Functional interaction of hepatitis C Virus NS5B with Nucleolin GAR domain.
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.
Kusakawa
Takashi
T
Division of Signal Transduction, Cancer Research Institute, University Hospital, Kanazawa University, Takara-Machi, Kanazawa, Ishikawa, Japan.
Shimakami
Tetsuro
T
Kaneko
Shuichi
S
Yoshioka
Katsuji
K
Murakami
Seishi
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry; D019556:COS Cells; D002522:Chlorocebus aethiops; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D000097796:Nucleolin; D012324:RNA-Dependent RNA Polymerase
D000596:Amino Acids; D001120:Arginine; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D014364:Tryptophan; D017361:Viral Nonstructural Proteins; D000097796:Nucleolin; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase