Fig 2: Inhibition of HSV-1 replication blocks cGAS release from the chromatin.a RAW 264.7 cells were mock-infected or infected with 1 MOI of HSV-1 KOS d109 for 16 h. Cell lysates were fractionated and blotted as indicated. b RAW 264.7 cells were pretreated without or with 8 μg/mL of acyclovir for 16 h, followed by infection with 1 MOI of HSV-1 McKrae for 12 h. Cell lysates were fractionated and blotted as indicated. The arrow indicates nuclear soluble cGAS. c RAW 264.7 macrophages were treated with dimethylformamide (DMF) as a vehicle mock control, 50 μM cisplatin for 4 h, or infected with HSV-1 McKrae for 12 h. Cell lysates were fractionated and blotted as indicated. The arrow indicates nuclear soluble cGAS. STING: membrane marker; α-tubulin: cytosolic marker; H3: nuclear marker; ICP8: HSV-1 viral protein; γ-H2A.X: DNA damage marker. The arrow indicates nuclear soluble cGAS. d RAW 264.7 macrophages were treated with DMF (mock control), 50 μM cisplatin or infected with HSV-1 McKrae for indicated times. Then, cell viability was determined by MTT assays. Data represent means ± s.d. of three independent experiments. The P-value was calculated by two-way ANOVA followed by Sidak’s multiple comparisons test.

Fig 3: PCBP2 attenuates the cGAS condensation.a HEK293T cells were transfected with the indicated expression plasmids. After 24 h of transfection, cell lysates were subjected to Co-IP with anti-Flag beads, followed by immunoblotting. b HEK293T cells were transfected with the indicated expression plasmids. After 24 h of transfection, cell lysates were resolved with SDD-AGE or SDS-PAGE, followed by immunoblotting. c Pcbp2+/+ and Pcbp2-/- L929 cells were transfected with HT-DNA (2 µg/ml) for 6 h, and the cell lysates were resolved with SDD-AGE or SDS-PAGE, followed by immunoblotting. d Recombinant cGAS and PCBP2 wild-type or its KH3-deleted mutant proteins were incubated with or without HT-DNA (200 µg/ml) at 25 °C for 1.5 h and then resolved by SDD-AGE and SDS-PAGE, followed by immunoblotting. e HEK293A cells were transfected with GFP-cGAS, together with mCherry-tagged wild-type PCBP2 or its KH3-deleted mutant or mCherry for 24 h. The cells were fixed, stained with DAPI (blue), and observed by confocal microscopy. Scale bars, 10 µm. The number of cGAS granules per cell was quantified (bottom), at least 100 cells from each group were analyzed. p < 0.0001; p < 0.0001 in sequence. P2: PCBP2. f Pcbp2+/+ and Pcbp2-/- L929 cells stably expressing mouse cGAS-GFP were transfected with or without HT-DNA (2 µg/ml) for 6 h. The cells were then fixed, stained with DAPI (blue), and observed by confocal microscopy. Scale bars, 10 µm. The number of cGAS granules per cell was quantified (bottom), at least 100 cells from each group were analyzed. p < 0.0001. g–i Recombinant GFP-cGAS proteins (5 µM) were incubated with 2 µM of GST-tagged wild-type PCBP2 or its KH3-deleted mutant or GST proteins in the presence of 2.5 µM Cy3-ISD (g), Cy3-VACV70 (h), or HSV120 (i) for 5 min. Confocal images are representative of cGAS condensates of all fields. Scale bars, 10 µm. Data shown in e, f are from one representative experiment of at least three independent experiments (mean ± SD, n = 107 cells in e, and n = 133 cells in f). Two-tailed Student’s t-test was used to analyze statistical significance. ***p < 0.001. Source data are provided as a Source data file.

Fig 4: PCBP2 specifically targets cGAS to antagonize its enzyme activity.a THP-1 cells were transfected with or without HT-DNA (2 µg/ml), and cell lysates were subjected to Co-IP with a PCBP2 antibody or control IgG, followed by immunoblotting. b–e HEK293T cells were transfected with IFNß-Luc and expression vectors encoding cGAS and STING (b), STING (c), TBK1 (d), or IRF3-5D (e), together with the increased amount of PCBP2 or an empty vector. After 24 h of transfection, the cells were lysed for luciferase reporter assays (upper panel) and immunoblotting assays (lower panels). b p = 0.0258; p = 0.0004 in sequence. f PCBP2+/+ and PCBP2-/- THP-1 cells were permeabilized with Perfringolysin O (PFO; 300 ng/ml), then treated with or without cGAMP (0.2 µg/ml) for 4 h, followed by immunoblotting. g THP-1 cells were infected with a lentivirus expressing PCBP2 or an empty vector for 48 h, followed by infection with HSV-1 for 9 h. Cytoplasmic cGAMP was extracted and quantified with a cGAMP ELISA kit. p = 0.0155. h PCBP2-knockout and control THP-1 cells were transfected with HT-DNA for 6 h and then harvested to measure the abundance of cGAMP with a cGAMP ELISA kit. p = 0.0256. i Pcbp2+/+ and Pcbp2+/- MEFs were transfected with HT-DNA for 6 h, and the cGAMP abundance in cytoplasmic extracts was measured with a cGAMP ELISA kit. p = 0.0027. j In vitro enzyme activity assays of cGAS were conducted by incubating recombinant human cGAS protein and GST, GST-PCBP2, or GST-PCBP2-?KH3 with HT-DNA (50 ng/µl) at 37 °C for 2 h. The mixtures were then incubated with benzonase at 37 °C for 30 min, heated at 95 °C for 5 min, followed by centrifugation. The heat-resistant supernatants were used to determine the abundance of cGAMP using L929-ISRE cells. p < 0.0001. P2: PCBP2. Data shown in b–e, g–j are from one representative experiment of at least 2 biological independent experiments (mean ± SD, n = 2 independent samples). Two-tailed Student’s t-test was used to analyze statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant versus the control groups. Source data are provided as a Source data file.

Fig 5: Nuclear soluble cGAS inhibits HSV-1 infection.a cGAS KO, KO(mcGAS), KO(LL/RK-NLS), and KO(LL/RK-NLS)/D307A cells were treated with Dox for 24 h and then infected with 1 MOI of HSV-1-GFP for 16 h. Cells expressing GFP were examined and counted under a fluorescence microscope. The relative infection was determined by the ratio of positive cells. Data represent means ± s.d. of three independent experiments (> 200 cells were counted in each field and five fields were counted per experiment). The P-value was calculated by one-way ANOVA followed by Dunnett’s multiple comparisons test. b The cGAS KO, KO(mcGAS), KO(LL/RK-NLS) RAW264.7 cells were treated with Dox for 24 h and then infected with 1 MOI of HSV-1 McKrae for 16 h. Then, cells were collected for RNA extraction. The RNA levels of HSV-1 VP16 were determined by real-time PCR. All experiments were repeated three times. The P-value was calculated by two-way ANOVA followed by Dunnett’s multiple comparisons test. c The cGAS KO, KO(mcGAS), KO(LL/RK-NLS) RAW264.7 macrophages were treated with Dox for 24 h and then infected with 1 MOI of HSV-1 McKrae for indicated times. Cell lysates were collected and blotted as indicated. d The cGAS KO, KO(mcGAS), KO(LL/RK-NLS) RAW264.7 macrophages were treated with Dox for 24 h and then infected with 0.01 MOI of HSV-1 McKrae for the indicated days. HSV-1 titers were determined in Vero cells. Data represent means ± s.d. of three independent experiments. The P-value was calculated by two-way ANOVA followed by Tukey’s multiple comparisons test. e The cGAS KO, KO(mcGAS), KO(LL/RK-NLS) RAW264.7 macrophages were treated with Dox for 24 h and then infected with HSV-1-Luc, VACV-Luc, VSV-Luc, or IAV-Gluc for 16 h. Luciferase activities were measured to determine the relative infection activity. Data represent means ± s.d. of three independent experiments. The P-value was calculated by two-way ANOVA followed by Dunnett’s multiple comparisons test. f Model of nuclear soluble cGAS sensing DNA virus infection.