State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
Virus Res. 2021 Apr 2;295:198318. doi: 10.1016/j.virusres.2021.198318. Epub 2021 Jan 22.
Dengue virus NS3 is a prototypical DEx(H/D) helicase that binds and hydrolyzes NTP to translocate along and unwind double-stranded nucleic acids. NS3 and NS4B are essential components of the flavivirus replication complex. Evidences showed that NS4B interacted with NS3 and modulated the helicase activity of NS3. Despite important insights into structural, mechanistic, and cellular aspects of the NS3 function, there is still a gap in understanding how it coordinates the helicase activities within the replicase complex for efficient replication. Here, using the DENV2 as a model, we redefined the critical region of NS4B required for NS3 function by pull-down and MST assays. The FRET-based unwinding assay showed that NS3 would accelerate unwinding duplex nucleic acids in the presence of NS4B (51-83). The simulated NS3-NS4B complex models based on the rigid-body docking delineated the potential interaction sites located in the conserved motif within the core domain of NS3. Mutations in motif I (I190A) and motif III (P319L) of NS3 interfered with the unwinding activity stimulated by NS4B. Upon binding to the NS3 helicase, NS4B assisted NS3 to dissociate from single-stranded nucleic acid and enabled NS3 helicase to keep high activity at high ATP concentrations. These results suggest that NS4B probably serves as an essential cofactor for NS3 to coordinate the ATP cycles and nucleic acid binding during viral genome replication.
登革病毒 NS3 是一种典型的 DEx(H/D)解旋酶，它结合并水解 NTP，沿着双链核酸移动并解旋。NS3 和 NS4B 是黄病毒复制复合物的重要组成部分。有证据表明，NS4B 与 NS3 相互作用并调节 NS3 的解旋酶活性。尽管对 NS3 的结构、机制和细胞方面有了重要的了解，但对于它如何协调复制酶复合物中的解旋酶活性以实现有效的复制，仍存在理解上的差距。在这里，我们使用 DENV2 作为模型，通过下拉和 MST 测定重新定义了 NS4B 对 NS3 功能所必需的关键区域。基于 FRET 的解旋测定表明，在 NS4B（51-83）存在的情况下，NS3 会加速双链核酸的解旋。基于刚性对接的模拟 NS3-NS4B 复合物模型描绘了位于 NS3 核心结构域内保守基序中的潜在相互作用位点。NS3 中的 motif I（I190A）和 motif III（P319L）突变干扰了 NS4B 刺激的解旋活性。当与 NS3 解旋酶结合时，NS4B 协助 NS3 从单链核酸解离，并使 NS3 解旋酶在高 ATP 浓度下保持高活性。这些结果表明，NS4B 可能作为 NS3 的必需辅助因子，协调病毒基因组复制过程中的 ATP 循环和核酸结合。
