Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between a positively charged patch on the protease module and NS3 function. The surface patch is composed of four basic residues, R50, K74 and K94 in the NS3 protease domain and H24 in the structurally integrated cofactor NS4APCS. Single-residue or simultaneous four-residue substitutions in the patch to alanine or aspartic acid had little effect on ATPase activity. However, single substitutions of R50, K94 or H24 or a simultaneous four-residue substitution resulted in apparent changes in the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, a single substitution at K94 or a simultaneous four-residue substitution (Qua_A or Qua_D) impaired the production of infectious virus. Furthermore, the replication efficiency of the CSFV variants was partially correlated with the helicase activity of NS3 in vitro. Our results suggest that the conserved positively charged patch on NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.

中文翻译：

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瘟病毒NS3蛋白酶模块上带正电荷的表面补丁在调节NS3解旋酶活性和病毒产生中起重要作用。

瘟病毒非结构蛋白3（NS3）是一种多功能蛋白，具有蛋白酶和解旋酶活性，对于病毒复制至关重要。在这项研究中，我们结合了生物化学和遗传学方法来研究蛋白酶模块上带正电荷的贴剂与NS3功能之间的关系。表面补丁由四个基本残基组成，分别是NS3蛋白酶域中的R50，K74和K94和结构整合的辅助因子NS4APCS中的H24。补丁中的单残基或同时四残基取代为丙氨酸或天冬氨酸对ATPase活性的影响很小。但是，R50，K94或H24的单次取代或同时的四个残基取代导致NS3的解旋酶活性和RNA结合能力发生明显变化。当将这些突变引入经典猪瘟病毒（CSFV）cDNA克隆中时，在K94处的单取代或在同时存在的四个残基取代（Qua_A或Qua_D）会损害传染性病毒的产生。此外，在体外，CSFV变体的复制效率与NS3的解旋酶活性部分相关。我们的结果表明，NS3上保守的带正电荷的贴片在体外调节NS3解旋酶活性和CSFV产生中起重要作用。