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) core protein has been shown to interact with the death domain (DD) of tumor necrosis factor receptor-1 (TNFR1). In this study, we further examined the interaction of the core protein with the signaling molecules of TNFR1, including FADD, TRADD, and TRAF2, in a human embryonic kidney cell line, HEK-293, that overexpresses the HCV core protein. This core protein-expressing cell line exhibited enhanced sensitivity to TNF-induced apoptosis. By in vitro binding and in vivo coimmunoprecipitation assays, we showed that the HCV core protein interacted with the DD of FADD and enhanced apoptosis induced by FADD overexpression. This enhancement could be blocked by a dominant-negative mutant of FADD. In contrast, the core protein did not directly interact with the DD of TRADD, but could disrupt the binding of TRADD to TNFR1. TRAF2 recruitment to the TNFR1 signaling complex was also disrupted by the core protein. Correspondingly, TRAF2-dependent activation of the protein kinase JNK was suppressed in the core protein-expressing cells. However, NF kappa B activation by TNF was not significantly altered by the HCV core protein, suggesting the existence of TRAF2-independent pathways for NF kappa B activation. These results combined indicate that the HCV core protein sensitizes cells to TNF-induced apoptosis primarily by facilitating FADD recruitment to TNFR1. The inhibition of JNK activation by the HCV core protein may also contribute to the increased propensity of cells for apoptosis. These results, in comparison with other published studies, suggest that the effects of the HCV core protein and their underlying mechanisms vary significantly among cells of different origins.
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PMID:Hepatitis C virus core protein enhances FADD-mediated apoptosis and suppresses TRADD signaling of tumor necrosis factor receptor. 1133 43

Interferons (IFNs) induce an antiviral state in the cell through complex and indirect mechanisms, which culminate in a direct inhibition of viral replication and stimulation of the host adaptive responses. Viruses often counteract with elaborate strategies to interfere with the induction as well as action of IFN effector molecules. This evolutionary battle between viruses and IFN components is a subject of intense research aimed at understanding the immunopathogenesis of viruses and the molecular basis of IFN signaling and action. In the case with hepatitis C virus (HCV), this may have profound implications for the therapeutic use of recombinant IFN in treating chronic hepatitis C. Depending on the subtype of HCV, current IFN-based treatment regimens are effective for only a small subset of chronic hepatitis C patients. Thus, one of the Holy Grails in HCV research is to understand the mechanisms by which the virus may evade IFN antiviral surveillance and establish persistent infection, which may eventually provide insights into new avenues for better antiviral therapy. Despite the lack of an efficient tissue culture system and an appropriate animal model for HCV infection, several mechanisms have been proposed based on clinical studies and in vitro experiments. This minireview focuses on the HCV NS5A nonstructural protein, which is implicated in playing a role in HCV tolerance to IFN treatment, possibly in part through its ability to inhibit the cellular IFN-induced PKR protein kinase.
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PMID:How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A. 1135 62

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-DeltaN110), 222 (NS5A-DeltaN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-DeltaN110 and NS5A-DeltaN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-kappaB and AP-1 were important in NS5A-DeltaN222 transactivation in the presence of tumor necrosis factor alpha and that NF-IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.
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PMID:Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response. 1139 Jun 11

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.
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PMID:The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis. 1141 75

Interferon therapy may decrease the risk of hepatocellular carcinoma in patients with hepatitis C virus (HCV)-related liver cirrhosis. Interaction of the cellular protein kinase PKR with the PKR-binding domain (PKR-bd) of HCV-NS5A protein may affect cellular growth control and viral resistance to interferon therapy. Mutations within the PKR-bd, which comprises the interferon sensitivity determining region (ISDR), have been associated with interferon sensitivity. To determine whether or not there is an association between HCV heterogeneity and the presence of hepatocellular carcinoma, HCV-1b genomic regions were amplified and directly sequenced from serum samples obtained from 82 patients with liver cirrhosis, 53 with, and 29 without hepatocellular carcinoma. None of them had received antiviral therapy. When compared with the deduced consensus sequence, the median number of amino acid changes in the PKR-bd was higher among samples from patients with (4.22) than from those without hepatocellular carcinoma (1.62; P <.001), and isolates with 3 or more amino acid changes were significantly more common among the former (60%) than among the later (6%, P <.001). No such differences were observed in other viral regions, including Core, E2-HVR-1, E2-PePHD, NS3, and the 5' and 3' PKR-bd flanking regions. In addition, amino acid variation in viral regions other than HVR-1 did not accumulate over time in the analyzed sequential serum samples obtained from patients with or without hepatocellular carcinoma. Therefore, a mutated HCV-PKR-bd phenotype is very common in cirrhotic patients with hepatocellular carcinoma.
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PMID:High amino acid variability within the NS5A of hepatitis C virus (HCV) is associated with hepatocellular carcinoma in patients with HCV-1b-related cirrhosis. 1143 47

Two genomic regions of hepatitis C virus (HCV), the interferon sensitivity-determining region (ISDR) of the non-structural 5A gene (NS5A) and the protein kinase-RNA activated (PKR)-eukariotic transcription factor (eIF2-alpha) phosphorylation homology domain (PePHD) of the structural E2 gene, interact in vitro with the interferon-inducible cellular PKR protein kinase. Mutations within these regions might, therefore, influence the response to interferon therapy. Viral load at baseline and sequence heterogeneity of HCV in NS5A and E2 regions was studied in 74 HCV-1b and in 12 HCV-3a infected patients with chronic hepatitis C who were treated with interferon. As previously reported by us, in a smaller series of patients in which the ISDR region was analyzed [Saiz et al. (1998) Journal Infectious Diseases 177:839-847], in the present study a low viral load and a high number of amino acid mutations within the ISDR, but not within the PePHD region, were significantly associated with long-term response to interferon among HCV-1b infected patients. No relationship between these viral features and response to therapy was disclosed in patients infected with HCV-3a.
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PMID:Influence of the genetic heterogeneity of the ISDR and PePHD regions of hepatitis C virus on the response to interferon therapy in chronic hepatitis C. 1150 41

Hepatitis C virus (HCV) core protein is known to repress the transcription of p21(waf1) directly in a p53-independent manner. In this study, the region of HCV core protein responsible for the transcriptional repression of p21 promoter was determined. N-terminal half of core protein almost completely lost the ability to repress p21 promoter, indicating that the domain required for the majority of p21 repression is located between amino acid positions 84 and 191. The trans-repression activity of HCV core mutant S99L on p21 gene expression was similar to that of wild type core protein whereas mutation of the 116th amino acid Ser into either Ile or Ala completely abolished the repressive ability of HCV core protein. In addition, the trans-repression activity of HCV core mutant S116D was similar to that of wild type core protein, suggesting that an acidic aspartate residue can mimic the effect of phosphorylation. When treated with a protein kinase A (PKA) inhibitor, H-89, the inhibitory activity of wild-type HCV core protein was dose-dependently decreased and was completely lost at the concentration of 5 microM. On the contrary, the repression activity of HCV core protein was increased by treatment with a PKA activator, dibutyryl-cAMP, indicating that the p21 repressive activity of HCV core protein is regulated by phosphorylation at S-116 by protein PKA
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PMID:The repressive activity of hepatitis C virus core protein on the transcription of p21(waf1) is regulated by protein kinase A-mediated phosphorylation. 1155 51

The hepatitis C virus (HCV) envelope protein E2 has been shown to accumulate in the lumen of the endoplasmic reticulum (ER) as a properly folded glycoprotein as well as large aggregates of misfolded proteins. In the present study, we have identified an additional unglycosylated species, with an apparent molecular mass of 38 kDa (E2-p38). In contrast to the glycosylated E2, E2-p38 is significantly less stable and is degraded through the proteasome pathway. Correspondingly, E2-p38 is found to be ubiquitinated. E2-p38 is localized mostly in the cytosol, in contrast to the glycosylated form, which is exclusively membrane associated. Alpha interferon (IFN-alpha) treatment or overexpression of the double-stranded RNA-activated protein kinase (PKR) significantly increased the stability of E2-p38, consistent with a previous report (D. R. Taylor, S. T. Shi, P. R. Romano, G. N. Barber, and M. M. Lai, Science 285:107-110, 1999) that E2 interacts with PKR and inhibits its kinase activity. Direct interaction between PKR and E2-p38, but not the glycosylated form of E2, was also observed. These results show that E2-p38 is the form of E2 that interacts with PKR in the cytosol and may contribute to the resistance of HCV to IFN-alpha. Thus, an ER protein can exist in the cytosol as an unglycosylated species and impair cellular functions.
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PMID:Detection of a novel unglycosylated form of hepatitis C virus E2 envelope protein that is located in the cytosol and interacts with PKR. 1177 2

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5'-nontranslated region (5'NTR). We investigated the effect of interferon alfa (IFN-alpha) on the IRES-directed translation of HCV, using two stably transformed cell lines, RCF-1 and RCF-26, of Huh7 cells derived from human hepatocellular carcinoma that express dicistronic reporter proteins, Renilla luciferase (RL) and firefly luciferase (FL), separated by HCV-IRES. After the administration of IFN-alpha or poly(I)-poly(C), HCV-IRES-directed translation was inhibited in a dose-dependent manner. The relative HCV-IRES activity (F/L) decreased to 60% at 5,000 IU/mL of IFN-alpha and 45% at 40 microg/mL of poly(I)-poly(C). Thus, IFN-alpha or poly(I)-poly(C) inhibited HCV-IRES-directed translation more efficiently than a cellular cap-dependent translation. 2',5'-oligoadenylate synthetase (2',5'AS) protein level in cells analyzed significantly increased after the administration of IFN-alpha, but not upon poly(I)-poly(C). Overexpression of double-stranded RNA-activated protein kinase (PKR) gene did not mimic the selective inhibition of HCV-IRES-directed translation in the transformant cells, suggesting that neither the 2',5'AS nor the PKR system are involved in this selective inhibition. Interestingly, the expression of the autoantigen, La, which has been reported to enhance HCV-IRES-directed translation, was significantly reduced after the administration of IFN-alpha and poly(I)-poly(C) in a dose-dependent manner. Transient expression of La protein completely restored the selective inhibition of HCV-IRES-directed translation by IFN-alpha and poly(I)-poly(C). These findings suggested a new antiviral mechanism induced by IFN-alpha in that IFN-alpha or poly(I)-poly(C) selectively inhibited HCV-IRES-directed translation compared with the eukaryotic cap-dependent translation through the reduction of La protein.
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PMID:Inhibition of internal ribosomal entry site-directed translation of HCV by recombinant IFN-alpha correlates with a reduced La protein. 1178 77

The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2). The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity.
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PMID:Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells. 1183


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