SV40 Large T Antigen Is Not Responsible for the Loss of STING in 293T Cells but Can Inhibit cGAS-STING Interferon Induction

Abstract

Several DNA viruses have evolved antagonists to inhibit the cyclic GMP–AMP synthase (cGAS)-stimulator of interferon genes (STING) DNA-sensing immune pathway. This includes DNA viral oncogenes that antagonize the cGAS-STING pathway by binding STING through the LxCxE motif. The 293T human cells are widely used in biology studies as they are highly transfectable. While parental 293 cells express high levels of STING, 293T cells lack STING and are unable to induce interferon antiviral responses to cytosolic DNA. Additionally, 293T cells express the SV40 polyomavirus large T antigen (LT) which enhances the replication of transfected DNA plasmids carrying the SV40 origin of replication. Since SV40 LT also encodes the LxCxE motif, the lack of STING expression in 293T cells is commonly assumed to be due to SV40 large T antigen. We find that SV40 LT does not alter exogenously expressed and endogenous levels of STING protein. We show that STING transcription is suppressed in 293T cells but is not driven by SV40. This study also revealed that SV40 LT does indeed inhibit cGAS-STING interferon induction, but through a mechanism distinct from other DNA virus oncogenes. Collectively, these results indicate that while SV40 LT can inhibit cGAS-STING interferon induction, it does so in an unanticipated manner.

Effective delivery of STING agonist using exosomes suppresses tumor growth and enhances antitumor immunity

“…STING protein was not detected in HEK293T cell lysates or exosomes ( Fig. S1 , A and B ) ( 28 , 29 , 30 ). Previous studies identified the folate receptor SLC19A1 as a transporter of STINGa into cells ( 31 ).…”

Disruption of DNA polymerase ζ engages an innate immune response

“…Our experiments were performed with MEFs immortalized by large T-antigen, which allowed cellular survival in the absence of pol z. In addition to blunting p53 activity and inactivating Rb, large T-antigen has been implicated in impairing an interferon response to nucleic acids in primary MEFs and suppressing cGAS-STING activation in human 293 cells through an unknown mechanism or mechanisms (Lau et al, 2015;Reus et al, 2020). Which functions of the large T-antigen enable cells to tolerate Rev3l deletion is an interesting but unexplored question.…”

Transcriptomic Analysis of the Innate Immune Response toin vitroTransfection of Plasmid DNA

“…Many other groups have also observed the high transfection efficiency exhibited by HEK-293T cells, which has led to their widespread adoption in industry and academia. [31][32][33] The high transfection efficiency and lack of CSG expression in HEK-293T cells may be due to the SV40 large T antigen, which was integrated into the parental HEK-293 cell genome to create the HEK-293T cell line. 34 The SV40 large T antigen has previously been shown to improve the replication efficiency of DNA viruses 35 and inhibit the induction of interferon expression by the cGAS-STING signaling pathway in other cell lines.…”

“…34 The SV40 large T antigen has previously been shown to improve the replication efficiency of DNA viruses 35 and inhibit the induction of interferon expression by the cGAS-STING signaling pathway in other cell lines. 33 Likewise, proteins expressed by the human papilloma virus (HPV) have also been shown to inhibit STING, 36 while the absence of STING in hepatocytes has been correlated with a lack of cytokine expression during hepatitis B virus (HBV) infection. 37 In regards to nonviral gene delivery, the inhibition of STING with small molecule inhibitors (C176, C178) has been shown to enhance transgene expression by up to 3-fold in a variety of cell lines, including primary T cells.…”