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Journal of Virology, January 2001, p. 205-214, Vol. 75, No. 1
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.1.205-214.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Mutations That Affect Dimer Formation and Helicase Activity of the Hepatitis C Virus Helicase

Yee-Ling Khu,1 Esther Koh,1 Siew Pheng Lim,1 Yin Hwee Tan,1 Sydney Brenner,2 Seng Gee Lim,1,3 Wan Jin Hong,1 and Phuay-Yee Goh1,*

Collaborative Anti-Viral Research Group, Institute of Molecular and Cell Biology, Singapore 117609,1 and Department of Medicine, National University Hospital, Singapore 119074,3 Singapore, and The Molecular Sciences Institute Inc., Berkeley, California 947042

Received 17 July 2000/Accepted 4 October 2000

Interaction between viral proteins is necessary for viral replication and viral particle assembly. We used the yeast two-hybrid assay to identify interactions among all the mature proteins of the hepatitis C virus. The interaction between NS3 and NS3 was one of the strongest viral protein-protein interactions detected. The minimal region required for this interaction was mapped to a specific subdomain of 174 amino acids in the N terminus of the helicase region. Random mutations in the minimal region were generated by PCR, and mutants that failed to interact with a wild-type minimal fragment were isolated using the yeast two-hybrid assay as a screen. Three of these mutations resulted in a reduction or a loss of interaction between helicases. Analytical gel filtration showed that in the presence of an oligonucleotide, wild-type helicases form dimers whereas the mutants remain mostly monomeric. All three mutants were partially or almost inactive when assayed for helicase activity in vitro. Mixing a mutant helicase (Y267S) with wild-type helicase did not dramatically affect helicase activity. These data indicate that dimerization of the helicase is important for helicase activity. The mutations that reduce self-association of the helicase may define the key residues involved in NS3-NS3 dimerization.

* Corresponding author. Mailing address: Collaborative Anti-Viral Research Group, Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore. Phone: (65) 874 3387 or 874 7820. Fax: (65) 779 1117. E-mail: mcbgohpy{at}imcb.nus.edu.sg.

Journal of Virology, January 2001, p. 205-214, Vol. 75, No. 1
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.1.205-214.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.