The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis-cleavage event. Although this conformation is incompatible with protease trans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein.IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states.

中文翻译：

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瘟病毒NS3中蛋白酶反式切割和解旋酶活性的解偶联。

黄病毒科的非结构蛋白NS3是一种多功能蛋白，包含N端蛋白酶和C端解旋酶，在病毒多蛋白加工和基因组复制中起着至关重要的作用。在这里，我们报道了以2.35Å分辨率解析其经典猪瘟病毒（CSFV）NS3及其NS4A蛋白酶辅因子片段（PCS）的全长晶体结构。该结构揭示了以前未鉴定的〜2,200-Å2分子内蛋白酶-解旋酶界面，该界面包含三个相互作用簇，代表与NS3-NS4A顺式裂解事件有关的“闭合”全局构象。尽管此构象与蛋白酶的反式切割不兼容，但它在功能上似乎很重要，并且对解旋酶活性有益，由于旨在干扰这种构象的突变会破坏体外解旋酶的活性和体内病毒的产生，因此我们的工作揭示了瘟病毒NS3中蛋白酶-解旋酶协调作用的重要特征，并为不同构象状态如何显着促进其功能提供了关键基础。重要说明：许多RNA病毒编码解旋酶，通过解开结构化RNA来帮助其RNA基因组复制和转录。由黄病毒科病毒编码的NS3解旋酶与参与病毒多蛋白加工的蛋白酶天然融合，因此具有独特性。因此，这两个酶模块如何在单个多肽中协调特别令人关注。在这里，我们报告了瘟病毒NS3与其NS4A蛋白酶辅因子区段（PCS）复杂的先前未鉴定的构象。该构象状态与蛋白酶的顺式切割事件有关，并且对于解旋酶的功能而言是最佳的。这项工作为理解NS3的不同酶活性如何通过蛋白酶和解旋酶之间通过不同构象状态的配合而实现提供了重要依据。