Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase
Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase
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DOI：
10.1074/jbc.m103358200
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发表时间：
2001-11-23
影响因子：
4.8
通讯作者：
Moradpour, D
中科院分区：
文献类型：
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作者：
Schmidt-Mende, J;Bieck, E;Moradpour, D
丙型肝炎病毒（HCV）RNA依赖性RNA聚合酶（RdRp），以非结构蛋白5 B（NS 5 B）为代表，被认为与其他非结构蛋白和尚未鉴定的宿主组分一起形成膜相关RNA复制复合物。然而，这种必需的病毒酶的膜结合的决定因素尚未确定。通过双标记免疫荧光分析，发现NS 5 B在内质网（ER）或ER样修饰的隔室中单独表达或在整个HCV多蛋白的背景下表达。羧基末端的21个氨基酸残基是必需的，足以将NS 5 B或异源蛋白靶向ER膜的胞质侧。该疏水结构域在分析的269个HCV分离株中高度保守，并预测形成跨膜α-螺旋。NS 5 B与ER膜的结合是通过不依赖ATP的翻译后机制进行的。这些特征将HCV RdRp定义为尾锚定蛋白家族的新成员，尾锚定蛋白家族是一类通过羧基末端插入序列在转录后被膜靶向的完整膜蛋白。因此，HCV复制复合物的形成涉及膜结合的特定决定因素，这些决定因素代表了抗病毒干预的潜在靶点。
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane a-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.