Blay-Cadanet, J.; Pedersen, M. L.; Pedersen, A.; Hemberg, M.; Twayana, K.; Storgaard, J.; Bach-Nielsen, C. S.; Thielke, A. L.; Poulsen, C.; Kollmann, C. F.; Farahani, E.; Lappe, M.; Iversen, M. B.; Wu, Q.; Nielsen, C. B.; Johannsen, M.; Fenton, R. A.; Olagnier, D.; Lin, L.; Hansen, A. L.; Holm, C. K.

Metabolic reprogramming is a key component of antiviral immunity, yet how metabolite transport regulates innate immune signaling remains incompletely understood. Here, we show that infection with herpes simplex virus 1 (HSV-1) and stimulation with cytosolic DNA induce the cellular export of glutamate via the xCT (SLC7A11) transporter and that inhibition of xCT reduces cellular resistance to viral replication. Mechanistically, xCT inhibition impaired cGAS-STING signaling by reducing DNA-induced cGAMP production, thereby diminishing type I interferon (IFN?/?) responses and downstream induction of interferon-stimulated genes. Interestingly, modulating intracellular glutamate levels through inhibition of other glutamate pathways, e.g., glutaminolysis or glutamate import, also affected cellular IFN responses, suggesting that glutamate is a central control knob for DNA sensing. Finally, we demonstrate that HSV-1 suppresses xCT expression via a mechanism dependent on the immediate early viral protein ICP27, thereby promoting viral replication by limiting cGAS-dependent IFN induction. Together, these findings identify xCT-dependent glutamate transport as a critical metabolic regulator of cGAS-STING-mediated antiviral immunity.