Protein Kinase Regulated by dsRNA Downregulates the Interferon Production in Dengue Virus- and dsRNA-Stimulated Human Lung Epithelial Cells
Protein Kinase Regulated by dsRNA Downregulates the Interferon Production in Dengue Virus- and dsRNA-Stimulated Human Lung Epithelial Cells
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dsRNA 调节的蛋白激酶下调登革热病毒和 dsRNA 刺激的人肺上皮细胞中干扰素的产生
DOI：
10.1371/journal.pone.0054369
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发表时间：
2013-01-25
期刊：
影响因子：
3.7
通讯作者：
Huang, Xi
中科院分区：
文献类型：
--
作者：
Li, Yuye;Xie, Jiong;Huang, Xi
背景：登革病毒(DENV)在热带和亚热带地区发现，每年感染数百万人。目前，还没有针对登革热病毒的特定疫苗或抗病毒治疗。天然免疫已被证明对宿主抵抗DENV感染具有重要作用。尽管双链RNA(PKR)调控的蛋白激酶(PKR)可以促进多种病毒感染后的先天信号转导，但其在DENV感染后的先天反应中的作用仍不清楚。我们的研究旨在探讨PKR在DENV诱导的先天免疫反应中的作用。方法/主要研究结果：通过RNAi，沉默PKR显著增强DENV感染的人肺上皮细胞A549细胞中干扰素-β的表达。Western印迹和免疫荧光显微镜数据显示，在DENV2感染后，PKR基因敲除上调了包括p38和JNK丝裂原激活蛋白激酶(MAPKs)、干扰素调节因子-3和核因子-kappaB在内的固有信号级联反应的激活。同样，在Poly(IC)攻击的细胞中，也观察到PKR对干扰素产生的负调控作用。此外，RIG-I或IPS-1沉默可减弱PKR基因敲除介导的干扰素诱导。最后，一个催化失活的PKR突变体(K296R)的过度表达，而不是缺乏dsRNA结合活性的突变体(K64E)或双突变体(K64EK296R)的过表达，逆转了PKR敲除介导的干扰素诱导，提示PKR需要dsRNA结合活性来下调干扰素的产生。结论：PKR在DENV和Poly(IC)诱导的干扰素诱导中起负调控作用，这一调节依赖于其dsRNA结合活性。这些发现揭示了PKR在先天性免疫中的一个新的调节作用，表明PKR可能是抗DENV治疗的一个有前途的靶点。
Background: Dengue virus (DENV) is found in the tropical and subtropical regions and affects millions of people annually. Currently, no specific vaccine or antiviral treatment against dengue virus is available. Innate immunity has been shown to be important for host resistance to DENV infection. Although protein kinase regulated by double-stranded RNA (PKR) has been found to promote the innate signaling in response to infection by several viruses, its role in the innate response to DENV infection is still unclear. Our study aimed to investigate the role of PKR in DENV-induced innate immune responses.Methodology/Principal Findings: By RNAi, silencing of PKR significantly enhanced the expression of interferon (IFN)-beta in DENV infected human lung epithelial A549 cells. Western blot and immunofluorescence microscopy data showed that PKR knockdown upregulated the activation of innate signaling cascades including p38 and JNK mitogen-activated protein kinases (MAPKs), interferon regulatory factor-3 and NF-kappa B, following DENV2 infection. Likewise, a negative regulatory effect of PKR on the IFN production was also observed in poly(IC) challenged cells. Moreover, the PKR knockdown-mediated IFN induction was attenuated by RIG-I or IPS-1 silencing. Finally, overexpression of a catalytically inactive PKR mutant (K296R), but not of a mutant lacking dsRNA binding activity (K64E) or the double mutant (K64EK296R), reversed the IFN induction mediated by PKR knockdown, suggesting that the dsRNA binding activity is required for PKR to downregulate IFN production.Conclusions/Significance: PKR acts as a negative regulator of IFN induction triggered by DENVs and poly(IC), and this regulation relies on its dsRNA binding activity. These findings reveal a novel regulatory role for PKR in innate immunity, suggesting that PKR might be a promising target for anti-DENV treatments.