Functional Interaction of Hepatitis C Virus NS5B with Nucleolin GAR Domain

• Published in: Journal of biochemistry (Tokyo) Vol. 141; no. 6; pp. 917 - 927
• Main Authors: Kusakawa, Takashi, Shimakami, Tetsuro, Kaneko, Shuichi, Yoshioka, Katsuji, Murakami, Seishi
• Format: Journal Article
• Language: English
• Published: England Japanese Biochemical Society 01.06.2007; Oxford University Press
• Subjects: Amino Acid Sequence; Amino Acids - chemistry; Animals; Arginine - chemistry; Chlorocebus aethiops; COS Cells; Hepatitis C virus; Molecular Sequence Data; Mutation; Nucleolin; Phosphoproteins - chemistry; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; RNA-Binding Proteins - chemistry; Sequence Homology, Amino Acid; Tryptophan - chemistry; Viral Nonstructural Proteins - chemistry; HCV; RGG; nucleoli; NS5B; nucleolin
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvm102

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.