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Journal of Virology, August 1999, p. 7044-7049, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Specific Interaction between the Hepatitis C Virus NS5B RNA Polymerase and the 3' End of the Viral RNA

Ju-Chien Cheng,1,dagger Ming-Fu Chang,2 and Shin C. Chang1,*

Institutes of Microbiology1 and Biochemistry,2 National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China

Received 26 January 1999/Accepted 3 May 1999

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.

* Corresponding author. Mailing address: No. 1, Sec. 1, Jen-Ai Rd., Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China. Phone: 886-2-23970800, ext. 8290. Fax: 886-2-23915293. E-mail: scchang{at}ha.mc.ntu.edu.tw.

dagger Present address: School of Medical Technology, China Medical College, Taichung, Taiwan.

Journal of Virology, August 1999, p. 7044-7049, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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