Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase
Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase
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DOI：
10.1128/jvi.77.7.4149-4159.2003
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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：
Gao, L;Tu, H;Lai, MMC
为了确定丙型肝炎病毒(HCV)RNA依赖的RNA聚合酶(NS5B)的潜在细胞调控因子，我们通过酵母双杂交筛选人肝细胞cDNA文库，寻找与NS5B蛋白相互作用的细胞蛋白。我们鉴定了一种泛素样蛋白hPLIC1(人类连接整合相关蛋白和细胞骨架的同源蛋白1)，它在肝脏中表达(M.F.Kleijnen，A.H.Shih，P.周，S.Kumar，R.E.Soccio，N.L.KederashG.Gill，和P.M.Howley，Moll.牢房6：409-419,2000)。体外结合实验和体内免疫共沉淀实验证实了hPLIC1与NS5B之间的相互作用，这种相互作用是通过hPLIC1蛋白C端的泛素相关结构域实现的。由于hPLIC已被证明与两个E3泛素蛋白连接酶以及蛋白酶体物理上相关(Kleijnen等人，Moll.细胞6：409-419,2000)，我们研究了hPLIC1是否影响NS5B的稳定性和翻译后修饰。脉冲追逐标记实验表明，过表达hPLIC1，而不是缺失NS5B结合域的突变体，显著缩短了NS5B的半衰期，并增强了NS5B的多泛素化。此外，在表达丙型肝炎病毒亚基因组复制子的Huh7细胞中，由于hPLIC1的过表达，NS5B和复制子RNA的数量都减少了。因此，hPLIC1可能通过与NS5B的相互作用而调节丙型肝炎病毒的RNA复制。
To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA pollymerase (NS5B), we searched-for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Moll. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Moll. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.