Novel ATP-independent RNA annealing activity of the dengue virus NS3 helicase.
Novel ATP-independent RNA annealing activity of the dengue virus NS3 helicase.
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DOI：
10.1371/journal.pone.0036244
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Gamarnik AV
中科院分区：
文献类型：
--
作者：
Gebhard LG;Kaufman SB;Gamarnik AV
黄病毒非结构蛋白3（NS 3）具有多种酶活性，是抗病毒干预的一个有吸引力的靶点。NS 3在N-末端含有病毒丝氨酸蛋白酶，在C-末端含有ATP酶、RT-PCR和解旋酶活性。这些活动对于病毒复制是必不可少的;然而，NS 3解旋酶在病毒生命周期中RNA重塑的生物学作用仍不清楚。存在于病毒基因组中的二级和三级RNA结构对于病毒复制至关重要。在这里，我们使用来自登革病毒的NS 3蛋白来研究NS 3与RNA结构变化相关的功能。使用不同的NS 3变体，我们表征了一个跨越残基171至618的结构域，该结构域显示出与全长蛋白质所观察到的类似的ATP酶和RNA解旋活性。有趣的是，我们发现，除了RNA解旋活性，登革病毒NS 3大大加速了互补RNA链与病毒或非病毒序列的退火。这种新的活性被发现是ATP不依赖的。确定缺乏ATP酶活性的突变的NS 3保留全RNA退火活性。使用ATP再生系统和不同的ATP浓度，我们观察到NS 3在RNA解旋和退火之间建立了一个ATP依赖的稳态，允许通过ATP浓度调节这种酶的两种相反的活性。此外，我们观察到NS 3增强了代表病毒基因组末端的分子之间的RNA-RNA相互作用，这些分子已知是病毒RNA合成所必需的。我们认为，根据ATP的可用性，NS 3可以调节病毒RNA结构的折叠或解折叠。
The flavivirus nonstructural protein 3 (NS3) bears multiple enzymatic activities and represents an attractive target for antiviral intervention. NS3 contains the viral serine protease at the N-terminus and ATPase, RTPase, and helicase activities at the C-terminus. These activities are essential for viral replication; however, the biological role of RNA remodeling by NS3 helicase during the viral life cycle is still unclear. Secondary and tertiary RNA structures present in the viral genome are crucial for viral replication. Here, we used the NS3 protein from dengue virus to investigate functions of NS3 associated to changes in RNA structures. Using different NS3 variants, we characterized a domain spanning residues 171 to 618 that displays ATPase and RNA unwinding activities similar to those observed for the full-length protein. Interestingly, we found that, besides the RNA unwinding activity, dengue virus NS3 greatly accelerates annealing of complementary RNA strands with viral or non-viral sequences. This new activity was found to be ATP-independent. It was determined that a mutated NS3 lacking ATPase activity retained full-RNA annealing activity. Using an ATP regeneration system and different ATP concentrations, we observed that NS3 establishes an ATP-dependent steady state between RNA unwinding and annealing, allowing modulation of the two opposing activities of this enzyme through ATP concentration. In addition, we observed that NS3 enhanced RNA-RNA interactions between molecules representing the ends of the viral genome that are known to be necessary for viral RNA synthesis. We propose that, according to the ATP availability, NS3 could function regulating the folding or unfolding of viral RNA structures.
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