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 function and the possible mechanism of cyclic GMP-AMP synthase (cGAS), a DNA sensor, in HeLa cells during human T cell leukemia virus type 1 (HTLV-1) in-fection.Methods HeLa cells were co-cultured with MT2 cells (HTLV-1-positive T cells) and then detec-ted by immunoblot assay to analyze the changes in the expression of cGAS .A hemagglutinin ( HA)-tagged cGAS plasmid was constructed and transfected into HeLa cells .Twenty-four hours after transfection , these cells were co-cultured with MT2 cells for another 24 hours.Immunoblot assay was used to detect the expres-sion of HTLV-1 proteins Tax and p19.Real-time PCR was performed to measure the expression of HTLV-1 Tax, p19, Env, HBZ and px at mRNA level .Immunoblot assay was also used to analyze the phosphorylation of interferon regulatory factor 3 (IRF3) and p65.Expression of interferon (IFN)-β, IFN-gamma-inducible protein 10 ( IP-10 ) , RANTES ( regulated on activation , normal T cell expressed and secreted ) and tumor necrosis factor (TNF)-αwas detected by real-time PCR assay.Results Expression of cGAS was enhanced in HeLa cells after co-cultured with MT2 cells.Compared with control cells , the HeLa cells that were trans-fected with cGAS plasmid showed lower levels of Tax and p 19 proteins, suppressed expression of HTLV-1 Tax, p19, Env, HBZ and px at mRNA level , enhanced phosphorylation of IRF 3 and p65, and higher levels of IFN-β, IP-10, RANTES and TNF-αafter co-cultured with MT2 cells.Conclusion cGAS might promote the innate immune response and inhibit HTLV-1 replication in HTLV-1-infected HeLa cells .