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Journal of Virology, September 2009, p. 8379-8395, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00891-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes {triangledown}

Tung Phan,1 Rudolf K. F. Beran,1 Christopher Peters,1 Ivo C. Lorenz,2,{dagger} and Brett D. Lindenbach1*

Section of Microbial Pathogenesis, Yale University School of Medicine, 295 Congress Ave., New Haven, Connecticut 06536,1 Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Ave., New York, New York 100212

Received 3 June 2009/ Accepted 4 June 2009

The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.

* Corresponding author. Mailing address: Section of Microbial Pathogenesis, Yale University School of Medicine, 354C BCMM, 295 Congress Ave., New Haven, CT 06536. Phone: (203) 785-4705. Fax: (203) 737-2630. E-mail: brett.lindenbach{at}yale.edu

{triangledown} Published ahead of print on 10 June 2009.

{dagger} Present address: International AIDS Vaccine Initiative, AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220.

Journal of Virology, September 2009, p. 8379-8395, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00891-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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