Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts

Abstract

The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly characterized. Our previous study has shown that HCV RNA synthesis occurs on a lipid raft membrane structure [J. Virol. 77 (2003) 77 4160]. In this study, we further characterized these replication complexes (RCs) in Huh-7 cells that support active RNA replication of a subgenomic HCV replicon. Biochemical analysis showed that these membrane structures were resistant to Nonidet P-40 or Triton X-100 (TX-100) at 4 degrees C while solubilized by beta-octylglucoside at 4 degrees C or Triton TX-100 at 37 degrees C, characteristic of lipid rafts. Cholesterol sequestration assay further demonstrated the association between HCV nonstructural (NS) proteins and cholesterol-rich lipid rafts. The RCs contained both minus- and plus-strand HCV RNA, with the plus-stranded RNA being approximately 10-fold more abundant than the minus-strand. Furthermore, the HCV RNA and NS proteins were resistant to RNase and protease digestion, respectively, but became sensitive after treatment with the raft-disrupting agents. These results suggested that the HCV RCs are protected within lipid rafts. Detergent-resistant membrane (DRM) fractions containing NS proteins and viral RNA were capable of HCV RNA synthesis using the endogenous HCV RNA template. NS proteins were distributed in both the ER and the Golgi, but the majority of the active RCs were detected in the Golgi-derived membrane. Depletion of cellular cholesterol selectively reduced HCV RNA replication. These findings provide further insights into the mechanism of HCV replication in vivo.

Serum cholesterol and statin use predict virological response to peginterferon and ribavirin therapy

“…This complex involves an isoprenoid protein called geranylgeranyl pyrophosphate, which is attached to specific G proteins via a process called protein prenylation. 31 In vitro studies of various statins have shown depletion of specific nonsterol isoprenoids (particularly geranylgeranyl pyrophosphate) that are generated via mevalonate metabolism. The availability of fewer lipid rafts for replication seems to be a plausible explanation for at least some of the anti-HCV properties of statins.…”

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

“…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 membrane flotation assay was performed as previously described [31]. Briefly, Cells were first lysed in 1 ml of hypotonic buffer (10 mM Tris-HCV [pH 7.5], 10 mM KCl, 5 mM MgCl 2 ) and passed through a 25-gauge needle 20 times.…”

“…Proteinase digestion assay was performed as described previously [31]. Briefly, after the membrane flotation assay, the membrane fraction was treated without or with 1% TX-100 at 4 or 37°C for 1 h. Samples were then incubated with or without trypsin (10, 100 lg/ml) at 4°C for 5 min.…”

“…To determine the quantity of RNA by real-time PCR, we performed a single-tube reaction by using the TaqMan EZ RT-PCR Core Reagents (Applied Biosystems, Foster City, CA) as described previously [31]. Values were normalized to that of GAPDH (Applied Biosystems).…”