Key Points DENV infection modulates EV cargo to influence neighboring and distant cells. miR-148a transferred via EVs suppresses USP33/ATF3 axis in human microglia. Dengue virus (DENV) infection disrupts host innate immune signaling at various checkpoints. Cellular levels and stability of intermediate signaling molecules are a crucial hijacking point for a successful viral pathogenesis. Stability and turnover of all the cellular proteins including intermediate signaling molecules are principally regulated by proteasomal degradation pathway. In this study, we show that how DENV infection and particularly DENV-NS1 can modulate the host extracellular vesicle (EV) cargo to manipulate the deubiquitination machinery of the human microglial cell (CHME3). We have performed EV harvesting, size analysis by nanoparticle tracking analysis, identification of cargo microRNA via quantitative PCR, microRNA target validation by overexpression, and knockdown via mimics and anti-miRs, immunoblotting, dual luciferase reporter assay, in vivo ubiquitination assay, chase assay, and promoter activity assay to reach the conclusion. In this study, we show that DENV-infected monocytes and DENV-NS1–transfected cells release high amounts of EVs loaded with miR-148a. These EVs get internalized by human microglial cells, and miR-148a suppresses the ubiquitin-specific peptidase 33 (USP33) protein expression levels via binding to its 3′ untranslated region. Reduced USP33 in turn decreases the stability of cellular ATF3 protein via deubiquitylation. ATF3 acts as a suppressor of major proinflammatory gene expression pathways of TNF-α, NF-κB, and IFN-β. Our mechanistic model explains how DENV uses the EV pathway to transfer miR-148a for modulating USP33 and downstream ATF3 levels in human microglial cells and contributes in neuroinflammation within the CNS.

中文翻译：

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登革热病毒通过细胞外囊泡降解 USP33-ATF3 轴以激活人类小胶质细胞

关键点 DENV 感染调节 EV 货物以影响邻近和远处的细胞。通过 EV 转移的 miR-148a 抑制人类小胶质细胞中的 USP33/ATF3 轴。登革热病毒 (DENV) 感染会在各个检查点破坏宿主先天免疫信号。中间信号分子的细胞水平和稳定性是成功病毒发病机制的关键劫持点。包括中间信号分子在内的所有细胞蛋白质的稳定性和周转主要受蛋白酶体降解途径的调节。在这项研究中，我们展示了 DENV 感染，尤其是 DENV-NS1 如何调节宿主细胞外囊泡 (EV) 货物以操纵人类小胶质细胞 (CHME3) 的去泛素化机制。我们已经通过纳米粒子跟踪分析进行了电动汽车收集、尺寸分析、通过定量 PCR 鉴定货物 microRNA、通过过表达进行 microRNA 靶标验证、通过模拟物和抗 miRs 进行敲低、免疫印迹、双荧光素酶报告基因检测、体内泛素化检测、追踪检测和启动子活性检测，以得出结论。在这项研究中，我们表明 DENV 感染的单核细胞和 DENV-NS1 转染的细胞释放大量装载有 miR-148a 的 EV。这些 EV 被人类小胶质细胞内化，而 miR-148a 通过与其 3' 非翻译区结合来抑制泛素特异性肽酶 33 (USP33) 蛋白表达水平。减少的 USP33 反过来通过去泛素化降低细胞 ATF3 蛋白的稳定性。ATF3 作为 TNF-α、NF-κB 和 IFN-β 主要促炎基因表达途径的抑制剂。