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Journal of Virology, March 2000, p. 2293-2304, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Subcellular Localization, Stability, and trans-Cleavage Competence of the Hepatitis C Virus NS3-NS4A Complex Expressed in Tetracycline-Regulated Cell Lines

Benno Wölk,1 Domenico Sansonno,2 Hans-Georg Kräusslich,3 Franco Dammacco,2 Charles M. Rice,4 Hubert E. Blum,1 and Darius Moradpour1,*

Department of Medicine II, University of Freiburg, D-79106 Freiburg,1 and Heinrich-Pette-Institute, D-20251 Hamburg,3 Germany; Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, I-70124 Bari, Italy2; and Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-10934

Received 7 July 1999/Accepted 6 November 1999

A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3'-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.

* Corresponding author. Mailing address: Department of Medicine II, University Hospital Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany. Phone: 49-761-270-3510. Fax: 49-761-270-3610. E-mail: moradpou{at}ruf.uni-freiburg.de.

Journal of Virology, March 2000, p. 2293-2304, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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