistic role of NS5A in promoting viral persistence, pathogenesis and, indirectly, viral-related hepatocarcinogenesis indicates its key role in HCV pathobiology.
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PMID:The nonstructural NS5A protein of hepatitis C virus: an expanding, multifunctional role in enhancing hepatitis C virus pathogenesis. 1206 93

Interferon (IFN) therapy is used worldwide as the best available treatment for hepatitis C virus (HCV) infection; however, little is known about how IFN or other drugs work against liver diseases. The effect of 6 drugs (glycyrrhizin, ursodeoxycholic acid, ribavirin, methylprednisolone, IFN-alpha, and IFN-beta) on HCV RNA translation from the HCV internal ribosome entry site (IRES) was investigated, using a bicistronic reporter containing the HCV IRES. IFNs suppressed both cap-dependent and HCV IRES-dependent translation, with HCV IRES-dependent translation being more significantly suppressed. In contrast to HCV IRES, IFN did not suppress either foot-and-mouth disease virus IRES-dependent or encephalomyocarditis virus IRES-dependent translation more than it suppressed cap-dependent translation. Moreover, dominant inhibition of HCV IRES-dependent over cap-dependent translation depended neither on the double-stranded RNA-activated protein kinase activation nor on La protein function. These results indicate a novel antiviral effect of IFNs against HCV.
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PMID:Interferons specifically suppress the translation from the internal ribosome entry site of hepatitis C virus through a double-stranded RNA-activated protein kinase-independent pathway. 1213 50

Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.
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PMID:Subversion of cell signaling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and P85 phosphatidylinositol 3-kinase. 1218 4

Hepatitis C virus (HCV) core protein can form capsid-like particles and is believed to be the viral capsid protein. Besides its structural functions, this protein is also known to possess multiple regulatory functions. In this article, we have studied the possible phosphorylation of HCV core protein in two different human liver-derived cell lines Huh7 and HepG2. Our results indicated that the HCV core protein could be phosphorylated, albeit inefficiently, independent of its downstream E1 protein in these two cell lines. Two of the basal phosphorylation sites were identified to be serine-53 and serine-116. The phosphorylation of the core protein could be enhanced by the PKC activator phorbol 12-myristic 13-acetate (PMA), and the PKA activator forskolin, and these enhancements could be abolished by the respective inhibitors of PKC and PKA, indicating that the core protein is a substrate of these two kinases. While both serine-53 and serine-116 served as the PKC phosphorylation sites, serine-116 appeared to be the major PKA phosphorylation site. Further analyses using serine-to-alanine mutation to mimic dephosphorylation and serine-to-aspartic acid mutation to mimic phosphorylation revealed that the conversion of serine-116 to aspartic acid led to an enhanced nuclear localization of the core protein. This observation indicates that one function of phosphorylation may be to regulate the nuclear localization of the core protein.
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PMID:Phosphorylation of hepatitis C virus core protein by protein kinase A and protein kinase C. 1220 2

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) may repress the interferon (IFN)-induced protein kinase R (PKR). High variability of different regions in the carboxy-terminal half of NS5A implicated in the interaction with PKR (particularly the interferon sensitivity determining region (ISDR)) may be a predictor of response to IFN in patients infected with genotype 1b of HCV. We examined pretreatment serum samples from 17 HCV-1b infected patients included in the same schedule of IFN therapy. Seven patients were a rare series of sustained responders (SR) with a post-treatment follow-up of 5-7 years, while ten were nonresponders (NR). The carboxy-terminal half of the NS5A gene was amplified and directly sequenced in all 17 cases. In addition, the entire NS5A gene and the part of the HCV E2 gene coding for the hypervariable region 1 (HVR1) were amplified, cloned and sequenced in six cases (three NR and three SR). No difference in number and distribution of amino acid mutations was observed between isolates from SR and NR in any portion of the protein, including the ISDR region. Analysis of full length NS5A confirmed no difference between the two groups. The NS5A gene sequence was different among the six cases cloned although it appeared to be conserved in each individual patient independently of the quasispecies complexity evaluated through HVR1 examination. These data indicate that pretreatment analysis of theNS5A genomic variability has no value in predicting long-lasting response to IFN therapy in HCV-1b-infected patients, and that the HCV NS5A gene has high quasispecies homology.
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PMID:Is investigation of hepatitis C virus NS5A gene heterogeneity a tool for predicting long-lasting response to interferon therapy in patients with HCV-1b chronic hepatitis? 1222 31

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2alpha phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2alpha phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-alpha is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2alpha phosphorylation is induced by IFN-alpha in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2alpha phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2alpha phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3' untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.
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PMID:PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone. 1236 6


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