Viral protease binds to nucleosomal DNA and cleaves nuclear cGAS that attenuates type I interferon

Fig 1. SVV infection induces protease 3C translocation into the nucleus and causes nuclear DNA leakage. (A) qPCR analysis of 18S in the cytoplasm of HEK-293T cells infected with SVV at an MOI of 10 for 0, 6, 9, 12, 15, or 18 h. (B) qPCR analysis of nuclear DNA in the cytoplasm of ST cells infected with SVV at an MOI of 10 for 0, 6, 7, 8, 9, and 10 h. (C) qPCR analysis of 18S in the cytoplasm of HEK-293T cells mock transfected or transfected with a plasmid expressing Flag-3C for 15, 18, 21, or 24 h. (D) qPCR analysis of nuclear DNA in the cytoplasm of ST cells (MOI 10). (E) Confocal microscopy analysis of DNA in mock ST cells or ST cells infected with SVV-GFP at an MOI of 10 for 9 h. The graph in the right panel shows the quantification of cytosolic DNA. (F) Localization analysis of 3C or 2C in HeLa cells transfected with a plasmid expressing 3C or 2C for 18 h using confocal microscopy. (G) Confocal microscopy analysis of 3C localization in ST cells infected with SVV-GFP (MOI 10) for 9 h. (H) Western blot analysis of 3C or 2C in HeLa nuclear and cytoplasmic fractions corresponding to (F). The nuclear and cytoplasmic fractions were separated using Nuclear and Cytoplasmic Extraction…

Fig 2. SVV protease 3C specifically cleaves H2A. (A–D) Western blot analysis of the expression levels of different histone proteins in HEK-293T cells transfected with a plasmid encoding 3C, together with a plasmid encoding H2A (A), H2B (B), H3 (C), or H4 (D) for 24 h. (E) Western blot analysis of H2A cleavage by transiently expressing H2A and 3C (wild-type or mutant 3C) in HEK-293T cells. (F–I) SDS-PAGE analysis of in vitro cleavage of H2A (F), H2B (G), H3 (H), and H4 (I) in a 25 µL reaction containing 10 µg of Histone-6×His recombinant protein and varying amounts of purified recombinant 3C protein (1, 5, 10, 15, or 20 µg) incubated for 2 h at 37°C, followed by protein sequence analysis of the cleaved H2A band using mass spectrometry. (J and K) Western blot analysis of endogenous H2A in ST (J) or HEK-293T (K) cells treated with or without SVV (MOI 10) for 6, 9, or 12 h. (L) Western blot analysis of endogenous H2A in HEK-293T cells transfected with a plasmid encoding Flag-3C.

Fig 3. SVV protease 3C binds to the nucleosomal DNA. (A) DNA-binding analysis of recombinant protein 3C in a reaction containing different amounts of purified recombinant 3C protein (10, 15, 20, or 25 µg) with the nucleosomes extracted from ST cells for 10 min on ice followed by electrophoretic mobility shift assay (EMSA). (B) Detection of the efficiency of Enzymatic Shearing Cocktail (ESC) digestion for nucleosomes. Nucleosomes were extracted from mock ST cells or ST cells infected with SVV (MOI 20) for 9 h and then incubated with ESC for 0, 5, 10, or 15 min at 37°C. The digested nucleosomes were purified, and the nucleosomal DNA was detected using agarose gel electrophoresis. The proportion of mononucleosome produced by ESC digestion was quantified with ImageJ. (C) HSV60 and the recombinant protein 3C were mixed at molar ratios of 1:2, 1:4, 1:8, 1:16, and 1:32, followed by incubation with ESC at 37°C for 5 min, after which EDTA was added to stop the reaction. The reaction products were treated with Protease K and then subjected to agarose gel electrophoresis. (D) Diagram illustrating how SVV 3C limits ESC digestion of linker DNA. (E and F) DNA-binding analysis of recombinant 3C…

Fig 4. SVV 3C co-localize with and cleaves nuclear cGAS within its N-terminal domain. (A and B) Confocal microscopy analysis of co-localization between 3C and pcGAS in the nucleus of HeLa cells stably expressing GFP-pcGAS, either transfected with a plasmid encoding Flag-3C for 18 h (A) or infected with SVV (MOI 10) for 12 h (B). Scale bars, 10 µm. (C) Detection of pcGAS cleavage in the nucleus of HEK-293T cells stably expressing Myc-pcGAS-Flag transfected with a plasmid encoding WT or mutant 3C for 24 h by Western blot analysis. (D) Detection of pcGAS cleavage in the nucleus of HEK-293T cells stably expressing Myc-pcGAS-Flag infected with SVV at an MOI of 20 for 0, 6, 9, 12, and 15 h by Western blot analysis. (E) Detection of pcGAS expression in the nucleus of PAMs infected with SVV at an MOI of 20 for 0, 9, and 12 h by Western blot analysis. The asterisk (*) in the figure denotes a non-specific band.

Fig 5. Double-stranded DNA significantly enhances the cleavage activity of SVV 3C. (A–C) SDS-PAGE analysis of the cleavage of SUMO-pcGASFL in reactions containing SUMO-pcGASFL and 3C, together with ISD45 (A), ISD90 (B), or nucleosomal DNA (C) at increasing DNA concentrations incubated at 37°C for 2 h. The graph in the bottom panel shows the content of pcGASFL. (D) SDS-PAGE analysis of H2A cleavage in a reaction containing H2A-6×His and 3C, together with ISD45 incubated at 37°C for 2 h. The graph in the bottom panel shows the content of H2AFL. (E) SDS-PAGE analysis of the cleavage of SUMO-pcGASFL in a reaction containing SUMO-pcGASFL and mutant 3C, together with ISD45 at increasing concentrations incubated at 37°C for 2 h. The graph in the bottom panel shows the content of pcGASFL. (F) Schematic diagram illustrating the DNA-enhanced cleavage of pcGAS by 3C protease.

Fig 6. SVV 3C cleaves the N-terminus of nuclear cGAS to evade immune restriction. (A) The localization of pcGAS in ST cells stably expressing pcGAS-Flag (pcGAS-Flag-ST cells) treated with or without SVV (MOI 10) for 4 h followed by mock transfection or transfection with poly(dA:dT) for 6 h. Scale bars, 10 µm. (B) Relative ratio of pcGAS in the cytoplasm to the nucleus corresponding to (A). (C) The localization of pcGAS C-terminus in ST cells stably expressing pcGASCT(139-495aa)-HA (pcGASCT-HA-ST cells) following mock-transfection or transfection with poly(dA:dT) for 6 h. Scale bars, 10 µm. (D) Relative ratio of pcGAS C-terminus in the nucleus to whole cells corresponding to (C). (E and F) Western blot analysis of pcGAS in the nucleus and cytoplasm of ST cell line stably expressing the full-length (E) or pcGAS C-terminus (139-495 aa) (F). (G) qRT-PCR analysis of the mRNA level of IFNB1 (relative to GAPDH) in pcGAS-Flag-ST or pcGASCT-HA-ST cells following mock transfection or transfection with poly(dA:dT) for 6 h. (H) qRT-PCR analysis of the mRNA level of IFNB1 (relative to GAPDH) in pcGAS-Flag-ST or pcGASCT-HA-ST cells treated with or without SVV (MOI 10) for 9 h. (I) Schematic for the…

Fig 7. Schematic model of SVV protease 3C-mediated cleavage of nuclear pcGAS to attenuate type I interferon. SVV infection results in the translocation of 3C proteins into the nucleus, where they bind to nucleosomal DNA. Concurrently, 3C cleaves nuclear cGAS, impairing its export and inhibiting the immune response.