Phosphoinositide Interactions Position cGAS at the Plasma Membrane to Ensure Efficient Distinction between Self- and Viral DNA

Abstract

The presence of DNA in the cytosol of mammalian cells is an unusual event that is often associated with genotoxic stress or viral infection. The enzyme cGAS is a sensor of cytosolic DNA that induces interferon and inflammatory responses that can be protective or pathologic, depending on the context. Along with other cytosolic innate immune receptors, cGAS is thought to diffuse throughout the cytosol in search of its DNA ligand. Herein, we report that cGAS is not a cytosolic protein but rather localizes to the plasma membrane via the actions of an N-terminal phosphoinositide-binding domain. This domain interacts selectively with PI(4,5)P₂, and cGAS mutants defective for lipid binding are mislocalized to the cytosolic and nuclear compartments. Mislocalized cGAS induces potent interferon responses to genotoxic stress, but weaker responses to viral infection. These data establish the subcellular positioning of a cytosolic innate immune receptor as a mechanism that governs self-nonself discrimination.

Keywords: STING; cGAS; innate immunity; interferon; localization; macrophages; phosphoinositides.

Figure 1.. cGAS Associates with the Plasma Membrane

(A) Subcellular fractionation of THP1 cells in the presence or absence of benzonase. Western blot analysis was used to probe the cytosolic fraction (S100), membrane fraction (P100), and nuclear (P25) fractions for the indicated proteins. (B) Membrane floatation assays of THP1 post-nuclear lysates on a 10%–45% Optiprep gradient in the presence or absence of benzonase. Fractions 1–24 were taken from the top to the bottom of the gradient, and western blot analysis was used to probe these fractions for the indicated proteins. (C) Membrane floatation assays of THP1 post-nuclear lysates on a 10%–45% Optiprep gradient in the presence of benzonase in the presence or absence of 1% Triton X-100. (D) Confocal micrographs cGAS in WT and cGAS^−/− THP1 cells treated with 1,000 U/mL recombinant IFN-β1 for 4 h. In diagrams to the left of micrographs, red dashed lines indicate the plane in view (not to scale). (E) Subcellular fractionation of THP1-expressing cGAS-HA cells in the presence of benzonase. (F) Confocal micrographs of various cell lines expressing cGAS-HA. Experiments shown are representative of n = 3 biological replicates. See also Figure S1.

Figure 2.. The N Terminus of cGAS Is Necessary and Sufficient for Plasma Membrane Association

(A) Schematic of human cGAS domain architecture and truncation mutants. (B) Western blot analysis of mutant cGAS expression in THP1 cells. (C) Confocal micrograph of THP1 cells expressing human cGAS N or cGASΔN. (D) Western blot analysis of murine mutant cGAS expression in iBMDMs. (E) Confocal micrographs of iBMDMs expressing murine cGAS N or cGASΔN. (F) Confocal micrographs of L929 and HeLa cells expressing cGAS N. (G) Heatmap showing the relative pI of the N- and C-terminal domains of cGAS from various species (n = 10) alongside innate immune adaptors TIR-domain-containing adaptor protein (TIRAP) and TRIF-related adaptor molecule (TRAM) with N-terminal PIP-binding domains. Experiments shown are representative of n = 3 biological replicates. See also Figure S2.

Figure 3.. cGAS Binds PI(4,5)P₂ via Its N Terminus

(A) PIP strip analysis of recombinant cGAS. Recombinant cGAS was incubated with a membrane spotted with the indicated lipids and analyzed by far western. (B) Cosedimentation assay for cGAS interactions with liposomes containing 18% of the specified PIPs on a 3:1 PC:PE backbone. Recombinant cGAS was incubated with liposomes, after which the liposomes were isolated by ultracentrifugation and probed by western blot for cGAS association. (C) Fluorescent liposome pull-down assay. 6xHis-tagged cGAS was incubated with fluorescent liposomes containing the specified PIPs. cGAS was isolated by nickel affinity resin, and cGAS-lipid interaction was measured by the amount of fluorescence pulled down with the resin.. (D) Same as (C), but 6x-His-tagged cGAS, cGASΔN, and cGAS N were probed for binding to PI(4,5)P₂. (E) Confocal micrographs of electroporated primary BMDMs expressing cGAS N-HA with PLCδ1-PH-YFP. (F) Confocal micrographs of electroporated primary BMDMs expressing gp91^phox-PX-YFP. (G) Confocal micrograph of an iBMDM expressing cGAS N phagocytosing a fluorescent zymosan particle. Experiments shown are representative of or averages of n = 3 biological replicates. Data are represented as a mean ± SEM, and statistical analysis was performed using a Student’s t test with asterisk coding as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 4.. Loss of the cGAS N Terminus Leads to Heightened Basal IFN Signaling and Increased Responses to Genotoxic Stress

(A) qPCR analysis of IFNβ1 expression in THP1 cell lines at steady state. (B) qPCR analysis of RSAD2 expression in THP1 cell lines at steady state. (C) qPCR analysis of IFNβ1 expression following a 6-h treatment with 500 μM H₂O₂ in the indicated THP1 cell lines. (D) Western blot analysis for the indicated proteins before and after 6-h treatment with 500 μM H₂O₂. (E) qPCR analysis of IFNβ1 expression following a 14-h treatment with 500 nM Doxorubicin in the indicated THP1 cell lines. (F) Viability of THP1 cell lines following a 14-h treatment with 500 nM Doxorubicin, shown as a percentage of untreated controls, as measured by intracellular ATP content. (G) qPCR analysis of IFNβ1 and RSAD2 expression at the indicated time points following treatment with 50 ng/mL PMA in the indicated THP1 cell lines. (H) Viability of THP1 cell lines following overnight treatment with 50 ng/mL PMA, as described in (F). (I) qPCR analysis of IFNβ1 expression at the indicated time points following infection with MVA-Ova (MOI 3) in the indicated THP1 cell lines. (J) Western blot analysis of the indicated proteins before and after infection with MVA-Ova (MOI 3) in the indicated cell lines. H.P.I., hours post-infection. Experiments shown are representative of or averages of n = 3 biological replicates. Data are shown as a mean ± SEM. Statistical analysis comparing two data points was performed using a Student’s t test, and statistical analysis of time-course experiment trends in (G) and (I) were performed using two-way ANOVA with asterisk coding as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. See also Figure S3.

Figure 5.. The cGAS R71/75E Mutant Does Not Bind PI(4,5)P₂ and Does Not Associate with the Plasma Membrane

(A) Left: schematic of truncation mutants screened in the cGAS N terminus in HeLa cells and their ability to associate with the plasma membrane. Bottom center: residues identified as essential for cGAS membrane association with mutated residues indicated in red and the indicated point mutations. Right: alignment of mutated residues in several species (same as Figure 2G) with R71 and R75 boxed. (B) Fluorescent liposome pull-down assay with the indicated mutants, as described in Figure 3C. (C) Western blot analysis of expression of indicated cGAS mutants in transiently transfected 293T cells and BMDMs stably expressing mutants via lentiviral transduction. (D) ISRE luciferase assay of 293Ts expressing the indicated constructs 24 h post-transfection. – indicates cells transfected with the ISRE reporter construct alone. All cGAS constructs were co-transfected with STING to enable pathway signaling. (E) Confocal micrographs of indicated cell lines expressing cGAS R71/75E C396/7A. Experiments shown are representative of or averages of n = 3 biological replicates. Data are shown as a mean ± SEM, and statistical analysis was performed using a Student’s t test with asterisk coding as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 6.. Artificial Localization of cGASΔN to the Plasma Membrane through the TIRAP PI(4,5)P₂ Binding Domain Rescues Localization and Prevents Hyperresponsiveness to Genotoxic Stress

(A) Schematic of the synthetic TIR domain containing adaptor protein (TIRAP) PI(4,5)P₂-binding domain fused to cGASΔN. (B) Western blot analysis of THP1 cells stably expressing the indicated mutants. (C) Confocal micrograph of THP1 cells stably expressing TIRAP-ΔN-HA. (D) qPCR analysis of IFNβ1 expression 3 h post-treatment with 50 ng/mL PMA in the indicated THP1 cell lines. (E) Viability of THP1 cell lines following overnight treatment with 50 ng/mL PMA, shown as a percentage of untreated controls, as measured by intracellular ATP content. (F) Schematic of the synthetic Fyn dual-acylation motif fused to cGASΔN. (G) Confocal micrograph of THP1 cells stably expressing Fyn-ΔN-HA. (H) ISRE luciferase assay of 293Ts expressing the indicated constructs 24 h post-transfection, as described in Figure 5D. (I) Western blot analysis of 293T cells expressing indicated constructs. Experiments shown are representative of or averages of n = 3 biological replicates. Data are shown as a mean ± SEM, and statistical analysis was performed using a Student’s t test with asterisk coding as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.