Hirano Masaaki, Kaneko Shuichi, Yamashita Tatsuya, Luo Hong, Qin Weiping, Shirota Yukihiro, Nomura Takahiro, Kobayashi Kenichi, Murakami Seishi
Department of Molecular Oncology, Cancer Research Institute, Ishikawa 920-0934, Japan.
J Biol Chem. 2003 Feb 14;278(7):5109-15. doi: 10.1074/jbc.M207629200. Epub 2002 Nov 9.
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 NS5B 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 NS5B retains the ability to bind nucleolin. The interaction between nucleolin and NS5B 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 NS5B. Scanning clustered alanine substitution mutants library of NS5B revealed two sites on NS5B that binds nucleolin. NS5B 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 NS5B, which is a prerequisite for the RdRP activity. C-terminal nucleolin inhibited the NS5B RdRP activity in a dose-dependent manner. Taken together, this indicates the binding ability of nucleolin may be involved in NS5B functions.
丙型肝炎病毒（HCV）NS5B是一种依赖RNA的RNA聚合酶（RdRP），是HCV复制过程中的核心催化酶。在研究缺乏C末端膜锚定结构域的NS5B突变体NS5Bt的亚细胞定位时，我们发现绿色荧光蛋白（GFP）融合形式的表达仅定位于核仁。有趣的是，在表达GFP-NS5B的细胞中，内源性核仁素的分布发生了很大变化，除了核仁外，核仁素在核周区域与GFP-NS5B共定位，这表明NS5B保留了与核仁素结合的能力。通过谷胱甘肽-S-转移酶（GST）下拉实验证实了核仁素与NS5B之间的相互作用。GST下拉实验结果表明，核仁素的C末端区域对其与NS5B的结合很重要。扫描NS5B的丙氨酸取代突变体聚类文库揭示了NS5B上两个与核仁素结合的位点。发现NS5B的208-214位氨基酸和500-506位氨基酸对于与核仁素的结合都是不可或缺的。我们报道，后一个序列对于NS5B的寡聚化至关重要，而寡聚化是RdRP活性的前提条件。C末端核仁素以剂量依赖的方式抑制NS5B的RdRP活性。综上所述，这表明核仁素的结合能力可能参与了NS5B的功能。
