Kundharapu Satyamurthy, Chowdary Tirumala Kumar
School of Biological Sciences, National Institute of Science Education and Research, An OCC of Homi Bhabha National Institute, Bhubaneswar 752050, India.
Viruses. 2022 Aug 3;14(8):1712. doi: 10.3390/v14081712.
Dengue virus replicates its single-stranded RNA genome in membrane-bound complexes formed on the endoplasmic reticulum, where viral non-structural proteins (NS) and RNA co-localize. The NS proteins interact with one another and with the host proteins. The interaction of the viral helicase and protease, NS3, with the RNA-dependent RNA polymerase, NS5, and NS4b proteins is critical for replication. In vitro, NS3 helicase activity is enhanced by interaction with NS4b. We characterized the interaction between NS3 and NS4b and explained a possible mechanism for helicase activity modulation by NS4b. Our bacterial two-hybrid assay results showed that the N-terminal 57 residues region of NS4b is enough to interact with NS3. The molecular docking of the predicted NS4b structure onto the NS3 structure revealed that the N-terminal disordered region of NS4b wraps around the C-terminal subdomain (CTD) of the helicase. Further, NS3 helicase activity is enhanced upon interaction with NS4b. Molecular dynamics simulations on the NS4b-docked NS3 crystal structure and intrinsic tryptophan fluorescence studies suggest that the interaction results in NS3 CTD domain motions. Based on the interpretation of our results in light of the mechanism explained for NS3 helicase, NS4b-NS3 interaction modulating CTD dynamics is a plausible explanation for the helicase activity enhancement.
登革病毒在形成于内质网的膜结合复合物中复制其单链RNA基因组，病毒非结构蛋白（NS）和RNA在此共定位。NS蛋白彼此之间以及与宿主蛋白相互作用。病毒解旋酶和蛋白酶NS3与RNA依赖性RNA聚合酶NS5以及NS4b蛋白的相互作用对复制至关重要。在体外，NS3解旋酶活性通过与NS4b相互作用而增强。我们对NS3和NS4b之间的相互作用进行了表征，并解释了NS4b调节解旋酶活性的可能机制。我们的细菌双杂交试验结果表明，NS4b的N端57个残基区域足以与NS3相互作用。将预测的NS4b结构与NS3结构进行分子对接显示，NS4b的N端无序区域环绕解旋酶的C端亚结构域（CTD）。此外，NS3解旋酶活性在与NS4b相互作用时增强。对NS4b对接的NS3晶体结构进行分子动力学模拟以及对色氨酸固有荧光的研究表明，这种相互作用导致NS3 CTD结构域运动。根据我们的结果并结合对NS3解旋酶机制的解释，NS4b-NS3相互作用调节CTD动力学是解旋酶活性增强的一个合理原因。
