Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA.
Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3' viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5' noncoding region and very poorly by the 3' conserved 98-nucleotide tail. A 3' coding region with conserved stem-loop structures rather than the 3' noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3' coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication.
Institutes of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China.
J C
JC
Cheng
M F
MF
Chang
S C
SC
Chang
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D004279:DNA, Viral; D015870:Gene Expression; D016679:Genome, Viral; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase