Schmidt-Mende J, Bieck E, Hugle T, Penin F, Rice C M, Blum H E, Moradpour D
Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany.
J Biol Chem. 2001 Nov 23;276(47):44052-63. doi: 10.1074/jbc.M103358200.
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 alpha-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.
丙型肝炎病毒（HCV）的RNA依赖性RNA聚合酶（RdRp），以非结构蛋白5B（NS5B）为代表，被认为与其他非结构蛋白以及尚未确定的宿主成分一起形成膜相关的RNA复制复合体。然而，这种重要病毒酶的膜结合决定因素尚未明确。通过双标记免疫荧光分析，发现单独表达或在整个HCV多聚蛋白背景下表达时，NS5B存在于内质网（ER）或类似ER的修饰区室中。羧基末端的21个氨基酸残基对于将NS5B或异源蛋白靶向到ER膜的胞质侧是必要且充分的。该疏水结构域在分析的269株HCV分离株中高度保守，并预测形成跨膜α螺旋。NS5B与ER膜的结合通过一种不依赖ATP的翻译后机制发生。这些特征将HCV RdRp定义为尾锚定蛋白家族的新成员，这是一类整合膜蛋白，通过羧基末端插入序列在翻译后靶向膜。因此，HCV复制复合体的形成涉及膜结合的特定决定因素，这些因素代表了抗病毒干预的潜在靶点。
