Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein.
Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein.
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DOI：
10.1006/viro.1999.9893
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发表时间：
1999-10
期刊：
影响因子：
3.7
通讯作者：
Hong Tu;Lu Gao;Stephanie T. Shi;Deborah R. Taylor;Tao Yang;A. Mircheff;Yumei Wen;A. Gorbalenya;S. Hwang;Michael M. C. Lai
中科院分区：
文献类型：
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作者：
Hong Tu;Lu Gao;Stephanie T. Shi;Deborah R. Taylor;Tao Yang;A. Mircheff;Yumei Wen;A. Gorbalenya;S. Hwang;Michael M. C. Lai
丙型肝炎病毒（HCV）NS 5A是一种具有隐蔽的反式激活活性的磷蛋白。为了研究NS 5A蛋白在病毒复制中的潜在作用，我们利用酵母双杂交技术从人肝细胞cDNA文库中筛选与NS 5A蛋白相互作用的细胞蛋白。我们鉴定了一种新发现的可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体样蛋白，称为人囊泡相关膜蛋白相关蛋白33 kDa（hVAP-33）。体外结合实验和体内免疫共沉淀实验证实了hVAP-33与NS 5A之间的相互作用。有趣的是，hVAP-33还显示与病毒RNA依赖性RNA聚合酶NS 5 B相互作用。NS 5A和NS 5 B结合hVAP-33的不同结构域：NS 5A结合hVAP-33的C-末端，而NS 5 B结合hVAP-33的N-末端。免疫荧光染色显示hVAP-33与NS 5A和NS 5 B蛋白均存在显著的共定位。hVAP-33在其C-末端含有卷曲螺旋结构域，随后是跨膜结构域。细胞分级分离分析显示，hVAP-33主要与ER，高尔基复合体，和前溶酶体膜，其在细胞内膜运输的潜在作用一致。这些相互作用为HCV RNA复制复合物的膜缔合提供了机制，并进一步表明NS 5A是病毒RNA复制复合物的一部分。
Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.