ABSTRACT
Objective To investigate whether cyclic GMP-AMP synthase ( cGAS ) , a cytosolic DNA sensor, could recognize the reverse transcription intermediate and induce the subsequent signaling path-way 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 interac-tion 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 ex-pression 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.
ABSTRACT
Objective To investigate the role of cyclic GMP-AMP synthase (cGAS),a cytosolic DNA sensor,in regulating innate immune responses induced by reverse transcription intermediate of human T cell leukemia virus type 1 (HTLV-1). Methods (1)ssDNA90,the reverse transcription intermediate of HTLV-1,was transfected into HeLa cells to observe changes in the expression pattern of cGAS in transfected-HeLa cells with immunoblot assay. (2) HeLa cells were firstly transfected with cGAS-encoding plasmid and then ssDNA90 24 hours later. Real-time PCR was used to measure the expression of interferon ( IFN)-β, IFN-gamma-inducible protein 10 ( IP-10 ), regulated on activation, normal T cell expressed and secreted (RANTES) and tumor necrosis factor (TNF)-α. Immunoblot assay was performed to measure phosphorylated interferon regulatory factor 3 (IRF3) and p65. (3)cGAS expression was silenced by siRNA in HeLa and phorbol-12-myristate-13-acetate (PMA)-treated THP1 (PMA-THP1) cells and then ssDNA90 was transfect-ed into these cells 24 hours later. Real-time PCR was used to measure the expression of IFN-β,IP-10,RAN-TES and TNF-α. Immunoblot assay was performed to measure phosphorylated IRF3 and p65. Results Ex-pression of cGAS was increased in HeLa cells after ssDNA90 transfection. Compared with control cells, cGAS-transfected HeLa cells showed increased expression of IFN-β, IP-10, RANTES and TNF-α and en-hanced phosphorylation of IRF3 and p65 after ssDNA90 transfection. Compared with control cells,both HeLa and PMA-THP1 cells with silenced expression of cGAS showed impaired production of IFN-β,IP-10,RAN-TES and TNF-α after ssDNA90 transfection. Moreover,ssDNA90-induced phosphorylation of IRF3 and p65 were decreased after cGAS gene-knockdown. Conclusion cGAS might promote HTLV-1 RTI ssDNA90-in-duced innate immune responses.