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Journal of Virology, January 2003, p. 546-559, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.546-559.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Role of the Asialoglycoprotein Receptor in Binding and Entry of Hepatitis C Virus Structural Proteins in Cultured Human Hepatocytes

Bertrand Saunier,1,2,3* Miriam Triyatni,4,{dagger} Luca Ulianich,2 Padma Maruvada,5 Paul Yen,5 and Leonard D. Kohn1,2

Edison Biotechnology Institute and College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701,1 Cell Regulation Section,2 Liver Diseases Section,4 Clinical Endocrinology Branch, National Institute of Diabetes, Digestive and Kidney Diseases,5 Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208923

Received 23 April 2002/ Accepted 25 September 2002

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.

* Corresponding author. Mailing address: Konneker Research Laboratories, Rm. 077, Edison Biotechnology Institute, The Ridges, Ohio University, Athens, OH 45701. Phone: (740) 593-0926. Fax: (740) 597-1468. E-mail: saunier{at}ohiou.edu.

{dagger} Present address: Molecular Structure Section, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.

Journal of Virology, January 2003, p. 546-559, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.546-559.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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