title: Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.
abstract: Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.
authors.affiliation: Department of Molecular Oncology, Kanazawa University, 13-1 Takara-Machi, Kanazawa 920-0934, Ishikawa, Japan.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Shirota
authors.forename: Hong
authors.initials: H
authors.lastname: Luo
authors.forename: Weiping
authors.initials: W
authors.lastname: Qin
authors.forename: Shuichi
authors.initials: S
authors.lastname: Kaneko
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yamashita
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Seishi
authors.initials: S
authors.lastname: Murakami
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D010957:Plasmids; D011485:Protein Binding; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase