Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase (Retracted Article)
Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase (Retracted Article)
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DOI：
10.1074/jbc.m106880200
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发表时间：
2002-01-18
影响因子：
4.8
通讯作者：
Murakami, S
中科院分区：
文献类型：
--
作者：
Qin, WP;Luo, H;Murakami, S
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，具有“手掌和手指”的亚结构。我们最近鉴定了五种对RdRP活性至关重要的新残基（Qin，W.，山下，T.，Shirota，Y.，Lin，Y.，魏，W。，和Murakami，S.（2001）Hepatology 33，728-737）。其中，GLU-18和His-502，远离催化中心，可能参与RdRP活性的构象变化，如在一些棕榈和林格酶中所述。我们研究了NS 5 B寡聚化的可能性，并且我们可以检测两种不同标记的NS 5 B蛋白在体外和在哺乳动物细胞中瞬时表达之间的相互作用。通过扫描27个聚簇，然后点丙氨酸取代。Glu-18和His-502在体内和体外的同聚体相互作用中是关键的，强烈地表明NS 5 B的寡聚化和RdRP活性之间的密切关系。所有在这两个残基上具有取代的突变体都不能结合野生型NS 5 B，然而E18 H在体外和体内与H502 E相互作用。有趣的是，具有E18 H或H502 E的NS 5 B蛋白没有表现出RdRP活性，但两种突变蛋白的混合物表现出RdRP活性。这些结果清楚地表明，HCV NS 5 B的两个残基对于作为RdRP活性的先决条件的寡聚化是关键的。
HCV NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication, which has the "palm and fingers" substructure. We recently identified five novel residues critical for RdRP activity (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). Among them, GLU-18 and His-502, far from the catalytic center, may be involved in conformational change(s) for RdRP activity as addressed in some palm and ringers enzymes. We examined the possibility that NS5B is oligomerized, and we could detect the interaction between two different tagged NS5B proteins in vitro and transiently expressed in mammalian cells. By scanning 27 clustered and then point alanine substitutions in. vivo and in vitro, Glu-18 and His-502 were found to be critical for the homomeric interaction in vivo and in vitro, strongly suggesting a close relationship between the oligomerization and RdRP activity of NS5B. All mutants with substitutions at these two residues failed to bind wild type NS5B, however E18H interacted with H502E in vitro and in vivo. Interestingly, the NS5B protein with E18H or H502E did not exhibit RdRP activity, but a mixture of the two mutant proteins did. These results clearly indicate that two residues of HCV NS5B are critical for the oligomerization that is prerequisite to RdRP activity.