Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase
Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase
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DOI：
10.1073/pnas.1900656116
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发表时间：
2019-05-07
影响因子：
11.1
通讯作者：
Luo, Dahai
中科院分区：
文献类型：
--
作者：
Law, Yee-Song;Utt, Age;Luo, Dahai
基孔肯雅病毒（CHIKV）通过蚊子传播给人类，引起基孔肯雅热。非结构蛋白2（nsP 2）具有病毒RNA复制和转录所需的蛋白酶和RNA解旋酶活性。与C-末端蛋白酶不同，N-末端RNA解旋酶（nsP 2 h）的结构尚未确定。在这里，我们报告了与CHIKV基因组的保守3 '端14个核苷酸结合的nsP 2 h和不可水解的过渡态核苷酸类似物ADP-AlF 4的晶体结构。总体而言，结构分析表明，nsP 2 h采用独特折叠的N-末端结构域，随后是超家族1 RNA解旋酶折叠。保守的解旋酶基序与RNA骨架建立极性接触。有三个疏水残基（Y161、F164和F287）与RNA碱基形成堆积相互作用，从而使RNA骨架弯曲。一个F287 A取代，破坏这些堆叠的相互作用增加了基础ATP酶的活性，但降低了RNA结合亲和力。此外，F287 A取代通过减弱亚基因组RNA合成来降低病毒感染性。通过假回复（A287 V）或RecA 2解旋酶结构域中的适应性突变（T358 S或V410 I）恢复突变病毒的复制。Y161 A和/或F164 A取代，其被设计为破坏与RNA分子的相互作用，不影响ATP酶活性，但完全消除了病毒RNA的复制和转录以及CHIKV的感染性。我们的研究揭示了RNA解旋酶区域在病毒复制中的作用，并提供了可能适用于甲病毒和其他RNA病毒的见解。
Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) exhibits the protease and RNA helicase activities that are required for viral RNA replication and transcription. Unlike for the C-terminal protease, the structure of the N-terminal RNA helicase (nsP2h) has not been determined. Here, we report the crystal structure of the nsP2h bound to the conserved 3'-end 14 nucleotides of the CHIKV genome and the nonhydrolyzable transitionstate nucleotide analog ADP-AlF4. Overall, the structural analysis revealed that nsP2h adopts a uniquely folded N-terminal domain followed by a superfamily 1 RNA helicase fold. The conserved helicase motifs establish polar contacts with the RNA backbone. There are three hydrophobic residues (Y161, F164, and F287) which form stacking interactions with RNA bases and thereby bend the RNA backbone. An F287A substitution that disrupted these stacking interactions increased the basal ATPase activity but decreased the RNA binding affinity. Furthermore, the F287A substitution reduced viral infectivity by attenuating subgenomic RNA synthesis. Replication of the mutant virus was restored by pseudoreversion (A287V) or adaptive mutations in the RecA2 helicase domain (T358S or V410I). Y161A and/or F164A substitutions, which were designed to disrupt the interactions with the RNA molecule, did not affect the ATPase activity but completely abolished the replication and transcription of viral RNA and the infectivity of CHIKV. Our study sheds light on the roles of the RNA helicase region in viral replication and provides insights that might be applicable to alphaviruses and other RNA viruses in general.