Specific Interaction of DDX6 with an RNA Hairpin in the 3′ UTR of the Dengue Virus Genome Mediates G1 Phase Arrest
Specific Interaction of DDX6 with an RNA Hairpin in the 3′ UTR of the Dengue Virus Genome Mediates G1 Phase Arrest
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DOI：
10.1128/jvi.00510-21
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发表时间：
2021-09-01
影响因子：
5.4
通讯作者：
Chimnaronk, Sarin
中科院分区：
文献类型：
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作者：
Choksupmanee, Opas;Tangkijthavorn, Worapol;Chimnaronk, Sarin
病毒基因组 RNA 与宿主因子相互作用以及对宿主反应和疾病发病机制的贡献程度尚不清楚。在这里，我们报告人类 RNA 解旋酶 DDX6 与登革热 3' UTR 中 A3 元件中病毒最保守的 RNA 发夹特异性结合，具有纳摩尔亲和力。 DDX6 CLIP 证实了登革热病毒血清型 2 感染的 HuH-7 细胞中的相互作用。这种相互作用需要三个保守残基 - Lys(307)、Lys(367) 和 Arg(369) - 以及 DDX6 C 端结构域中的非结构化延伸。有趣的是，这三个基本残基的丙氨酸取代导致了不依赖于RNA的ATP酶活性，这表明RNA结合和ATP酶活性在DEAD盒解旋酶中耦合的机制。此外，我们应用跨组学基因富集方法表明 DDX6 在功能上与细胞周期调节和病毒致病性相关。事实上，受感染的细胞表现出细胞周期停滞在 G1 期和早期 S 期减少。完整 DDX6 的外源表达（而非 A3 结合缺陷突变体）通过拯救 DNA 前起始复合物表达来减轻这些影响。在登革热和寨卡减毒活疫苗株中发现了 DDX6 结合位点的破坏。我们的研究结果表明，登革热病毒进化出了针对 DDX6 的 RNA 适体来改变宿主细胞状态，并将 DDX6 定义为 G1/S 转换的新调节因子。 重要性 登革热病毒 (DENV) 通过蚊子传播给人类，每年在全球感染 3.9 亿人。约20%的感染患者表现出一系列临床表现，从轻度流感样综合征到登革热，再到危及生命的严重登革热疾病，包括登革出血热和登革休克综合征。目前，登革热疾病尚无特效治疗方法，而且登革热发病机制的分子机制仍知之甚少。在这项研究中，我们结合了生化、生物信息学、高内涵分析和 RNA 测序方法，以表征感染细胞中 DENV RNA 基因组与名为 DDX6 的人类因子的高度保守界面。我们研究的意义在于确定病毒策略改变宿主细胞命运的机制，这可能使我们能够生成减毒活疫苗模型并设计登革热疾病的新治疗试剂。
The extent to which viral genomic RNAs interact with host factors and contribute to host response and disease pathogenesis is not well known. Here, we report that the human RNA helicase DDX6 specifically binds to the viral most conserved RNA hairpin in the A3 element in the dengue 3' UTR, with nanomolar affin-ities. DDX6 CLIP confirmed the interaction in HuH-7 cells infected by dengue virus serotype 2. This interaction requires three conserved residues-Lys(307), Lys(367), and Arg(369)-as well as the unstructured extension in the C- terminal domain of DDX6. Interestingly, alanine substitution of these three basic residues resulted in RNA-independent ATPase activity, suggesting a mechanism by which RNA-binding and ATPase activities are coupled in DEAD box helicases. Furthermore, we applied a cross-omics gene enrichment approach to suggest that DDX6 is functionally related to cell cycle regulation and viral pathogenicity. Indeed, infected cells exhibited cell cycle arrest in G1 phase and a decrease in the early S phase. Exogenous expression of intact DDX6, but not A3-binding-deficient mutants, alleviated these effects by rescue of the DNA preinitiation complex expression. Disruption of the DDX6-binding site was found in dengue and Zika live-attenuated vaccine strains. Our results suggested that dengue virus has evolved an RNA aptamer against DDX6 to alter host cell states and defined DDX6 as a new regulator of G1/S transition.IMPORTANCE Dengue virus (DENV) is transmitted by mosquitoes to humans, infecting 390 million individuals per year globally. About 20% of infected patients shows a spectrum of clinical manifestation, ranging from a mild flu-like syndrome, to dengue fever, to life-threatening severe dengue diseases, including dengue hemorrhagic fever and dengue shock syndrome. There is currently no specific treatment for dengue diseases, and the molecular mechanism underlying dengue pathogenesis remains poorly understood. In this study, we combined biochemical, bioinformatics, high-content analysis and RNA sequencing approaches to characterize a highly conserved interface of the RNA genome of DENV with a human factor named DDX6 in infected cells. The significance of our research is in identifying the mechanism for a viral strategy to alter host cell fates, which conceivably allows us to generate a model for live-attenuated vaccine and the design of new therapeutic reagent for dengue diseases.