背景:RNA解旋酶正在成为调节宿主-病毒相互作用的关键因素。DEAD-boxATP依赖性RNA解旋酶DDX5在细胞RNA生物学的许多方面发挥着重要作用,还发现在感染几种RNA病毒时促进或抑制病毒复制。这里,我们的目的是研究DDX5对辛德毕斯病毒(SINV)感染的影响.
方法:我们使用成像和RNA免疫沉淀方法分析了DDX5和病毒RNA之间的相互作用。通过质谱测定模拟和SINV感染的细胞中DDX5的相互作用。我们在存在或不存在RNase处理的情况下通过共免疫沉淀验证了DDX17和病毒衣壳之间的相互作用。我们通过联合免疫荧光确定了DDX5,其辅因子DDX17和病毒衣壳蛋白的亚细胞定位。最后,我们研究了DDX5耗竭和过表达对病毒蛋白的SINV感染的影响,RNA和感染性颗粒积累水平。DDX17的贡献也通过敲除实验进行测试。
结果:在这项研究中,我们证明了DDX5在感染过程中与SINVRNA相互作用。此外,对模拟和SINV感染的HCT116细胞中DDX5相互作用组的蛋白质组学分析鉴定了新的细胞和病毒伴侣,并证实了DDX5和DDX17之间的相互作用.DDX5和DDX17在SINV感染后从细胞核重新定位到细胞质,并与病毒衣壳蛋白相互作用。我们还表明DDX5耗竭对病毒复制周期产生负面影响,而其过度表达具有病毒前作用。最后,我们观察到DDX17耗竭减少了SINV感染,在DDX5耗尽的背景中更明显的效果,提示DDX5和DDX17蛋白对SINV的协同性前病毒作用。
结论:这些结果不仅揭示了DDX5作为SINV生命周期的一种新颖且重要的宿主因子,但也扩大了我们对DDX5和DDX17作为病毒感染调节因子的作用的理解。
RNA helicases are emerging as key factors regulating host-virus interactions. The DEAD-box ATP-dependent RNA helicase
DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of
DDX5 on Sindbis virus (SINV) infection.
We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of
DDX5 in mock- and SINV-infected cells was determined by mass spectrometry. We validated the interaction between DDX17 and the viral capsid by co- immunoprecipitation in the presence or absence of an RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein by co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation level. The contribution of DDX17 was also tested by knockdown experiments.
In this study we demonstrate that
DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between
DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect which is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV.
These results not only shed light on DDX5 as a novel and important host factor to the SINV life cycle, but also expand our understanding of the roles played by DDX5 and DDX17 as regulators of viral infections.