PDF(Pubmed)
Abstract：
The non-structural protein (NSs) and nucleoprotein (NP) of the severe fever with thrombocytopenia syndrome virus (SFTSV) encoded by the S segment are crucial for viral pathogenesis. They reside in viroplasm-like structures (VLS), but their interaction and their significance in viral propagation remain unclear. Here, we investigated the significance of the association between NSs and NP during viral infection through in-silico and in-vitro analyses. Through in-silico analysis, three possible binding sites were predicted, at positions C6S (Cystein at 6th position to Serine), W61Y (Tryptophan 61st to Tyrosine), and S207T (Serine 207th to Threonine), three mutants of NSs were developed by site-directed mutagenesis and tested for NP interaction by co-immunoprecipitation. NSsW61Y failed to interact with the nucleoprotein, which was substantiated by the conformational changes observed in the structural analyses. Additionally, molecular docking analysis corroborated that the NSW61Y mutant protein does not interact well compared to wild-type NSs. Over-expression of wild-type NSs in HeLa cells increased the SFTSV replication by five folds, but NSsW61Y exhibited 1.9-folds less viral replication than wild-type. We demonstrated that the W61Y alteration was implicated in the reduction of NSs-NP interaction and viral replication. Thus, the present study identified a critical NSs site, which could be targeted for development of therapeutic regimens against SFTSV.
摘要：
由S段编码的严重发热伴血小板减少综合征病毒（SFTSV）的非结构蛋白（NSs）和核蛋白（NP）对于病毒的发病机理至关重要。它们存在于病毒质样结构(VLS)中，但它们的相互作用及其在病毒繁殖中的意义仍不清楚。这里,我们通过计算机模拟和体外分析研究了病毒感染期间NSs和NP之间关联的意义.通过硅分析，预测了三个可能的结合位点，在C6S位置（丝氨酸第6位的半胱氨酸），W61Y（色氨酸61至酪氨酸），和S207T（丝氨酸207至苏氨酸），通过定点诱变开发了NSs的三个突变体，并通过免疫共沉淀测试了NP相互作用。NSsW61Y未能与核蛋白相互作用，结构分析中观察到的构象变化证实了这一点。此外,分子对接分析证实，与野生型NSs相比，NSW61Y突变蛋白的相互作用不佳。野生型NSs在HeLa细胞中的过表达使SFTSV复制增加了五倍，但NSsW61Y的病毒复制比野生型少1.9倍。我们证明了W61Y改变与NSs-NP相互作用和病毒复制的减少有关。因此，本研究确定了一个关键的NSs位点，这可以作为开发针对SFTSV的治疗方案的目标。
