Viruses, cells, and reagents.

DMEM-F12 (Lonza) or DMEM (BioWhittaker) were supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin, and LPS-free heat-inactivated FCS (BioWhittaker). ProLong Gold, DAPI, and recombinant mTNF-α were from Invitrogen. Z-VAD-FMK, nigericin, murine 3′3-cAIMP, and human 2′-3′cGAMP were from InvivoGen. LPS and PI were both obtained from Sigma-Aldrich. Both HSV-1 (McKrae) and GFP-HSV-1 (HSV-1–expressing eGFP driven by the CMV promoter) were gifts provided by David Leib, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire, USA and were grown in Vero cells. The Vero cells used were from ATCC (CCL-81, RRID:CVCL_0059). The titers of the stocks used were 8 × 10⁹ to 14 × 10⁹ PFU/mL, determined by standard plaque assay on Vero cells. Beriglobin was used to neutralize extracellular HSV-1 (CSL Behring) and fully neutralized the virus in the dilutions used to calculate titers. The BV-2 murine microglial cell line (RRID:CVCL_0182) was a gift provided by Anni Paulina Kleino, Aarhus Institute of Advanced Studies, Aarhus, Denmark). The BV-2 cells were cultured in DMEM including 10% FCS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Primary mouse neurons, microglia, and astrocytes were isolated from cGas^–/– and WT neonatal mice as described previously (25). Spleen cells were harvested from age-matched 12-week-old female mice. Spleens were gently homogenized manually in a glass homogenizer in RPMI supplemented with 10% FCS. Erythrocytes were removed by hemolysis. The cells were passed through a 70 μm pore size filter, washed, counted, and seeded 24 hours prior to stimulation.