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Journal of Virology, April 2002, p. 3865-3872, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3865-3872.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase

Q. May Wang,1* Michelle A. Hockman,1 Kirk Staschke,1 Robert B. Johnson,1 Katharine A. Case,1 Jirong Lu,1 Steve Parsons,1 Faming Zhang,1 Radhakrishnan Rathnachalam,1 Karla Kirkegaard,2 and Joseph M. Colacino1

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285,1 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 943052

Received 19 December 2001/ Accepted 15 January 2002

The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, Kd, of {approx}22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.

* Corresponding author. Mailing address: Drop Code: 0438, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285. Phone: (317) 277-6975. Fax: (317) 276-1743. E-mail: qmwang{at}lilly.com.

Journal of Virology, April 2002, p. 3865-3872, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3865-3872.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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