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Journal of Virology, September 2004, p. 9257-9269, Vol. 78, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.17.9257-9269.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

Kevin C. Klein,1 Stephen J. Polyak,2 and Jaisri R. Lingappa1,3*

Department of Pathobiology,1 Departments of Laboratory Medicine and Microbiology,2 Department of Medicine, University of Washington, Seattle, Washington3

Received 12 November 2003/ Accepted 19 April 2004

The assembly of hepatitis C virus (HCV) is poorly understood, largely due to the lack of mammalian cell culture systems that are easily manipulated and produce high titers of virus. This problem is highlighted by the inability of the recently established HCV replicon systems to support HCV capsid assembly despite high levels of structural protein synthesis. Here we demonstrate that up to 80% of HCV core protein synthesized de novo in cell-free systems containing rabbit reticulocyte lysate or wheat germ extracts assembles into HCV capsids. This contrasts with standard primate cell culture systems, in which almost no core assembles into capsids. Cell-free HCV capsids, which have a sedimentation value of {approx}100S, have a buoyant density (1.28 g/ml) on cesium chloride similar to that of HCV capsids from other systems. Capsids produced in cell-free systems are also indistinguishable from capsids isolated from HCV-infected patient serum when analyzed by transmission electron microscopy. Using these cell-free systems, we show that HCV capsid assembly is independent of signal sequence cleavage, is dependent on the N terminus but not the C terminus of HCV core, proceeds at very low nascent chain concentrations, is independent of intact membrane surfaces, and is partially inhibited by cultured liver cell lysates. By allowing reproducible and quantitative assessment of viral and cellular requirements for capsid formation, these cell-free systems make a mechanistic dissection of HCV capsid assembly possible.

* Corresponding author. Mailing address: Department of Pathobiology, Box 357238, University of Washington, 1959 NE Pacific St., Seattle, WA 98195. Phone: (206) 616-9305. Fax: (206) 543-3873. E-mail: jais{at}u.washington.edu.

Journal of Virology, September 2004, p. 9257-9269, Vol. 78, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.17.9257-9269.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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