The protein–RNA interactions within the flavivirus replication complex (RC) are not fully understood. Our structure of dengue virus NS3 adenosine triphosphatase (ATPase)/helicase bound to the conserved 5′ genomic RNA 5′-AGUUGUUAGUCU-3′ reveals that D290 and R538 make specific interactions with G2 and G5 bases respectively. We show that single-stranded 12-mer RNA stimulates ATPase activity of NS3, however the presence of G2 and G5 leads to significantly higher activation. D290 is adjacent to the DEXH motif found in SF2 helicases like NS3 and interacts with R387, forming a molecular switch that activates the ATPase site upon RNA binding. Our structure guided mutagenesis revealed that disruption of D290–R387 interaction increases basal ATPase activity presumably as a result of higher conformational flexibility of the ATPase active site. Mutational studies also showed R538 plays a critical role in RNA interactions affecting translocation of viral RNA through dynamic interactions with bases at positions 4 and 5 of the ssRNA. Restriction of backbone flexibility around R538 through mutation of G540 to proline abolishes virus replication, indicating conformational flexibility around residue R538 is necessary for RNA translocation. The functionally critical sequence-specific contacts in NS3 RNA binding groove in subdomain III reveals potentially novel allosteric anti-viral drug targets.

中文翻译：

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登革热病毒的NS3解旋酶特异性识别病毒RNA序列，以确保最佳复制

黄病毒复制复合体（RC）中的蛋白质-RNA相互作用尚不完全清楚。我们与保守的5'基因组RNA 5'- AG UU G结合的登革热病毒NS3腺苷三磷酸酶（ATPase）/解旋酶的结构UUAGUCU-3'揭示D290和R538分别与G2和G5碱基发生特定的相互作用。我们显示单链12聚体RNA刺激NS3的ATPase活性，但是G2和G5的存在导致明显更高的激活。D290与SF2解旋酶（如NS3）中发现的DEXH基序相邻，并与R387相互作用，形成分子开关，在结合RNA后激活ATPase位点。我们的结构指导诱变表明，D290-R387相互作用的破坏可能会增加基础ATPase活性，这可能是由于ATPase活性位点具有更高的构象灵活性。突变研究还显示，R538通过与ssRNA 4和5位碱基的动态相互作用，在影响病毒RNA易位的RNA相互作用中起关键作用。通过将G540突变为脯氨酸来限制R538周围的骨架柔韧性可消除病毒复制，这表明R538残基周围的构象柔韧性对于RNA转运是必需的。亚域III中NS3 RNA结合槽中的功能关键序列特异性接触揭示了潜在的新型变构抗病毒药物靶标。