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

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.Hepatitis C virus (HCV) is an important human pathogen associated with non-A, non-B hepatitis and is the leading cause of chronic hepatitis and liver cirrhosis. As a member of the Flaviviridae family, HCV contains a positive-sense, singlestranded RNA genome of approximately 9.6 kb. The viral genome encodes a single polyprotein of about 3,010 amino acids, which is proteolytically processed by a combination of host-and virus-encoded proteases into 10 viral structural and nonstructural (NS) proteins arranged in the following order:The establishment of the HCV subgenomic replicon and the subsequent analysis of the adaptive mutations revealed that most of the HCV NS proteins, with the probable exception of NS2, are involved in HCV RNA replication (6,29,37). NS3 is a helicase and a serine protease, whose function is dependent on NS4A. It is conceivable that the enzymatic activities of these proteins are key components of the HCV replication complex. The function of NS4B is thus far unknown, although it has been implicated in inducing transformation (34) and intracellular membrane alterations (13); the latter may play a significant role in the formation of the HCV RNA replication complex. NS5A is known to be a multifunctional protein implicated in the pathogenesis and interferon resistance of HCV infection. However, it has become evident that NS5A also plays an indispensable role in the replication of the HCV subgenomic replicon (6), but the underlying mechanism has yet to be identified. HCV NS5B is an RNA-dependent RNA polymerase (RdRp). All of these NS proteins, together with host prote...

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

“…Previously, we have shown that HCV RNA replication occurs on speckle-like structures [29], which may be equivalent to the membranous web, as reported by Gosert et al [45]. The immunofluorescence staining study presented here showed that PTB in Huh-7 cells mainly existed in the nucleus, while PTB in the replicon cells localized in not only the nucleus but also distinct structures in the cytoplasm and colocalized with the newly synthesized HCV RNA (Figure 1).…”

“…Significantly, PTB was detected in this membrane fraction in the replicon-containing cells, but not in Huh-7 cells. As shown previously, caveolin-2, a known DRM-resident protein was detected in the DRM fraction of both Huh-7 and replicon cells (29,31; see also Figure 4). These results indicated that PTB, together with the HCV NS proteins and hVAP-33, was present as large complexes in the DRM fraction.…”

“…PTB was associated with a DRM structure containing HCV NS proteins and RNA Previous studies have shown that HCV RCs were resistant to detergent treatment [8,29,31].…”

“…The mouse monoclonal antibody against NS5B was made by NS5B protein expressed from a recombinant baculovirus and was a generous gift of Dr. Soon Hwang (Hallym University, Korea) [28,29]. The mouse monoclonal antibody against PTB isoforms 1, 2, and 4 (anti-PTB) was purified from culture medium of the mouse hybridoma cell, BB7, purchased from ATCC (Manassas, VA).…”

“…Furthermore, the newly synthesized HCV RNA and the viral NS proteins colocalized on a distinct speckle-like structure, which is made of the detergent-resistant membranes (DRMs), in the cytoplasm of the replicon cells, [29]. To test the possibility that PTB not only participates in HCV translation, but also is associated with the HCV replication complex, we first determined the intracellular localization of PTB in the HCV replicon-containing cells.…”