Direct interaction between nucleolin and hepatitis C virus NS5B
Direct interaction between nucleolin and hepatitis C virus NS5B
复制标题
DOI：
10.1074/jbc.m207629200
复制
发表时间：
2003-02-14
影响因子：
4.8
通讯作者：
Murakami, S
中科院分区：
文献类型：
--
作者：
Hirano, M;Kaneko, S;Murakami, S
丙型肝炎病毒（HCV）NS 5 B是一种RNA依赖性RNA聚合酶（RdRP），是HCV复制的中心催化酶。在研究缺乏C-末端膜锚定结构域的NS 513突变体（NS 5 Bt）的亚细胞定位时，我们发现绿色荧光蛋白（GFP）融合形式的表达仅为核仁。有趣的是，在表达GFP-NS 5 B的细胞中，内源性核仁素的分布发生了很大的变化，核仁素与GFP-NS 5 B共定位在除了核仁之外的核周区域，这表明NS 513保留了结合核仁素的能力。核仁素与NS 513之间的相互作用为。通过GST pull-down分析证实。GST pull-down分析结果表明核仁素C端区域对它与NS 513的结合是重要的。扫描成簇的NS 513丙氨酸取代突变体库，发现NS 513上有两个结合核仁素的位点。发现NS 513氨基酸208-214和500-506对于核仁素结合都是不可缺少的。我们报道，后者的序列是必不可少的寡聚化的NS 513，这是一个先决条件的RdRP活动。C-末端核仁素以剂量依赖性方式抑制NS 513 RdRP活性。综上所述，这表明核仁素的结合能力可能参与NS 513功能。
Hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme in HCV replication. While studying the subcellular localization of a NS513 mutant lacking the C-terminal membrane-anchoring domain, NS5Bt, we found that expression of the green fluorescent protein (GFP)-fused form was exclusively nucleolar. Interestingly, the distribution of endogenous nucleolin changed greatly in the cells expressing GFP-NS5B, with nucleolin colocalized with GFP-NS5B in perinuclear regions in addition to the nucleolus, suggesting that NS513 retains the ability to bind nucleolin. The interaction between nucleolin and NS513 was. demonstrated by GST pull-down assay. GST pull-down assay results indicated that C-terminal region of nucleolin was important for its binding to NS513. Scanning clustered alanine substitution mutants library of NS513 revealed two sites on NS513 that binds nucleolin. NS513 amino acids 208-214 and 500-506 were both found to be indispensable for the nucleolin binding. We reported that the latter sequence is essential for oligomerization of NS513, which is a prerequisite for the RdRP activity. C-terminal nucleolin inhibited the NS513 RdRP activity in a dose-dependent manner. Taken together, this indicates the binding ability of nucleolin may be involved in NS513 functions.