Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, 2 allée Konrad Roentgen, Strasbourg, 67084, France.
Institut de Biologie Moléculaire et Cellulaire du CNRS, Plateforme Protéomique Strasbourg - Esplanade, Université de Strasbourg, 2 allée Konrad Roentgen, Strasbourg, 67084, France.
Virol J. 2024 Mar 29;21(1):76. doi: 10.1186/s12985-024-02349-3.
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.
RNA 解旋酶作为调节宿主-病毒相互作用的关键因素而崭露头角。DEAD -box ATP 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，研究发现其在感染几种 RNA 病毒后，既能促进也能抑制病毒复制。在此，我们旨在研究 DDX5 对辛德比斯病毒（SINV）感染的影响。
我们通过成像和 RNA 免疫沉淀方法分析了 DDX5 与病毒 RNA 之间的相互作用。通过质谱法确定了 mock 和 SINV 感染细胞中 DDX5 的相互作用组。通过共免疫沉淀并在存在或不存在核糖核酸酶处理的情况下，验证了 DDX17 与病毒衣壳之间的相互作用。通过共免疫荧光法确定了 DDX5、其辅助因子 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 SINV 感染病毒蛋白、RNA 和感染性颗粒积累水平的影响。还通过敲低实验测试了 DDX17 的作用。
在本研究中，我们证明了 DDX5 在感染过程中与 SINV RNA 相互作用。此外，在 mock 和 SINV 感染的 HCT116 细胞中，对 DDX5 相互作用组的蛋白质组学分析鉴定了新的细胞和病毒伙伴，并证实了 DDX5 与 DDX17 之间的相互作用。DDX5 和 DDX17 在 SINV 感染后均从核内重新定位到细胞质，并与病毒衣壳蛋白相互作用。我们还表明，DDX5 耗竭会对病毒复制周期产生负面影响，而其过表达则具有促病毒作用。最后，我们观察到 DDX17 耗竭会降低 SINV 感染，在 DDX5 耗竭的背景下，这种影响更为明显，这表明 DDX5 和 DDX17 蛋白对 SINV 具有协同的促病毒作用。
这些结果不仅揭示了 DDX5 作为 SINV 生命周期中的一种新型重要宿主因子，还扩展了我们对 DDX5 和 DDX17 作为病毒感染调节剂的作用的理解。
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