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Yan-hwa Wu Lee - One of the best experts on this subject based on the ideXlab platform.
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Activation of RNA polymerase I transcription by hepatitis C virus Core Protein.
Journal of biomedical science, 2004Co-Authors: Chih-fei Kao, Shiow-yi Chen, Yan-hwa Wu LeeAbstract:The hepatitis C virus (HCV) Core Protein has been implicated in the transregulation of various RNA polymerase (Pol) II dependent genes as well as in the control of cellular growth and proliferation. In this study, we show that the Core Protein, whether individually expressed or produced as part of the HCV viral polyProtein, is the only viral product that has the potential to activate RNA Pol I transcription. Deletion analysis demonstrated that the fragment containing the N-terminal 1–156 residues, but not the 1–122 residues, of HCV Core Protein confers the same level of transactivation activity as the full-length Protein. Moreover, the integrity of the Ser116 and Arg117 residues of HCV Core Protein was found to be critical for its transregulatory functions. We used DNA affinity chromatography to analyze the human ribosomal RNA promoter associated transcription machinery, and the results indicated that recruitment of the upstream binding factor and RNA Pol I to the ribosomal RNA promoter is enhanced in the presence of HCV Core Protein. Additionally, the HCV Core Protein mediated activation of ribosomal RNA transcription is accompanied by the hyperphosphorylation of upstream binding factor on serine residues, but not on threonine residues. Moreover, HCV Core Protein is present within the RNA Pol I multiProtein complex, indicating its direct involvement in facilitating the formation of a functional transcription complex. Protein-Protein interaction studies further indicated that HCV Core Protein can associate with the selectivity factor (SL1) via direct contact with a specific component, TATA-binding Protein (TBP). Additionally, the HCV Core Protein in cooperation with TBP is able to activate RNA Pol II and Pol III mediated transcription, in addition to RNA Pol I transcription. Thus, the results of this study suggest that HCV has evolved a mechanism to deregulate all three nuclear transcription systems, partly through targeting of the common transcription factor, TBP. Notably, the ability of the HCV Core Protein to upregulate RNA Pol I and Pol III transcription supports its active role in promoting cell growth, proliferation, and the progression of liver carcinogenesis during HCV infection.
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Hepatitis C Virus Core Protein Interacts with Cellular Putative RNA Helicase
Journal of virology, 1999Co-Authors: Li-ru You, Chun-ming Chen, Tien-shun Yeh, Tzung-yuan Tsai, Ru-tsun Mai, Chi-hung Lin, Yan-hwa Wu LeeAbstract:The nucleocapsid Core Protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV Core Protein, a yeast two-hybrid cloning system was used for identification of Core Protein-interacting cellular Protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of Proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV Core Protein with this targeted Protein. Additionally, the Protein’s interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV Core Protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV Core Protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV Core Protein. Moreover, transient expression of HCV Core Protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV Core Protein promotes the trans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid Core Protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of Core Protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.
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Modulation of the trans-suppression activity of hepatitis C virus Core Protein by phosphorylation.
Journal of virology, 1995Co-Authors: Chwen-ming Shih, Chun-ming Chen, Shiow-yi Chen, Yan-hwa Wu LeeAbstract:We previously demonstrated that the Core Protein of hepatitis C virus (HCV) can suppress gene expression and replication of hepatitis B virus (HBV) in a human hepatoma cell line (HuH-7). In this study, we have characterized the phosphorylation property of HCV Core Protein and examined the effect of phosphorylation on its suppressive activity of HBV. Our results indicated that both the full-length HCV Core Protein (22 kDa) and its processed or degraded forms (14 to 18 kDa) were phosphorylated in insect cells. As demonstrated by using the glutathione S-transferase fusion Protein expression system and in vitro transcription and translation system, the phosphorylation of HCV Core Protein was carried out by Protein kinase A (PKA) and Protein kinase C (PKC) in vitro. In both kinase reactions, it was determined that the phosphorylated amino acid was a serine residue. The potential phosphorylated sites in Core Protein were identified as residues Ser-53 and Ser-116 for PKA and Ser-53 and Ser-99 for PKC. Comparison of the phosphorylation intensities of the wild type and Ser mutants suggested that Ser-99 and Ser-116 were the major phosphorylation sites for PKC and PKA, respectively. The phosphorylation of Ser-99 and Ser-116, but not Ser-53, in HCV Core Protein was essential for the suppressive activity of HCV Core Protein on HBV gene expression and replication in HuH-7 cells. Mutation of the former two serine residues to alanine or aspartate residues led to a drastic loss of the inhibitory effects of HCV Core Protein on HBV gene expression (both transcription and antigen production) and pregenomic RNA encapsidation, as well as the release of HBV virus particles. In contrast, the Ser-53 mutant conferred the same level of suppressive activity as the wild type did. This property is in accordance with the observation that Ser-99 and Ser-116 are the predominant phosphorylation sites in the HCV Core construct. All serine mutants (including those with mutations in PKA, PKC, and both kinase recognition sites) of HCV Core Protein retained the ability to translocate into the nucleus. Furthermore, wild-type HCV Core Protein diminished its suppressive activity when cells were treated with PKA or PKC inhibitor. In conclusion, HCV Core Protein is a phospho-Protein and in HuH-7 cells, its trans suppression of HBV gene expression and replication is positively regulated by PKA and PKC. The role of phosphorylation in the control of trans-suppressive activity cannot be reproduced by introducing an acidic residue.(ABSTRACT TRUNCATED AT 250 WORDS)
Yoshiharu Matsuura - One of the best experts on this subject based on the ideXlab platform.
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intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis c virus Core Protein and viral propagation
Journal of Virology, 2008Co-Authors: Kiyoko Okamoto, Kohji Moriishi, Yoshio Mori, Yasumasa Komoda, Toru Okamoto, Masayasu Okochi, Masatoshi Takeda, Tetsuro Suzuki, Yoshiharu MatsuuraAbstract:Hepatitis C virus (HCV) Core Protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the Core Protein has not been determined. The HCV Core Protein is cleaved off from a precursor polyProtein at the lumen side of Ala191 by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV Core Protein in the DRM and in viral propagation. The C terminus of the HCV Core Protein cleaved by SPP in 293T cells was identified as Phe177 by mass spectrometry. Mutations introduced into two residues (Ile176 and Phe177) upstream of the cleavage site of the Core Protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed Core Protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the Core Protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV Core Protein by SPP is required for the localization of the HCV Core Protein in the DRM and for viral propagation.
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Processing and pathogenicity of HCV Core Protein
Uirusu, 2008Co-Authors: Kohji Moriishi, Yoshio Mori, Yoshiharu MatsuuraAbstract:Hepatitis C virus (HCV) is a major causative agent of blood-borne hepatitis. Most of the HCV-positive individuals have been chronically infected with the virus for decades, leading to development of steatosis, cirrhosis and ultimately hepatocellular carcinoma. In addition, cryoglobulinemia and type 2 diabetes mellitus are associated with a chronic infection with HCV. Hepatocellular carcinoma induced by HCV infection is not caused by only the repeated inflammations but also the biological activity of HCV Proteins. HCV Core Protein has been reported as a component of the viral nucleocapsid as well as the pathogenic factor that could induce the production of oxidative stress and progression of cell growth. In this review, we summarize the current status of our knowledge regarding to the processing and pathogenicity of HCV Core Protein.
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Molecular Determinants for Subcellular Localization of Hepatitis C Virus Core Protein
Journal of virology, 2005Co-Authors: Ryosuke Suzuki, Kohji Moriishi, Shinichiro Sakamoto, Takeya Tsutsumi, Akiko Rikimaru, Keiko Tanaka, Takashi Shimoike, Takuya Iwasaki, Kiyohisa Mizumoto, Yoshiharu MatsuuraAbstract:Hepatitis C virus (HCV) Core Protein is a putative nucleocapsid Protein with a number of regulatory functions. In tissue culture cells, HCV Core Protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the Protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion Proteins involving various deletions and point mutations of the HCV Core combined with green fluorescent Proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the Core Protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic α-helix structure. With regard to the nuclear targeting of the Core Protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-α. Differences in the cellular trafficking of HCV Core Protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the Core Protein.
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Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein
Journal of Virology, 2004Co-Authors: Kiyoko Okamoto, Kohji Moriishi, Tatsuo Miyamura, Yoshiharu MatsuuraAbstract:Hepatitis C virus (HCV) Core Protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for Core Protein and as a signal sequence for E1 Protein. The signal sequence of Core Protein is further processed by signal peptide peptidase (SPP). We examined the regions of Core Protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV Core Protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of Core Protein. Precise mutation analyses indicated that replacement of Leu 139 , Val 140 , and Leu 144 of Core Protein by Ala inhibited processing by SPP, but cleavage at the Core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 Protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core Protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of Core Protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed Core Protein. These results indicate that Leu 139 , Val 140 , and Leu 144 in Core Protein play crucial roles in the ER retention and SPP cleavage of HCV Core Protein.
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hepatitis c virus Core Protein induces hepatic steatosis in transgenic mice
Journal of General Virology, 1997Co-Authors: Kyoji Moriya, Yoshiharu Matsuura, Tatsuo Miyamura, Hiroshi Yotsuyanagi, Yoshizumi Shintani, Hajime Fujie, Kotaro Ishibashi, Kazuhiko KoikeAbstract:Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, which finally leads to development of hepatocellular carcinoma. Chronic hepatitis C is characterized by several histological features in the liver which discriminate it from other forms of hepatitis: bile duct damage, lymphoid follicles and steatosis (fatty change). Little is known, however, about the role of HCV or its viral Proteins in the pathogenesis of hepatitis. Recently, the Core Protein of HCV has been suggested to have a transcriptional regulatory function, and thereby to be involved in inducing phenotypic changes in hepatocytes. To clarify whether or not the HCV Core Protein has an effect on pathological phenotypes in the liver, two independent transgenic mouse lines carrying the HCV Core gene were established. These mice developed progressive hepatic steatosis, indicating that the HCV Core Protein plays a direct role in the development of hepatic steatosis, which characterizes hepatitis C. This transgenic mouse system would be a good animal model for the study of pathogenesis in human HCV infection.
Tatsuo Miyamura - One of the best experts on this subject based on the ideXlab platform.
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Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein
Journal of Virology, 2004Co-Authors: Kiyoko Okamoto, Kohji Moriishi, Tatsuo Miyamura, Yoshiharu MatsuuraAbstract:Hepatitis C virus (HCV) Core Protein is suggested to localize to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that acts as a membrane anchor for Core Protein and as a signal sequence for E1 Protein. The signal sequence of Core Protein is further processed by signal peptide peptidase (SPP). We examined the regions of Core Protein responsible for ER retention and processing by SPP. Analysis of the intracellular localization of deletion mutants of HCV Core Protein revealed that not only the C-terminal signal-anchor sequence but also an upstream hydrophobic region from amino acid 128 to 151 is required for ER retention of Core Protein. Precise mutation analyses indicated that replacement of Leu 139 , Val 140 , and Leu 144 of Core Protein by Ala inhibited processing by SPP, but cleavage at the Core-E1 junction by signal peptidase was maintained. Additionally, the processed E1 Protein was translocated into the ER and glycosylated with high-mannose oligosaccharides. Core Protein derived from the mutants was translocated into the nucleus in spite of the presence of the unprocessed C-terminal signal-anchor sequence. Although the direct association of Core Protein with a wild-type SPP was not observed, expression of a loss-of-function SPP mutant inhibited cleavage of the signal sequence by SPP and coimmunoprecipitation with unprocessed Core Protein. These results indicate that Leu 139 , Val 140 , and Leu 144 in Core Protein play crucial roles in the ER retention and SPP cleavage of HCV Core Protein.
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cell cycle perturbation in a human hepatoblastoma cell line constitutively expressing hepatitis c virus Core Protein
Archives of Virology, 2003Co-Authors: Alessandro Ruggieri, Marina Murdolo, Tatsuo Miyamura, Takashi Harada, Maria RapicettaAbstract:Hepatitis C virus (HCV) is one of the major causes of chronic liver disease with the potential for development of hepatocellular carcinoma (HCC). The Core Protein of HCV has been shown to modulate expression of various cellular genes and to influence a number of cellular functions. We investigated the effect of constitutively expressed HCV Core Protein on cell cycle progression in HepG2 cell line, which is derived from a differentiated human hepatoblastoma and shows biosynthetic features similar to human hepatocytes. The results indicated that stable expression of the Core Protein in unsynchronized HepG2 cells induced a perturbation of the cell cycle with reduced cell doubling meantime and increased S phase fraction. Increase of c-myc Protein above the basal expression level was demonstrated with a significant increase of c-myc stability, as revealed by its prolonged intracellular half-life, in HepG2 expressing HCV Core Protein. In contrast, p53 and p21 levels were unchanged. These results suggest that HCV Core Protein may promote cell cycle progression in HepG2 cells possibly through increasing stability of c-myc oncoProtein. These results are in support of important role played by HCV Core Protein in virus-mediated pathogenesis in persistently infected hosts and in hepatocarcinogenesis.
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hepatitis c virus Core Protein induces hepatic steatosis in transgenic mice
Journal of General Virology, 1997Co-Authors: Kyoji Moriya, Yoshiharu Matsuura, Tatsuo Miyamura, Hiroshi Yotsuyanagi, Yoshizumi Shintani, Hajime Fujie, Kotaro Ishibashi, Kazuhiko KoikeAbstract:Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, which finally leads to development of hepatocellular carcinoma. Chronic hepatitis C is characterized by several histological features in the liver which discriminate it from other forms of hepatitis: bile duct damage, lymphoid follicles and steatosis (fatty change). Little is known, however, about the role of HCV or its viral Proteins in the pathogenesis of hepatitis. Recently, the Core Protein of HCV has been suggested to have a transcriptional regulatory function, and thereby to be involved in inducing phenotypic changes in hepatocytes. To clarify whether or not the HCV Core Protein has an effect on pathological phenotypes in the liver, two independent transgenic mouse lines carrying the HCV Core gene were established. These mice developed progressive hepatic steatosis, indicating that the HCV Core Protein plays a direct role in the development of hepatic steatosis, which characterizes hepatitis C. This transgenic mouse system would be a good animal model for the study of pathogenesis in human HCV infection.
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hepatitis c virus Core Protein shows a cytoplasmic localization and associates to cellular lipid storage droplets
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Giovanna Barba, Yoshiharu Matsuura, Michinori Kohara, F Harper, T Harada, S Goulinet, G Eder, Zs Schaff, M J Chapman, Tatsuo MiyamuraAbstract:There is now abundant evidence to substantiate an important role of hepatitis C virus (HCV) Core Protein in cellular gene expression as well as in the viral cycle. Thus the subcellular localization of this Protein has important implications. However, several studies have shown controversial results: the HCV Core has been, indeed, described as cytoplasmic or nuclear depending on the size of the Protein or on the genotype analyzed. We have studied the localization of the HCV Core Protein in two different cell lines, one nonhepatic (CHO) and the other hepatic (HepG2). Double immunofluorescence staining using a nuclear membrane marker and confocal analysis showed the Core Protein pattern to be cytoplasmic and globular. This pattern is not cell cycle-regulated. Electron microscopy analysis revealed the nature of the globular staining observed in immunofluorescence. The HCV Core Protein accumulated at the surface of lipid droplets that were also the unique morphological feature of nonhepatic Core transfected cells. The lipid droplets were isolated by sequential ultracentrifugation on the basis of their density; biochemical analysis revealed a prevalence of triglycerides. In addition the Core Protein colocalized with apolipoProtein AII at the surface of the lipid droplets as revealed by confocal microscopy. Moreover analysis of liver biopsies from chronically HCV-infected chimpanzees revealed that HCV Core is cytoplasmic and localized on the endoplasmic reticulum and on lipid droplets. These results clearly define the subcellular localization of the HCV Core Protein and suggest a relationship between the expression of the HCV Core Protein and cellular lipid metabolism.
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TRANS-SUPPRESSION OF GENE EXPRESSION BY HEPATITIS C VIRAL Core Protein
Japanese journal of medical science & biology, 1994Co-Authors: Dong Wan Kim, Ryosuke Suzuki, Takashi Harada, Izumu Saito, Tatsuo MiyamuraAbstract:We have demonstrated that the truncated hepatitis C (HCV) Core Protein with its C-terminal hydrophobic domains deleted is translocated to the nucleus of transfected cells (22). In this study, intact and truncated Core Proteins of HCV were transiently expressed in a human hepatoblastoma cell line, HepG2, and their effects on the expression of the chloramphenycol acethyl transferase (CAT) gene driven by viral and cellular promoters were examined. The intact Core Protein of 22 kDa which is localized in the cytoplasm of the transfected cells suppressed the expression in all of the promoters tested. They were promoters of the SV40 early region, the c-fos oncogene, the retinoblastoma susceptibility gene, the beta-interferon gene and the beta-actin gene. In contrast, the truncated HCV Core Protein located in the nucleus did not show such a suppressive activity. The HCV Core Protein appears to function not only as a viral structural Protein but as a regulator of gene expression and it might act as a suppressive factor for the cellular gene expression.
Chun-ming Chen - One of the best experts on this subject based on the ideXlab platform.
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Hepatitis C Virus Core Protein Interacts with Cellular Putative RNA Helicase
Journal of virology, 1999Co-Authors: Li-ru You, Chun-ming Chen, Tien-shun Yeh, Tzung-yuan Tsai, Ru-tsun Mai, Chi-hung Lin, Yan-hwa Wu LeeAbstract:The nucleocapsid Core Protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV Core Protein, a yeast two-hybrid cloning system was used for identification of Core Protein-interacting cellular Protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of Proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV Core Protein with this targeted Protein. Additionally, the Protein’s interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV Core Protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV Core Protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV Core Protein. Moreover, transient expression of HCV Core Protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV Core Protein promotes the trans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid Core Protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of Core Protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.
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Modulation of the trans-suppression activity of hepatitis C virus Core Protein by phosphorylation.
Journal of virology, 1995Co-Authors: Chwen-ming Shih, Chun-ming Chen, Shiow-yi Chen, Yan-hwa Wu LeeAbstract:We previously demonstrated that the Core Protein of hepatitis C virus (HCV) can suppress gene expression and replication of hepatitis B virus (HBV) in a human hepatoma cell line (HuH-7). In this study, we have characterized the phosphorylation property of HCV Core Protein and examined the effect of phosphorylation on its suppressive activity of HBV. Our results indicated that both the full-length HCV Core Protein (22 kDa) and its processed or degraded forms (14 to 18 kDa) were phosphorylated in insect cells. As demonstrated by using the glutathione S-transferase fusion Protein expression system and in vitro transcription and translation system, the phosphorylation of HCV Core Protein was carried out by Protein kinase A (PKA) and Protein kinase C (PKC) in vitro. In both kinase reactions, it was determined that the phosphorylated amino acid was a serine residue. The potential phosphorylated sites in Core Protein were identified as residues Ser-53 and Ser-116 for PKA and Ser-53 and Ser-99 for PKC. Comparison of the phosphorylation intensities of the wild type and Ser mutants suggested that Ser-99 and Ser-116 were the major phosphorylation sites for PKC and PKA, respectively. The phosphorylation of Ser-99 and Ser-116, but not Ser-53, in HCV Core Protein was essential for the suppressive activity of HCV Core Protein on HBV gene expression and replication in HuH-7 cells. Mutation of the former two serine residues to alanine or aspartate residues led to a drastic loss of the inhibitory effects of HCV Core Protein on HBV gene expression (both transcription and antigen production) and pregenomic RNA encapsidation, as well as the release of HBV virus particles. In contrast, the Ser-53 mutant conferred the same level of suppressive activity as the wild type did. This property is in accordance with the observation that Ser-99 and Ser-116 are the predominant phosphorylation sites in the HCV Core construct. All serine mutants (including those with mutations in PKA, PKC, and both kinase recognition sites) of HCV Core Protein retained the ability to translocate into the nucleus. Furthermore, wild-type HCV Core Protein diminished its suppressive activity when cells were treated with PKA or PKC inhibitor. In conclusion, HCV Core Protein is a phospho-Protein and in HuH-7 cells, its trans suppression of HBV gene expression and replication is positively regulated by PKA and PKC. The role of phosphorylation in the control of trans-suppressive activity cannot be reproduced by introducing an acidic residue.(ABSTRACT TRUNCATED AT 250 WORDS)
Shiow-yi Chen - One of the best experts on this subject based on the ideXlab platform.
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modulation of p53 transcription regulatory activity and post translational modification by hepatitis c virus Core Protein
Oncogene, 2004Co-Authors: Shiow-yi Chen, Jeouyuan ChenAbstract:Oncogenic virus Proteins often target to tumor suppressor p53 during virus life cycle. In the case of hepatitis C virus (HCV) Core Protein, it has been shown to affect p53-dependent transcription. Here, we further characterized the in vitro and in vivo interactions between HCV Core Protein and p53 and showed that these two Proteins colocalized in subnuclear granular structures and the perinuclear area. By use of a reporter assay, we observed that while low level of HCV Core Protein enhanced the transactivational activity of p53, high level of HCV Core Protein inhibited this activity. In both cases, however, HCV Core Protein increased the p53 DNA-binding affinity in gel retardation analyses, likely due to the hyperacetylation of p53 Lys373 and Lys382 residues. Additionally, HCV Core Protein, depending on its expression level, had differential effects on the Ser15 phosphorylation of p53. Moreover, HCV Core Protein could rescue p53-mediated suppressive effects on both RNA polymerase I and III transcriptions. Collectively, our results indicate that HCV Core Protein targets to p53 pathway via at least three means: physical interaction, modulation of p53 gene regulatory activity and post-translational modification. This feature of HCV Core Protein, may potentially contribute to the HCV-associated pathogenesis.
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Activation of RNA polymerase I transcription by hepatitis C virus Core Protein.
Journal of biomedical science, 2004Co-Authors: Chih-fei Kao, Shiow-yi Chen, Yan-hwa Wu LeeAbstract:The hepatitis C virus (HCV) Core Protein has been implicated in the transregulation of various RNA polymerase (Pol) II dependent genes as well as in the control of cellular growth and proliferation. In this study, we show that the Core Protein, whether individually expressed or produced as part of the HCV viral polyProtein, is the only viral product that has the potential to activate RNA Pol I transcription. Deletion analysis demonstrated that the fragment containing the N-terminal 1–156 residues, but not the 1–122 residues, of HCV Core Protein confers the same level of transactivation activity as the full-length Protein. Moreover, the integrity of the Ser116 and Arg117 residues of HCV Core Protein was found to be critical for its transregulatory functions. We used DNA affinity chromatography to analyze the human ribosomal RNA promoter associated transcription machinery, and the results indicated that recruitment of the upstream binding factor and RNA Pol I to the ribosomal RNA promoter is enhanced in the presence of HCV Core Protein. Additionally, the HCV Core Protein mediated activation of ribosomal RNA transcription is accompanied by the hyperphosphorylation of upstream binding factor on serine residues, but not on threonine residues. Moreover, HCV Core Protein is present within the RNA Pol I multiProtein complex, indicating its direct involvement in facilitating the formation of a functional transcription complex. Protein-Protein interaction studies further indicated that HCV Core Protein can associate with the selectivity factor (SL1) via direct contact with a specific component, TATA-binding Protein (TBP). Additionally, the HCV Core Protein in cooperation with TBP is able to activate RNA Pol II and Pol III mediated transcription, in addition to RNA Pol I transcription. Thus, the results of this study suggest that HCV has evolved a mechanism to deregulate all three nuclear transcription systems, partly through targeting of the common transcription factor, TBP. Notably, the ability of the HCV Core Protein to upregulate RNA Pol I and Pol III transcription supports its active role in promoting cell growth, proliferation, and the progression of liver carcinogenesis during HCV infection.
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Modulation of the trans-suppression activity of hepatitis C virus Core Protein by phosphorylation.
Journal of virology, 1995Co-Authors: Chwen-ming Shih, Chun-ming Chen, Shiow-yi Chen, Yan-hwa Wu LeeAbstract:We previously demonstrated that the Core Protein of hepatitis C virus (HCV) can suppress gene expression and replication of hepatitis B virus (HBV) in a human hepatoma cell line (HuH-7). In this study, we have characterized the phosphorylation property of HCV Core Protein and examined the effect of phosphorylation on its suppressive activity of HBV. Our results indicated that both the full-length HCV Core Protein (22 kDa) and its processed or degraded forms (14 to 18 kDa) were phosphorylated in insect cells. As demonstrated by using the glutathione S-transferase fusion Protein expression system and in vitro transcription and translation system, the phosphorylation of HCV Core Protein was carried out by Protein kinase A (PKA) and Protein kinase C (PKC) in vitro. In both kinase reactions, it was determined that the phosphorylated amino acid was a serine residue. The potential phosphorylated sites in Core Protein were identified as residues Ser-53 and Ser-116 for PKA and Ser-53 and Ser-99 for PKC. Comparison of the phosphorylation intensities of the wild type and Ser mutants suggested that Ser-99 and Ser-116 were the major phosphorylation sites for PKC and PKA, respectively. The phosphorylation of Ser-99 and Ser-116, but not Ser-53, in HCV Core Protein was essential for the suppressive activity of HCV Core Protein on HBV gene expression and replication in HuH-7 cells. Mutation of the former two serine residues to alanine or aspartate residues led to a drastic loss of the inhibitory effects of HCV Core Protein on HBV gene expression (both transcription and antigen production) and pregenomic RNA encapsidation, as well as the release of HBV virus particles. In contrast, the Ser-53 mutant conferred the same level of suppressive activity as the wild type did. This property is in accordance with the observation that Ser-99 and Ser-116 are the predominant phosphorylation sites in the HCV Core construct. All serine mutants (including those with mutations in PKA, PKC, and both kinase recognition sites) of HCV Core Protein retained the ability to translocate into the nucleus. Furthermore, wild-type HCV Core Protein diminished its suppressive activity when cells were treated with PKA or PKC inhibitor. In conclusion, HCV Core Protein is a phospho-Protein and in HuH-7 cells, its trans suppression of HBV gene expression and replication is positively regulated by PKA and PKC. The role of phosphorylation in the control of trans-suppressive activity cannot be reproduced by introducing an acidic residue.(ABSTRACT TRUNCATED AT 250 WORDS)