cGAS识别HTLV-1反转中间体并诱导STING依赖性的固有免疫应答

cGAS recognizes HTLV-1 RTI and induces STING-dependent innate immune responses

目的探讨DNA识别受体环鸟苷酸-腺苷酸合成酶(cGAS)在成人T淋巴细胞白血病病毒1型(HTLV-1)感染过程中对病毒反转中间体的识别以及诱导的下游信号通路。方法将生物素标记的HTLV-1的反转中间体ssDNA90转染入HeLa细胞,采用免疫共沉淀试验检测cGAS与ssDNA90的相互作用;将HeLa细胞与HTLV-1阳性细胞系MT2共培养,采用免疫共沉淀的方法检测cGAS与干扰素基因刺激蛋白(STING)的相互作用;采用siRNA的方法在HeLa细胞中敲减STING,24 h后再转染入cGAS表达质粒,24 h后与MT2细胞共培养24 h,real-time PCR实验检测HeLa细胞中干扰素(IFN)-β、调节活化正常T细胞表达和趋化因子(RANTES)、肿瘤坏死因子(TNF)-α、HTLV-1病毒蛋白Tax、p19以及HBZ的表达,免疫印迹检测HeLa细胞中干扰素调节因子3(IRF3)和p65的磷酸化水平。结果cGAS与ssDNA90存在相互作用;cGAS与STING存在相互作用,且作用部位在胞质中;在敲减STING的HeLa细胞中,cGAS过表达对IFN-β、RANTES和TNF-α的表达量无明显影响,对HTLV-1病毒蛋白Tax、p19和HBZ的表达量无明显影响,对IRF3和p65的磷酸化水平无明显影响。结论cGAS可结合HTLV-1的反转中间体ssDNA90并通过STING激活固有免疫应答。

Objective To investigate whether cyclic GMP-AMP synthase(cGAS),a cytosolic DNA sensor,could recognize the reverse transcription intermediate and induce the subsequent signaling pathway during the infection of human T cell leukemia virus type 1(HTLV-1).Methods Biotin-labeled ssDNA90,a reverse transcription intermediate of HTLV-1,was transfected into HeLa cells and the interaction between it and cGAS was detected by co-immunoprecipitation experiments.HeLa cells were co-cultured with HTLV-1-positive MT2 cells and the interaction between cGAS and stimulator of interferon genes(STING)was analyzed by co-immunoprecipitation experiments.The expression of STING in HeLa cells was silenced by siRNA.cGAS was transfected into the HeLa cells 24 h after the silencing and after 24 h,these cells were co-cultured with MT2 cells for another 24 h.Real-time PCR assay was used to measure the expression of IFN-β,RANTES(regulated upon activation,normal T-cell expressed,and secreted),TNF-α,HTLV-1 protein Tax,p19 and HBZ.Immunoblot assay was performed to evaluate the phosphorylation of IRF3 and p65 in HeLa cells.Results cGAS interacted with ssDNA90.cGAS interacted with STING in the cytoplasm.In STING-silenced HeLa cells,cGAS transfection had no influence on the expression of IFN-β,RANTES,TNF-α,Tax,p19 or HBZ,nor did it affect the phosphorylation of IRF3 or p65.Conclusions cGAS interacted with HTLV-1 RTI ssDNA90 and activated STING-dependent innate immune responses.