Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2.
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 degrees 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.
Shi
Stephanie T
ST
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
Lee
Ki-Jeong
KJ
Aizaki
Hideki
H
Hwang
Soon B
SB
Lai
Michael M C
MM
D051243:Caveolin 2; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D003902:Detergents; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000821:virology; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000821:virology; D008563:Membrane Lipids / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012995:Solubility; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014779:Virus Replication
D051243:Caveolin 2; D022461:Caveolins; D003902:Detergents; D008563:Membrane Lipids; D011500:Protein Synthesis Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40