Functional interaction of hepatitis C virus NS5B with nucleolin GAR domain
Functional interaction of hepatitis C virus NS5B with nucleolin GAR domain
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DOI：
10.1093/jb/mvm102
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发表时间：
2007-06-01
影响因子：
2.7
通讯作者：
Murakami, Seishi
中科院分区：
文献类型：
--
作者：
Kusakawa, Takashi;Shimakami, Tetsuro;Murakami, Seishi
丙型肝炎病毒（HCV）非结构蛋白是复制复合体的主要组成部分，受多种宿主因子的调控。我们以前报道过，核仁素，一个代表性的核仁标记物，通过两个独立的序列，氨基酸（aa）208-214和500-506与NS 5 B相互作用，并且前一段中的W208对于核仁素结合和HCV复制都是必需的。在这里，我们评估了WRHRARS的后一段氨基酸500-506在核仁结合和丙氨酸取代的簇状突变体（cm）或点突变体（pm）扫描的HCV复制中的作用。序列中的一个色氨酸和三个精氨酸残基被认为是必不可少的核仁结合在体内和HCV复制检测到的HCV亚基因组复制子转染到Huh 7细胞。核仁素的截短和成簇突变体进一步描绘了NS 5 B结合核仁素。富含甘氨酸精氨酸（GAR）结构域中的精氨酸-甘氨酸-甘氨酸（RGG）重复被定义为在体内和体外免疫检测中对NS 5 B结合不可或缺的，尽管RGG重复的短的内部截短对于NS 5 B结合是可容忍的。这些结果表明，核仁素是HCV复制的一个关键宿主因子，它通过与NS 5 B序列500-505位氨基酸残基的直接相互作用，以及核仁素C-末端包含RGG重复序列的长转角基序来实现。
Hepatitis C Virus (HCV) non-structural proteins are major components of replication complex that is modulated by several host factors. We previously reported that nucleolin, a representative nucleolar marker, interacts with the NS5B through two separated sequences, amino acids (aa) 208-214 and 500-506, and that W208 in the former stretch is essential for both nucleolin-binding and HCV replication. Here we evaluated the role of the latter stretch aa 500-506 of WRHRARS in nucleolin-binding and HCV replication scanned by alanine-substituted clustered mutant (cm) or point mutant (pm). One tryptophan and three arginine residues in the sequence were found to be essential both for nucleolin-binding in vivo and HCV replication detected with a HCV subgenomic replicon transfected into Huh7 cells. NS5B-binding of nucleolin was further delineated by truncation and clustered mutants of nucleolin. Arginine-glycine-glycine (RGG) repeat in the Glycine arginine rich (GAR) domain were defined to be indispensable for NS5B-binding immunologically detected in in vivo and in vitro although short internal-truncations of RGG repeat are tolerable for NS5B-binding. These results indicate that nucleolin is a critical host factor for HCV replication through the direct interaction between W208 and several residues at the sequence, aa 500-505, of NS5B, and the long-turn motif including RGG repeat at nucleolin C-terminal.