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Journal of Biochemistry 2007 141(6):917-927; doi:10.1093/jb/mvm102
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© 2007 The Japanese Biochemical Society.

Functional Interaction of Hepatitis C Virus NS5B with Nucleolin GAR Domain

Takashi Kusakawa1,3, Tetsuro Shimakami2, Shuichi Kaneko2, Katsuji Yoshioka1 and Seishi Murakami1,*

1Division of Signal Transduction, Cancer Research Institute; 2Department of Gastroenterology, University Hospital, Kanazawa University, Takara-Machi, Kanazawa, Ishikawa 920-0934; and 3Hokuto Science Industry, Takariya, Toyama 930-0897, Japan

*To whom correspondence should be addressed. Tel: +81-76-265-2713, Fax: +81-76-234-4501, E-mail: semuraka{at}kenroku.kanazawa-u.ac.jp

Received March 26, 2007; Accepted April 9, 2007


  Abstract

Hepatitis C Virus (HCV) non-structural proteins are major components of replication complex that is modulated by several host factors. We previously reported that nucleolin, a representative nucleolar marker, interacts with the NS5B through two separated sequences, amino acids (aa) 208–214 and 500–506, and that W208 in the former stretch is essential for both nucleolin-binding and HCV replication. Here we evaluated the role of the latter stretch aa 500–506 of WRHRARS in nucleolin-binding and HCV replication scanned by alanine-substituted clustered mutant (cm) or point mutant (pm). One tryptophan and three arginine residues in the sequence were found to be essential both for nucleolin-binding in vivo and HCV replication detected with a HCV subgenomic replicon transfected into Huh7 cells. NS5B-binding of nucleolin was further delineated by truncation and clustered mutants of nucleolin. Arginine-glycine-glycine (RGG) repeat in the Glycine arginine rich (GAR) domain were defined to be indispensable for NS5B-binding immunologically detected in in vivo and in vitro although short internal-truncations of RGG repeat are tolerable for NS5B-binding. These results indicate that nucleolin is a critical host factor for HCV replication through the direct interaction between W208 and several residues at the sequence, aa 500–505, of NS5B, and the long-turn motif including RGG repeat at nucleolin C-terminal.

Key Words: HCV, NS5B, nucleoli, nucleolin, RGG

Abbreviations: GAR, glycine arginine rich; HCV, hepatitis C virus; NS, non-structural; PBS, phosphate-buffered saline; PMSF, phenylmethylsulphonyl fluoride; RBD, RNA-binding domain; RdRp, RNA-dependent RNA polymerase; RGG, arginine-glycine-glycine


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