Effect Of Hepatitis C Virus Core Protein Research Articles

Cloning and identification of human gene 1 transactivated by hepatitis B virus X antigen

AIM: To clone and identify of human gene 2 transactivated by hepatitis C virus core protein. METHODS: To screen and clone the target genes transactivated by hepatitis C virus (HCV) core protein, we used suppression subtractive hybridization (SSH) and bioinformatics techniques. The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)-core and pcDNA3.1(-) empty vector, respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank, one of which was a new gene with unknown function. The new gene with no homology with known genes in this database was confirmed and electric polymerase chain reaction was conducted for the cloning of the full-length DNA for the new gene and in conjunction with Kozak role and the exist of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene, named TAHCCP2, from the mRNA of HepG2 cells transfected. The sequence for the TAHCCP2 gene was deposited into GenBank, and the accession number is AY039043. RESULTS: The full-length of TAHCCP2 was of 429 bp, and it was composed of 142 amino acid residues. The cloning of TAHCCP2 was correct and confirmed by sequencing. CONCLUSION: The cloning and identification of human gene 2 transactivated by HCV core protein will pave the way for the study of the molecular mechanism of the transactivating effects of HCV core protein and the development of new therapy for chronic hepatitis C. Wang JJ, Cheng J, Liu Y, Yang Q, Ji D, Dang XY, Wang CH. Cloning and identification of human gene 2 transactivated by hepatitis C virus core protein. Shijie Huaren Xiaohua Zazhi 2003;11(12):1893-1896

Hepatitis C virus and its roles in cell proliferation.

Hepatitis C virus (HCV) causes chronic hepatitis and is linked to the development of hepatocellular carcinoma (HCC). The role of HCV infection in the development of HCC remains to be clarified. We analyzed the effect of HCV core protein on modulation of cell proliferation. HCV core protein was shown to have at least two functions: activation of the Ras/Raf signaling pathway and anti-apototic function.

Hepatitis C Virus Core Protein Potentiates TNF-α-Induced NF-κB Activation through TRAF2-IKKβ-Dependent Pathway

Previous work has implicated that the core protein of hepatitis C virus (HCV) may play a modulatory effect on NF-κB activation induced by TNF-α. However, it is unclear how HCV core protein modulates TNF-α-induced NK-κB activation. Here we show that overexpression of HCV core protein potentiates NF-κB activation induced by TNF-α. Expression of dominant negative form of TRAF2 inhibits the synergistic effects of HCV core protein on NF-κB activation, suggesting that HCV core protein potentiates NF-κB activation through TRAF2. Moreover, we demonstrate that HCV core protein potentiates TRAF2-mediated NF-κB activation via IKKβ. In addition, HCV core protein associates with TNF-R1-TRADD-TRAF2 signaling complex, resulting in synergistically activation of NF-κB induced by TNF-α. Thus, these observations indicate that HCV core protein may play an important role in the regulation of the cellular inflammatory and immune responses through NF-κB.

Hepatitis C Virus Core Protein Activates Nuclear Factor κB-dependent Signaling through Tumor Necrosis Factor Receptor-associated Factor

Hepatitis C virus (HCV) core protein, a viral nucleocapsid, has been shown to affect various intracellular events including the nuclear factor kappaB (NF-kappaB) signaling supposedly associated with inflammatory response, cell proliferation, and apoptosis. In order to elucidate the effect of HCV core protein on the NF-kappaB signaling in HeLa and HepG2 cells, a reporter assay was utilized. HCV core protein significantly activated NF-kappaB signaling in a dose-dependent manner not only in HeLa and HepG2 cells transiently transfected with core protein expression plasmid, but also in HeLa cells induced to express core protein under the control of doxycycline. HCV core protein increased the DNA binding affinity of NF-kappaB in the electrophoretic mobility shift assay. Acetyl salicylic acid, an IKKbeta-specific inhibitor, and dominant negative form of IKKbeta significantly blocked NF-kappaB activation by HCV core protein, suggesting HCV core protein activates the NF-kappaB pathway mainly through IKKbeta. Moreover, the dominant negative forms of TRAF2/6 significantly blocked activation of the pathway by HCV core protein, suggesting HCV core protein mimics proinflammatory cytokine activation of the NF-kappaB pathway through TRAF2/6. In fact, HCV core protein activated interleukin-1beta promoter mainly through NF-kappaB pathway. Therefore, this function of HCV core protein may play an important role in the inflammatory reaction induced by this hepatotropic virus.

Hepatitis C Virus Core Protein Activates the MAPK/ERK Cascade Synergistically With Tumor Promoter TPA, But Not With Epidermal Growth Factor or Transforming Growth Factor α

Persistent hepatitis C virus (HCV) infection is associated with the development of human hepatocellular carcinoma (HCC), although the mechanism of HCV-related hepatocarcinogenesis remains unclear. Recently, however, the close relationships between the development of HCC and the mitogen-activated protein kinase (MAPK)/extracellular signal–regulated protein kinase (ERK) cascade have been described. In the present study, we investigated the effects of HCV core protein on this MAPK/ERK cascade. HCV core protein significantly activated the MAPK/ERK cascade, including Elk1. We also examined whether HCV core protein acted synergistically along with hepatocyte mitogen-mediated MAPK/ERK activation. Interestingly, Elk-1 activities were further enhanced by the tumor promoter, 12- O-tetradecanoyl phorbol 13-acetate (TPA), but not by hepatocyte mitogens (epidermal growth factor [EGF] and transforming growth factor α [TGF-α]) in NIH3T3 cells and HepG2 cells expressing HCV core protein. Moreover, the MAPK/ERK activation by HCV core protein was blocked in the presence of the specific MEK1 inhibitor, PD98059. These results indicate that ERK activation by HCV core protein may be independent of hepatocyte mitogen-mediated signaling but synergistic with TPA, and HCV core protein may function at MEK1 or farther upstream of that component. (Hepatology 2000;32:958-961.)

Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation.

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)

Mutational Analysis of Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) is the main blood-borne non-A, non-B hepatitis virus which plays a major role in development of chronic hepatitis and hepatocellular carcinoma. Our laboratory previously demonstrated that the core protein (191 amino acids) of HCV exhibits the suppressive activity to the gene expression and replication of hepatitis B virus (HBV) and this suppression effect is modulated by phosphorylation. Previous results also indicated that N-terminal 122 amino acid fragment, but not the 101-amino-acid fragment, of the HCV core protein retained the same suppressive effect as the full-length core protein. Thus, it is likely that the region between amino acids 101 and 122 of the HCV core polypeptide is responsible for the suppressive activity of the HCV core protein. This 21-amino acid segment of the HCV core polypeptide contains six arginine residues. In this study, we investigated the role of these six arginines residues in the suppressive activity. Based on the mutational analysis, results suggested that mutation of Arg117 did not interfere with the Irans-suppressive activity of the HCV core protein and the role of Arg101 in the trans-suppressive activity of the core protein was involved in phosphorylation of Ser99 by protein kinase C. Arg104 mutants blocked HBV encapsidation but did not confer any effect on the suppression of the HBV gene expression. However, single mutation at Arg113, Arg114 Arg115 led to the loss of both effects. Taken together, results suggests that the effects of HCV core protein on the transcription of HBV gene and viral encapsidation can be decoupled. Additionally, this study also demonstrates that the role of phosphoiylation in the control of trans-suppressive activity of the HCV core protein could not be mimic by introducing the acidic residues.