Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon
| Autor: | Tetsuro Shimakami, Takashi Kusakawa, Takayuki Murata, Masao Honda, Kunitada Shimotohno, Shuichi Kaneko, Seishi Murakami |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Gene Expression Regulation
Viral Small interfering RNA viruses Immunology RNA-dependent RNA polymerase RNA-binding protein Viral Nonstructural Proteins Biology Transfection Virus Replication Microbiology Cell Line chemistry.chemical_compound Virology Humans Point Mutation Amino Acid Sequence Replicon RNA Small Interfering NS5B Alanine RNA-Binding Proteins virus diseases RNA biochemical phenomena metabolism and nutrition Phosphoproteins RNA-Dependent RNA Polymerase Precipitin Tests Molecular biology digestive system diseases Genome Replication and Regulation of Viral Gene Expression Amino Acid Substitution Viral replication chemistry Insect Science Nucleolin |
| Zdroj: | Journal of Virology. 80:3332-3340 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.80.7.3332-3340.2006 |
| Popis: | We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication. |
| Databáze: | OpenAIRE |
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