CGAS enhances the HTLV-1 reverse transcription intermediate ssDNA90-induced 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 transfected into these cells 24 hours later.Real-time PCR was used to measure the expression of IFN-β, IP-10, RANTES and TNF-α.Immunoblot assay was performed to measure phosphorylated IRF3 and p65. Results Expression 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 enhanced 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, RANTES 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-induced innate immune responses. Key words: cGAS; HTLV-1; DNA sensor; Reverse transcription intermediate; Innate immune response