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REPLICATION

Hepatitis C Virus RNA Replication Occurs on a Detergent-Resistant Membrane That Cofractionates with Caveolin-2

Stephanie T. Shi, Ki-Jeong Lee, Hideki Aizaki, Soon B. Hwang, Michael M. C. Lai
Stephanie T. Shi
1Department of Molecular Microbiology and Immunology
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Ki-Jeong Lee
1Department of Molecular Microbiology and Immunology
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Hideki Aizaki
1Department of Molecular Microbiology and Immunology
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Soon B. Hwang
2Hallym Academy of Sciences, Hallym University, Chuncheon, Kangwon-Do 200-702, Korea
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Michael M. C. Lai
1Department of Molecular Microbiology and Immunology
3Howard Hughes Medical Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90033
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  • For correspondence: michlai@hsc.usc.edu
DOI: 10.1128/JVI.77.7.4160-4168.2003
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ABSTRACT

The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly understood. In this study, we labeled de novo-synthesized viral RNA in situ with bromouridine triphosphate (BrUTP) in Huh7 cells expressing an HCV subgenomic replicon. By immunofluorescence staining using an anti-BrUTP antibody and confocal microscopy, we showed that the newly synthesized HCV RNA was localized to distinct speckle-like structures, which also contain all of the HCV nonstructural (NS) proteins. These speckles are distinct from lipid droplets and are separated from the endoplasmic reticulum (ER), where some HCV NS proteins also reside. Membrane flotation analysis demonstrated that almost all of the NS5A and part of the NS5B proteins and all of the viral RNA were present in membrane fractions which are resistant to treatment with 1% NP-40 at 4°C. They were cofractionated with caveolin-2, a lipid-raft-associated intracellular membrane protein, in the presence or absence of the detergent. In contrast, the ER-resident proteins were detergent soluble. These properties suggest that the membranes on which HCV RNA replication occurs are lipid rafts recruited from the intracellular membranes. The protein synthesis inhibitors cycloheximide and puromycin did not inhibit viral RNA synthesis, indicating that HCV RNA replication does not require continuous protein synthesis. We suggest that HCV RNA synthesis occurs on a lipid raft membrane structure.

  • Copyright © 2003 American Society for Microbiology
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Hepatitis C Virus RNA Replication Occurs on a Detergent-Resistant Membrane That Cofractionates with Caveolin-2
Stephanie T. Shi, Ki-Jeong Lee, Hideki Aizaki, Soon B. Hwang, Michael M. C. Lai
Journal of Virology Apr 2003, 77 (7) 4160-4168; DOI: 10.1128/JVI.77.7.4160-4168.2003

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Hepatitis C Virus RNA Replication Occurs on a Detergent-Resistant Membrane That Cofractionates with Caveolin-2
Stephanie T. Shi, Ki-Jeong Lee, Hideki Aizaki, Soon B. Hwang, Michael M. C. Lai
Journal of Virology Apr 2003, 77 (7) 4160-4168; DOI: 10.1128/JVI.77.7.4160-4168.2003
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KEYWORDS

hepacivirus
RNA, Viral

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