Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to antagonize numerous cellular pathways, including the antiviral interferon-alpha response. However, the capacity of this protein to interact with the viral polymerase suggests a more direct role for NS5A in genome replication. In this study, we employed two bacterially expressed, soluble derivatives of NS5A to probe for novel functions of this protein. We find that NS5A has the capacity to bind to the 3'-ends of HCV plus and minus strand RNAs. The high affinity binding site for NS5A in the 3'-end of plus strand RNA maps to the polypyrimidine tract, an element known to be essential for genome replication and infectivity. NS5A has a preference for single-stranded RNA containing stretches of uridine or guanosine. Values for the equilibrium dissociation constants for high affinity binding sites were in the 10 nM range. Two-dimensional gel electrophoresis followed by Western blotting revealed the presence of unphosphorylated NS5A in Huh-7 cells stably expressing the subgenomic replicon. Moreover, RNA immunoprecipitation and NS5A pull-down experiments showed the capacity of replicon-derived NS5A to bind to synthetic RNA and the HCV genome, respectively. Deletion of all of the casein kinase II phosphorylation sites in NS5A supported stable replication of a subgenomic replicon in Huh-7. However, this derivative could not be labeled with inorganic phosphate, suggesting that extensive phosphorylation of NS5A is not required for the replication functions of NS5A. The discovery that NS5A is an RNA-binding protein defines a new functional target for development of agents to treat HCV infection and a new structural class of RNA-binding proteins.
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PMID:Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. 1612 20

The aim of this review is to describe the role of hepatitis C proteins, non-structural protein 5A and envelope protein E2, in resistance to interferon alpha. These proteins contain interferon induced-protein kinase R binding domains. The binding renders the kinase inactive; therefore the phosphorylation of translation factor eIF2 is inhibited. The studies indicate that phosphorylation of eIF4E is also inhibited. As a result, with the sufficient pool of active eIF2 in infected cell, synthesis of viral proteins proceeds while cap- and cap binding factors-, among them eIF4E, -dependent synthesis of host proteins is diminished. It seems this process is one of the molecular mechanisms responsible for the resistance of hepatitis C virus to interferon, persistence in infected cell and the resultant difficulties in treatment of infected individuals.
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PMID:[The mechanisms of hepatitis C virus resistance to interferon]. 1620 38

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infection associated with severe liver disease. HCV nonstructural protein 5A (NS5A) is essential for viral replication. Here, the kinase Raf-1 was identified as a novel cellular binding partner of NS5A, binding to the C-terminal domain of NS5A. Raf-1 colocalizes with NS5A in the HCV replication complex. The interaction of NS5A with Raf-1 results in increased Raf-1 phosphorylation at serine 338. Integrity of Raf-1 is crucial for HCV replication: inhibition of Raf-1 by the small-molecule inhibitor BAY43-9006 or downregulation of Raf-1 by siRNA attenuates viral replication.
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PMID:Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication. 1640 65

Some hepatitis C virus (HCV) proteins, including core protein, deregulate the cell cycle of infected cells, thereby playing an important role in the viral pathogenesis of HCC. Thus far, there are only few studies that have deeply investigated in depth the effects of the HCV core protein expression on the progression through the G1/S and G2/M phases of the cell cycle. To shed light on the molecular mechanisms by which the HCV core protein modulates cell proliferation, we have examined its effects on cell cycle in hepatocarcinoma cells. We show here that HCV core protein perturbs progression through both the G1/S and the G2/M phases, by modulating the expression and the activity of several cell cycle regulatory proteins. In particular, our data provided evidence that core-dependent deregulation of the G1/S phase and its related cyclin-CDK complexes depends upon the ERK1/2 pathway. On the other hand, the viral protein also increases the activity of the cyclin B1-CDK1 complex via the p38 MAPK and JNK pathways. Moreover, we show that HCV core protein promotes nuclear import of cyclin B1, which is affected by the inhibition of both the p38 and the RNA-dependent protein kinase (PKR) activities. The important role of p38 MAPK in regulating G2/M phase transition has been previously documented. It is becoming clear that PKR has an important role in regulating both the G1/S and the G2/M phase, in which it induces M phase arrest. Based on our model, we now show, for the first time, that HCV core expression leads to deregulation of the mitotic checkpoint via a p38/PKR-dependent pathway.
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PMID:Role of p38 MAPK and RNA-dependent protein kinase (PKR) in hepatitis C virus core-dependent nuclear delocalization of cyclin B1. 1644 63

Mutations in several subgenomic regions have been implicated in influencing response to interferon therapy; however, a comprehensive picture of Indian patients was lacking. Based on the viral load and clinical factors, 10 out of 15 patients were found to be complete responders, whereas 5 patients were nonresponders. The pretreatment viral RNA load of the patients was found to be between 5.20 and 6.13 log10 IU/ml, which eventually fell to 2.77 log10 IU/ml after 24 weeks of treatment, whereas in the case of nonresponders, the average was 5.38 log10 IU/ml. In order to study the influence of the hepatitis C virus genotype on the response to interferon therapy, the 5' untranslated region sequences of the samples were analyzed, which showed that genotype 3 patients responded better than genotype 1 patients. Additionally, the mutations in the interferon sensitivity-determining region (ISDR) of the NS5A protein and the double-stranded RNA-activated protein kinase-eukaryotic initiation factor 2 alpha phosphorylation homology domain (PePHD) of the E2 envelope protein, before and after treatment, were compared with nonresponder prototype J. Although, no clear correlation was found in the case of the mutated ISDR, some significant changes in residues were observed in the PePHD region, which could be helpful in understanding the molecular basis of resistance to therapy. Interestingly, analysis of the quasispecies variations showed a change in genotype in one sample during treatment, which might have contributed to the resistance. The results suggest that the mutations in different regions of the viral genome might have a concerted effect on the response to interferon therapy.
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PMID:Analysis of mutations within the 5' untranslated region, interferon sensitivity region, and PePHD region as a function of response to interferon therapy in hepatitis C virus-infected patients in India. 1651 43

RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.
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PMID:Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons. 1656 96

Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNA-dependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-alpha-induced NF-kappaB and JNK activation. In this study, we have demonstrated that TNF-alpha-induced NF-kappaB activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2- and IKK-induced NF-kappaB activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKKalpha. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKKalpha, and then TNF-alpha-mediated IKKalpha kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-alpha-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-alpha signaling pathways and may contribute to HCV pathogenesis.
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PMID:Hepatitis C virus nonstructural 5B protein regulates tumor necrosis factor alpha signaling through effects on cellular IkappaB kinase. 1658 80

The plasmid pET-21d-2c-5BDelta55 effectively expressing a C-terminally truncated form (NS5BDelta55) of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was constructed. It was derived from pET-21d-5BDelta55 plasmid and contained six mutations in the ATG-start codon region and an additional cistron upstream the target gene. The C-terminally His-tagged NS5BDelta55 protein was expressed in Rosetta(DE3) Escherichia coli strain bearing an additional pRARE plasmid encoding extra copies of rare tRNAs. The yield of the target enzyme exceeded by a factor of 29 the yield of NS5BDelta55 protein expressed from the parental pET-21d-5BDelta55 plasmid (5 mg/L). The increase in the protein yield could be explained by facilitated protein translation initiation, resulted from disruption of the stable secondary mRNA structure. The pET-21d-2c-5BDelta55 plasmid yielded one third amount of the protein when expressed in BL-21(DE3) strain, indicating that the pRARE plasmid is required for a high-level expression of NS5BDelta55 protein. The 29-fold enhancement of the protein yield was accompanied by only a 2.5-fold increase of the corresponding mRNA level. The expression of another HCV NS5A protein His-tagged at the C-terminus in the developed system yielded a similar amount of the protein (4 mg/L), whereas its N-terminally His-tagged counterpart was obtained in a 30 mg/L yield. The NS5A protein purified under denaturing conditions and renatured in solution inhibited the HCV RdRp and was a substrate for human casein kinase II.
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PMID:Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins. 1660 Jun 28

We analyzed protein kinase R (PKR)-binding domain sequences of hepatitis C virus (HCV) NS5A protein and the profile of HCV-specific antibodies from pretreatment sera of HCV-chronically infected patients. Results were compared with clinical data to verify their influence on the course and result of therapy. Of 9 patients enrolled in a 12-month treatment with pegylated interferon alpha (PEG-IFN-alpha) plus ribavirin (RBV), 6 patients responded to therapy, as assessed by the lack of HCV RNA in their sera, and 3 did not. Among 8 HCV-1b-infected patients, those who responded did not have significantly more mutations in the IFN sensitivity determining region (ISDR) compared to non-responders (P = 0.637). Similarly, in the remaining 26-amino acid region of the PKR-binding domain, behind ISDR, the number of mutations did not differ significantly between the two groups (P = 0.796). A correlation was found between the presence of envelope 2 (E2)-specific antibodies and the result of treatment (P = 0.048). This pilot study indicates that mutations in the PKR-binding domain of HCV genotype 1b do not correlate with outcome of PEG-IFN-alpha/RBV therapy. However, the presence of E2-specific antibodies in the pretreatment sera of HCV-chronically infected individuals could serve as a prognostic marker predicting the result of treatment, before its initiation.
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PMID:Mutations within protein kinase R-binding domain of NS5A protein of hepatitis C virus (HCV) and specificity of HCV antibodies in pretreatment sera of HCV-chronically infected patients and their effect on the result of treatment. 1663 8

Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspects of the virus life cycle are still a mystery. The HCV nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulating HCV replication. Using kinase inhibitors that specifically inhibit the formation of NS5A-p58 in cells, we identified the CKI kinase family as a target. NS5A-p58 increased upon overexpression of CKI-alpha, CKI-delta, and CKI-epsilon, whereas the RNA interference of only CKI-alpha reduced NS5A hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by CKI-alpha overexpression, and we demonstrated that the CKI-alpha isoform is targeted by NS5A hyperphosphorylation inhibitors in living cells. Finally, we showed that down-regulation of CKI-alpha attenuates HCV RNA replication.
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PMID:The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation. 1694 83


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