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Journal of Virology, August 2008, p. 7624-7639, Vol. 82, No. 15
0022-538X/08/$08.00+0     doi:10.1128/JVI.00724-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

NS3 Helicase Domains Involved in Infectious Intracellular Hepatitis C Virus Particle Assembly{triangledown}

Yinghong Ma, Jeremy Yates, Yuqiong Liang, Stanley M. Lemon,* and MinKyung Yi*

The Center for Hepatitis Research, Institute for Human Infections and Immunity, and Department of Microbiology & Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019

Received 1 April 2008/ Accepted 19 May 2008

A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.

* Corresponding author. Mailing address: Center for Hepatitis Research, The University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, TX 77555-1073. Phone for M. Yi: (409) 747-6866. Fax: (409) 747-7030. E-mail: miyi{at}utmb.edu. Phone for S. M. Lemon: (409) 747-7048. Fax: (409) 747-7030. E-mail: smlemon{at}utmb.edu

{triangledown} Published ahead of print on 28 May 2008.

Journal of Virology, August 2008, p. 7624-7639, Vol. 82, No. 15
0022-538X/08/$08.00+0     doi:10.1128/JVI.00724-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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