Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee-infectious HC-J4 isolate to replicate efficiently in Huh-7 cells
| Autor: | Chifumi Sato, Cheng-Hsin Chen, Nobuhiko Kanazawa, Yoko Tanabe, Nobuyuki Enomoto, Shinya Maekawa, Mamoru Watanabe, Hisayoshi Watanabe, E Ueda, Mina Nakagawa, T. Kohashi, Naoya Sakamoto, Masayuki Kurosaki, Tsuyoshi Yamashiro |
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| Rok vydání: | 2004 |
| Předmět: |
Untranslated region
Pan troglodytes viruses Molecular Sequence Data Mutant Genome Viral Hepacivirus Viral Nonstructural Proteins Biology Virus Replication medicine.disease_cause chemistry.chemical_compound Virology Tumor Cells Cultured medicine Animals Humans Amino Acid Sequence Replicon Cloning Molecular NS5A NS5B Subgenomic mRNA Genetics Mutation NS3 Hepatology Interferon-alpha virus diseases biochemical phenomena metabolism and nutrition digestive system diseases Infectious Diseases chemistry |
| Zdroj: | Journal of Viral Hepatitis. 11:394-403 |
| ISSN: | 1365-2893 1352-0504 |
| Popis: | Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. To extend the previous results to other isolated HCV clones, we constructed another HCV replicon from HC-J4, one of chimpanzee-infectious HCV clones. An HCV replicon derived from HC-J4 (RpJ4) consists of HCV-5' untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV-3' untranslated region. The adaptive mutations known to be required for HCV-Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4-S2197P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDR mutant and RpJ4-S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh-7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418-resistant cells were transduced efficiently by RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDR mutant, with the RpJ4-S2201del/ISDR mutant being most efficient. Hence the HCV replicon derived from HC-J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications. |
| Databáze: | OpenAIRE |
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