Biochemical analysis of NSs from different tospoviruses
Biochemical analysis of NSs from different tospoviruses
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DOI：
10.1016/j.virusres.2017.09.020
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发表时间：
2017-10-15
期刊：
影响因子：
5
通讯作者：
Kormelink, Richard
中科院分区：
文献类型：
--
作者：
Hedil, Marcio;de Ronde, Dryas;Kormelink, Richard
番茄斑萎病毒通过编码RNA沉默抑制子（NS）蛋白来抑制抗病毒RNA干扰。此前，使用含有NS的植物和昆虫细胞粗提物，通过电泳迁移率变化测定（EMSA）证明了NS对双链（ds）RNA分子的亲和力。来自番茄斑萎病毒（TSWV）和花生环斑病毒（GRSV）的NS能够结合小的和长的dsRNA分子，而来自番茄黄环病毒（TYRV）的NS，一种不同的亚洲番茄斑萎病毒，仅结合小的dsRNA。在此，使用来自GRSV和TYRV的细菌表达和纯化的NS，表明它们都能够结合小的和长的dsRNA。NS对siRNA的结合揭示了两个连续的变化，即在低NS浓度下的第一个变化，随后在较高浓度下的第二个较大的变化。当使用来自感染植物的提取物分析TSWV抗性诱导（RI）和抗性破坏（RB）分离物的NS时，仅观察到主要的siRNA变化。相反，含有各瞬时表达的NS蛋白质的植物提取物仅显示与NS的较低偏移（RI），但与NS没有偏移（RB）。观察到的RNA双链体的亲和力，以及两步的转变模式，讨论了光的NS作为沉默的抑制剂和它的重要性，番茄斑萎病毒感染。
Tospoviruses suppress antiviral RNA interference by coding for an RNA silencing suppressor (NSs) protein. Previously, using NSs-containing crude plant and insect cell extracts, the affinity of NSs for double-stranded (ds) RNA molecules was demonstrated by electrophoretic mobility shifts assays (EMSAs). While NSs from tomato spotted wilt virus (TSWV) and groundnut ringspot virus (GRSV) were able to bind small and long dsRNA molecules, the one from tomato yellow ring virus (TYRV), a distinct Asian tospovirus, only bound small dsRNA. Here, using bacterially expressed and purified NSs from GRSV and TYRV, it is shown that they are both able to bind to small and long dsRNA. Binding of siRNAs by NSs revealed two consecutive shifts, i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When NSs of TSWV resistance inducing (RI) and resistance breaking (RB) isolates were analyzed using extracts from infected plants only a major siRNA shift was observed. In contrast, plant extracts containing the respective transiently expressed NSs proteins showed only the lower shift with NSs(RI) but no shift with NSs(RB). The observed affinity for RNA duplexes, as well as the two-stepwise shift pattern, is discussed in light of NSs as a suppressor of silencing and its importance for tospovirus infection.