The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently
The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently
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DOI：
10.1074/jbc.m310630200
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发表时间：
2004-01-09
影响因子：
4.8
通讯作者：
Gu, BH
中科院分区：
文献类型：
--
作者：
Frick, DN;Rypma, RS;Gu, BH
丙型肝炎病毒编码的非结构 3 (NS3) 蛋白同时具有 N 端丝氨酸蛋白酶活性和 C 端 3'-5' 解旋酶活性。本研究通过比较全长 NS3 蛋白与截短版本（其中蛋白酶被删除或被多聚组氨酸（His 标签）或谷胱甘肽 S-转移酶融合蛋白（GST 标签）取代）的酶学特性来检查蛋白酶对解旋酶的影响。当 NS3 蛋白缺乏蛋白酶结构域时，它解旋 RNA 的速度会更慢，并且在存在过量核酸（充当酶陷阱）的情况下不会解旋 RNA。通过在截短的解旋酶的 N 末端添加 His 标签或 GST 标签，可以恢复部分但不是全部的 RNA 解旋酶活性，这表明蛋白酶的作用既具有特异性又具有非特异性。 DNA 解旋酶和易位测定中也观察到类似但较小的影响。当在 RNA（或 DNA）上易位时，全长蛋白水解 ATP 的速度比截短的蛋白慢，这表明蛋白酶可以更有效地利用 ATP。结合测定表明，全长蛋白质在单链 DNA 上组装为比截短片段更高阶的寡聚物，并且结合似乎更具合作性。数据表明，丙型肝炎病毒 RNA 解旋酶以及病毒复制可能会受到多蛋白加工过程中可能发生的蛋白酶结构域旋转的影响。
The nonstructural 3 (NS3) protein encoded by the hepatitis C virus possesses both an N-terminal serine protease activity and a C-terminal 3'-5' helicase activity. This study examines the effects of the protease on the helicase by comparing the enzymatic properties of the full-length NS3 protein with truncated versions in which the protease is either deleted or replaced by a polyhistidine (His tag) or a glutathione S-transferase fusion protein (GST tag). When the NS3 protein lacks the protease domain it unwinds RNA more slowly and does not unwind RNA in the presence of excess nucleic acid that acts as an enzyme trap. Some but not all of the RNA helicase activity can be restored by adding a His tag or GST tag to the N terminus of the truncated helicase, suggesting that the effects of the protease are both specific and nonspecific. Similar but smaller effects are also seen in DNA helicase and translocation assays. While translocating on RNA ( or DNA) the full-length protein hydrolyzes ATP more slowly than the truncated protein, suggesting that the protease allows for more efficient ATP usage. Binding assays reveal that the full-length protein assembles on single-stranded DNA as a higher order oligomer than the truncated fragment, and the binding appears to be more cooperative. The data suggest that hepatitis C virus RNA helicase, and therefore viral replication, could be influenced by the rotations of the protease domain which likely occur during polyprotein processing.