Fig 2: Endogenous cGAS is predominantly a nuclear protein.(A) Clonal lines of HeLa cells were generated using lentiCRISPR encoding either a non-targeting H1 control gRNA (top row) or a cGAS-targeted gRNA (cGAS KO). Cells were fixed with methanol, stained with antibodies to human cGAS and beta-tubulin, counter-stained with DAPI, and visualized by confocal microscopy. (B) We noted three reproducible patterns of cGAS localization in addition to the nucleus: condensed mitotic chromatin (top row), structures resembling micronuclei (middle row), and tendril-like bridges between cells. (C) Mouse Cgas+/+ and Cgas-/- primary bone marrow-derived macrophages were stained using a mouse antibody to cGAS and processed as in (A).

Fig 3: cGAS nuclear localization is independent of cell cycle phase or activation status.(A) HeLa cells were arrested at the G1/S border using double thymidine (DT) block, followed by release and harvest at the indicated time points for measurement of DNA content. (B) Cells from (A) were fractionated and cGAS localization was determined by western blot. (C) HeLa cells were transfected with Lipofectamine alone (Lipo) or with CT-DNA for 4 hr, followed by extraction, salt elution, and western blot for endogenous cGAS.

Fig 4: Effect of BRCA1 on IFI16 and H2B acetylation during KSHV de novo infection.(A) HMVEC-d cells electroporated with siC or siBRCA1 for 48 h were uninfected or infected by KSHV for 4 h and subjected to PLA using mouse anti-IFI16 and rabbit anti-acetyl lysine antibodies. The localization of IFI16-acetyl lysine (acetylation of IFI16) PLA spots are shown in red. (B) WCL from HMVEC-d cells electroporated with siC and siBRCA1 for 48 h followed by with or without KSHV infection for 30’ (30 min), 4 and 24 h were IP-ed using anti-IFI16 and anti-acetyl lysine antibodies and western blotted for IFI16, BRCA1, p300, and H2B. Bottom input control panels show the levels of BRCA1, IFI16, p300 and total protein acetylation. (C) Schematic model illustrating the essential role of histone H2B in IFI16-mediated viral DNA genome sensing and innate IFN-β production during KSHV or HSV-1 de novo infection. Soon after KSHV or HSV-1 DNA entry into the nucleus, IFI16 in complex with BRCA1-H2B or with BRCA1 recognizes the viral genome, leading into BRCA1 mediated p300 recruitment, interaction with IFI16, acetylation (Ac) of IFI16 and H2B by p300, and cytoplasmic transport of acetylated IFI16-H2B-BRCA1 via Ran GTP. C646 or LPT treatments abolish acetylation or cytoplasmic translocation of H2B and IFI16, respectively. The inflammasome (IFI16-BRCA1-ASC-procaspase-1) independent IFI16-H2B-BRCA1 complex in the cytoplasm associates with cGAS and STING to form a macromolecular complex leading into phosphorylation of TBK1 and IRF3, nuclear translocation of p-IRF3 and subsequent IFN-β production during KSHV or HSV-1 de novo infection. Viral genome recognition by IFI16-BRCA1 results in acetylation of IFI16 which interacts with ASC and procaspase-1 in the nucleus. This BRCA1-IFI16-ASC-procaspase-1 complex is transported to the cytoplasm via Ran-GTP resulting in caspase-1 formation, pro-IL-1β cleavage and IL-1β formation [1, 9]. These studies demonstrate that the innate nuclear foreign viral genome sensing responses are mediated by two IFI16 complexes, with recognition by the IFI16-H2B-BRCA1 complex resulting in IFN-β responses while recognition by the IFI16-BRCA1 complex results in inflammasome-IL-1β responses.

Fig 5: Association of IFI16, H2B, BRCA1, cGAS and STING during KSHV infection.(A) For a double PLA reaction, two independent reactions were performed. In the first PLA reactions, uninfected and KSHV (KS) infected (4 h) HMVEC-d cells were immunostained using mouse (ms) anti-IFI16 and goat (g) anti-H2B antibodies, and detected by DUOLink green detection agent. Cells were washed, blocked and subjected to the second PLA reactions using goat anti-H2B and rabbit (rb) anti-STING antibodies and visualized with red detection agents. Green and red dots indicate the localization of IFI16-H2B and H2B-STING, respectively. (B-E) Similarly, double PLA for different combinations were performed using BRCA1 (ms) + H2B (g) and H2B (g) + STING (rb); IFI16 (ms) + H2B (rb) and H2B (rb) + cGAS (g); BRCA1 (ms) + H2B (rb) and BRCA1 (ms) + cGAS (g); and H2B (ms) + STING (rb) and STING (rb) + cGAS (g). Red arrows indicate yellow dots representing the associations of three different proteins together such as IFI16-H2B-STING (A), BRCA1-H2B-STING (B), IFI16-H2B-cGAS (C), BRCA1-H2B-cGAS (D) and H2B-STING-cGAS (E) in the cytoplasm of KSHV infected cells. Nuclei were stained with DAPI. (F and G) Effect of BRCA1, cGAS and H2B on the interaction of IFI16-STING during KSHV de novo infection. (F) WCL from HMVEC-d cells electroporated with siC, siBRCA1, sicGAS and siH2B for 48 h then infected or uninfected with KSHV for 4 h were western blotted with anti-BRCA1, cGAS and H2B antibodies. (G) The WCL lysates from panel F were IP-ed with anti-IFI16 antibody and immunoblotted for STING and H2B. The bottom panels showed H2B knockdown efficiency and STING as an input control. (H and I) Role of ASC in the association of STING with IFI16 and H2B during KSHV de novo infection. WCL from HMVEC-d cells electroporated with siC or siASC for 48 h followed by with and without KSHV infections (4 and 24 h) were IP-ed with anti-STING antibody and immunoblotted for IFI16, H2B and STING. The bottom panels show the knockdown efficiency of ASC along with equal inputs of IFI16, H2B and STING by WB.