Landais Igor, Pelton Chantel, Streblow Daniel, DeFilippis Victor, McWeeney Shannon, Nelson Jay A
Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
Division of Biostatistics, Public Health and Preventive Medicine, Oregon Health and Science University, Portland, Oregon, United States of America.
PLoS Pathog. 2015 May 8;11(5):e1004881. doi: 10.1371/journal.ppat.1004881. eCollection 2015 May.
Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1β, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression.
人类巨细胞病毒（HCMV）编码多种微小RNA（miRNA），其功能才刚刚开始被揭示。通过计算机分析方法，我们确定Toll样受体（TLR）先天免疫途径是HCMV miRNA的一个可能靶点。荧光素酶报告基因检测筛选进一步确定TLR2是HCMV miR-UL112-3p的靶点。TLR2通过检测细菌和病毒配体（包括HCMV包膜蛋白gB和gH）在先天免疫反应中起主要作用。TLR2激活多种信号转导途径，包括NFκB途径。此外，TLR2在控制人类和小鼠的CMV感染中起重要作用。对用miR-UL112-3p模拟物转染的细胞进行免疫印迹分析表明，内源性TLR2被miR-UL112-3p下调，其效率与靶向TLR2的小干扰RNA（siTLR2）相似。接下来我们发现，在HCMV感染后期，TLR2蛋白水平下降，并且与成纤维细胞和单核细胞THP1细胞中miR-UL112-3p的积累相关。在感染缺乏miR-UL112-3p表达的HCMV突变体的细胞中未观察到内源性TLR2的下调，但在这些细胞中转染miR-UL112-3p可恢复TLR2的下调，从而证实了miR-UL112-3p的直接靶向作用。使用NFκB报告基因细胞系，我们发现转染miR-UL112-3p可显著抑制NFκB依赖的荧光素酶活性，其效率与siTLR2相似。与该观察结果一致，在用TLR2激动剂刺激后，转染miR-UL112-3p可显著降低多种细胞因子（IL-1β、IL-6和IL-8）的表达。最后，转染miR-UL112-3p可显著抑制TLR2诱导的IRAK1的翻译后激活，IRAK1是位于TLR2/NFκB信号轴上游的一种激酶。据我们所知，这是首次确定的HCMV调节TLR2的机制，也是病毒miRNA对TLR2进行功能靶向的首次报道。这些结果提供了一种新机制，通过该机制HCMV miRNA通过下调TLR-2表达来调节先天免疫反应。
