• Journal of Life Science
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• v.28 no.9
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• pp.1007-1015
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• 2018

The E6-associated protein (E6AP) is known to induce the ubiquitination and proteasomal degradation of HCV core protein and thereby directly impair capsid assembly, resulting in a decline in HCV replication. To counteract this anti-viral host defense system, HCV core protein has evolved a strategy to inhibit E6AP expression via DNA methylation. In the present study, we further explored the mechanism by which HCV core protein inhibits E6AP expression. HCV core protein upregulated both the protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b to inhibit E6AP expression via promoter hypermethylation in HepG2 cells but not in Hep3B cells, which do not express p53. Interestingly, p53 overexpression alone in Hep3B cells was sufficient to activate DNMTs in the absence of HCV core protein and thereby inhibit E6AP expression via promoter hypermethylation. In addition, upregulation of p53 was absolutely required for the HCV core protein to inhibit E6AP expression via promoter hypermethylation, as evidenced by both p53 knockdown and ectopic expression experiments. Accordingly, levels of the ubiquitinated forms of HCV core protein were lower in HepG2 cells than in Hep3B cells. Based on these observations, we conclude that HCV core protein evades ubiquitin-dependent proteasomal degradation in a p53-dependent manner.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.9-17
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• 1997

Hepatitis C virus (HCV) E2 protein is known to be one of putative envelope proteins. To develop a sensitive detection method for HCV infected tissues and cells, monoclonal antibodys (MAbs) to the E2 protein of HCV were prepared from mice immunized with recombinant baculovirus-expressing E2 protein (Bac-E2). Several hybridoma clones secreting various levels of MAb were isolated and isotypes of these MAb were determined. One clone (L.2.3.3) was used for ascites production and the E2-MAb was purified and characterized. The L.2.3.3 reacted well with both Bac-E2 and E. coli expressed glutathione-S-transferase-E2 (GST-E2) fusion proteins. Using HCV patient sera, E2 envelope protein was found to be localized in the cell membrane boundary both in CHO cells and insect cells which express HCV E2 protein. Similar result was obtained when same cells were treated with the MAb L.2.3.3. These results demonstrated that Bac-E2 protein is capable of eliciting high titer antibody production in mice.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.361-368
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• 1998

The E2 protein of HCV (hepatitis C virus) is thought to have a potential role in the development of subunit vaccines and diagnostics. To express it by the insect cell/baculovirus expression (Bacu) system, we constructed a recombinant Autographa californica nuclear polyhedrosis virus (AcIL3E2), determined the most appropriate expression conditions in terms of host cell line and culture medium, and characterized the expressed HCV E2 protein. A culture system using Trichoplusia ni BTI-TN5Bl-4 cells and SF 900IISFM medium expressed a relatively high level of HCV E2 protein. It was revealed that its glycosylation properties and subcellular localization were almost the same as the ones in the mammalian cell expression system previously reported, suggesting the recombinant HCV E2 protein derived from our Bacu system can be utilized for development of a subunit vaccine and diagnostics. Interestingly, HCV E2 protein was not degraded at all even at 43 h post-heat shock in the heat shock-induced necrotic cells, probably due to its integration into the microsomal membrane, indicating that heat shock can be employed to purify HCV E2 protein.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.105-113
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• 1997

The truncated $E1_{192-283}$ and $E2_{384-649}$ genes of hepatitis C virus (HCV) linked to the gene for glutathione S-transferase (GST) were constructed and their expressions were analyzed. The $GST-E1_{192-283}$ fusion gene overexpressed the fusion protein in E. coli as a soluble form, while the $GST-E1_{192-383}$ plasmid did not express expected fusion protein. The purified $GST-E1_{192-283}$ fusion protein was efficiently cleaved by thrombin. More than 90% pure, HCV $E1_{192-283}$ protein was obtained by GST-agarose chromatography. The truncated $GST-E2_{384-649}$ fusion gene expressed the fusion protein mainly as an insoluble form, whereas the $GST-E2_{384-740}$ did not express the fusion protein. The truncated $GST-E1_{182-283}$ and $GST-E2_{384-649}$ fusion proteins reacted specifically with an HCV patient serum. In addition, mice immunized with either the purified $E1_{192-283}$ or $GST-E2_{384-649}$ proteins generated specific antibodies to each antigen. The results suggested that hydrophobic carboxyl portions of the E1 and E2 proteins might affect expression levels as well as the solubility of each fusion protein in bacteria. Also, the truncated E1 protein with Tyr-192 to Ser-283 contained antigenic epitope(s) which could be specifically recognized by an HCV patient serum.

• BMB Reports
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• v.37 no.6
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• pp.735-740
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• 2004

Hepatitis C virus (HCV) is a causal agent of the chronic liver infection. To understand HCV morphogenesis, we studied the assembly of HCV structural proteins in insect cells. We constructed recombinant baculovirus expression vectors consisting of either HCV core alone, core-E1, or core-E1-E2. These structural proteins were expressed in insect cells and were examined to assemble into particles. Neither core-E1 nor core-E1-E2 was capable of assembling into virus-like particles (VLPs). It was surprising that the core protein alone was assembled into core-like particles. These particles were released into the culture medium as early as 2 days after infection. In our system, HCV structural proteins including envelope proteins did not assemble into VLPs. Instead, the core protein itself has the intrinsic capacity to assemble into amorphous core-like particles. Furthermore, released core particles were associated with HCV RNA, indicating that core proteins were assembled into nucleocapsids. These results suggest that HCV may utilize a unique core release mechanism to evade the hosts defense mechanism, thus contributing to the persistence of HCV infection.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

• Journal of Life Science
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• v.13 no.4
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• pp.541-550
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• 2003

E2 glycoprotein of hepatitis C virus (HCV) comprises a surface of viral particle together with E1 glycoprotein, and is thought to be involved in the attachment of HCV viral particle to receptor (s) on the permissible cells including hepatocytes, B cells, T cells, and monocytes. We constructed a phage library expressing cellular proteins of hepatocytes on the phage surface, which turned out to be 8.8${\times}$$10^5$ cfu of diversity and carried inserts in 95% of library. We screened both cDNA phage library and 12-mer peptide library to identify the cellular proteins binding to E2 protein. Some intracellular proteins including tensin and membrane band 4.1 which are involved in signal transduction of survival and cytoskeleton organization, were selected from cDNA phage library through several rounds of panning and screening. On the contrary, membrane proteins such as CCR7, CKR-L2, and insulin-like growth factor-1 receptor were identified through screening of peptide library. Phages expressing peptides corresponding to those membrane proteins were bound to E2 protein specifically as determined by neutralization of binding assay. Since it is well known that HCV can infect T cells as well as hepatocytes, we examined to see if E2 protein can bind to CCR7, a member of C-protein coupled receptor family expressed on T cells, using CCR7 transfected tells. Human CCR7 cDNA was cloned into pcDNA3.1(-) vector and transfected into human embryonic kidney cell, 293T, and expressed on the surface of the cell as shown by flow cytometer. Binding assay of E2 protein using CCR7 transfected cells indicated that E2 protein bound to CCR7 by dose-dependent mode, giving rise to the possibility that CCR7 might be a putative cellular receptor for HCV.

• Journal of the Korean Society of Tobacco Science
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• v.23 no.2
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• pp.103-108
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• 2001

Hog cholera virus(HCV) was purified from virus infected Bovine kidney cells. From this virus, total protein was analyzed by SDS-PAGE gel electrophoresis and about 55 kDa band of E2 envelope protein was detected. The viral RNA was purified and E2 cDNA was amplified by RT-PCR. E2 cDNA fragment was cloned to PCRII-TOPO cloning vector and named pE2. The analysis of nucleotide sequence showed that this E2 cDNA fragment inserted into pE2 was 1191 nucleotides long and coded 397 amino acids.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1769-1771
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• 2010

The envelope glycoprotein E2 of hepatitis C virus (HCV) binds to various cell surface receptors for viral infection. We performed biopanning against this protein and selected peptides from phage display peptide libraries. Two short peptides, pep7-1 and pep12-1, were selected and their ability to inhibit the infection process was investigated. When pep7-1 was present, the infectivity of HCV particles in cell culture was notably decreased. This decrease was demonstrated by Western blot analysis, immunofluorescence assay, and reverse transcription PCR assay. However, pep12-1 showed little inhibitory effect on HCV infection.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.353-359
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• 2008

Hepatitis C virus (HCV) is known as the causative agent of blood transmitted hepatitis. Two viral proteins, E2 and NS5A, are known to exert interferon resistance of HCV via PKR pathway. Here, we report a third protein, the RNA-dependent RNA polymerase (NS5B) of HCV, induced interferon resistance inhibiting p56 pathway. p56 was shown to interact with p48 subunit of eukaryotic initiation factor 3 (eIF3). This interaction inhibited formation of ternary complex in translation initiation. Using dual reporter assay system, we observed that the translation decreased when interferon alpha was added to the culture. But, in the presence of HCV NS5B, the translation partly recovered. NS5B and p48 subunit of eIF3 were shown to interact. This interaction seems to inhibit the interaction between p48 and p56. This is the first report that a virus exerts interferon resistance via p56 pathway.