Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that coffactionates with caveolin-2
Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that coffactionates with caveolin-2
复制标题
DOI：
10.1128/jvi.77.7.4160-4168.2003
复制
发表时间：
2003-04-01
影响因子：
5.4
通讯作者：
Lai, MMC
中科院分区：
文献类型：
--
作者：
Shi, ST;Lee, KJ;Lai, MMC
丙型肝炎病毒（HCV）RNA复制的机制和机制仍然知之甚少。在这项研究中，我们标记从头合成的病毒RNA原位与三磷酸溴尿苷（BrUTP）在Huh 7细胞表达HCV亚基因组复制子。通过使用抗BrUTP抗体和共聚焦显微镜的免疫荧光染色，我们发现，新合成的HCV RNA被定位到不同的斑点状结构，其中也包含所有的HCV非结构（NS）蛋白。这些斑点与脂滴不同，并与内质网（ER）分离，其中一些HCV NS蛋白也存在。膜浮选分析表明，几乎所有的NS 5A蛋白和部分NS 5 B蛋白以及所有的病毒RNA都存在于膜组分中，这些膜组分在4 ℃下对1%NP-40的处理具有抗性。在存在或不存在洗涤剂的情况下，将它们与小窝蛋白-2（一种脂筏相关的细胞内膜蛋白）共分级分离。相反，ER驻留蛋白是去污剂可溶的。这些性质表明，HCV RNA复制发生的膜是从细胞内膜募集的脂筏。蛋白质合成抑制剂放线菌酮和嘌呤霉素不抑制病毒RNA的合成，表明HCV RNA复制不需要连续的蛋白质合成。我们认为HCV RNA的合成发生在脂筏膜结构上。
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 4degrees. 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.