Specific interaction of hepatitis C virus protease/helicase NS3 with the 3′-terminal sequences of viral positive- and negative-strand RNA
Specific interaction of hepatitis C virus protease/helicase NS3 with the 3′-terminal sequences of viral positive- and negative-strand RNA
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DOI：
10.1128/jvi.75.4.1708-1721.2001
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发表时间：
2001-02-01
影响因子：
5.4
通讯作者：
Dasgupta, A
中科院分区：
文献类型：
--
作者：
Banerjee, R;Dasgupta, A
丙型肝炎病毒 (HCV) 编码的蛋白酶/解旋酶 NS3 可能参与病毒 RNA 复制。我们表达并纯化了重组 NS3（蛋白酶和解旋酶结构域）和 Delta pNS3（仅解旋酶结构域），并检查了它们与 HCV RNA 正链和负链 3' 端序列相互作用的能力。选择这些 RNA 区域是因为 RNA 合成的起始可能发生在 3' 非翻译区 (UTR) 处或附近。这里给出的结果表明 NS3（和 Delta pNS3）与正链和负链 RNA 的 3' 末端序列有效且特异地相互作用，但不与相应的互补 5' 末端 RNA 序列相互作用。 NS3 与 S' 末端负链 [称为 3'(-) UTR127] 的相互作用是特异性的，因为只有同源 RNA 才能有效竞争结合反应。存在于负链 RNA 3' 末端（距离 3' 末端第 5 至 20 个核苷酸）的预测茎环结构似乎对于 NS3 与负链 UTR 的结合很重要。茎环结构的删除几乎完全损害了 NS3（和 Delta pNS3）结合。额外的诱变表明茎内的三个 G-C 对对于解旋酶-RNA 相互作用至关重要。这里提供的数据还表明，茎中的双链结构和 3'-近端鸟苷残基是蛋白质结合的重要决定因素。与 3'(-) UTR 结合相对严格的要求相反，NS3（或 Delta pNS3）与正链 RNA [3'(+) UTR] 3' 末端序列的特异性相互作用似乎需要 HCV 的整个 3'(+) UTR。删除 98 个核苷酸的 3' 末端保守区或含有可变区和聚 (U) 和/或聚 (UC) 序列的 5' 半序列会显着损害 RNA-蛋白质相互作用。讨论了 NS3 与病毒正链和负链 RNA 3' 端序列结合对病毒复制的影响。
The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that NS3 (and Delta pNS3) interacts efficiently and specifically with the 3' terminal sequences of both positive- and negative strand RNA but not with the corresponding complementary 5'-terminal RNA sequences. The interaction of NS3 with the S'-terminal negative strand [called 3'(-) UTR127] was specific in that only homologous land not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3' terminus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3'-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3'(-) UTR binding, specific interaction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the positive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV. Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3'-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.