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Journal of Virology, July 2004, p. 7400-7409, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7400-7409.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes{dagger}

Darius Moradpour,1,2 Matthew J. Evans,1,3 Rainer Gosert,2 Zhenghong Yuan,1,{ddagger} Hubert E. Blum,2 Stephen P. Goff,4 Brett D. Lindenbach,1 and Charles M. Rice1*

Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10021,1 Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany,2 Integrated Program in Cellular, Molecular, and Biophysical Studies,3 Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 100324

Received 29 December 2003/ Accepted 28 March 2004

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

* Corresponding author. Mailing address: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-7046. Fax: (212) 327-7048. E-mail: ricec{at}rockefeller.edu.

{dagger} This study is dedicated to Walter Siegenthaler on the occasion of his 80th birthday.

{ddagger} Present address: Shanghai Medical College, Fudan University, Shanghai 200032, China.

Journal of Virology, July 2004, p. 7400-7409, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7400-7409.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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