Circovirus Rep evades immune restriction by disrupting cGAS oligomerization and phase separation

Figures

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  Fig 1. PCV2d infection suppresses dsDNA-mediated IFN- β induction. (A, B and C) RT-PCR analysis of Ifnb (A) and downstream Isg54 (B) and Isg56 (C) mRNA expression in iPAMs infected with PCV2d (MOI = 1) for 0, 24, 36. 48 and 72 h. (D, E and F) iPAMs were infected with PCV2d (MOI = 1) for indicated time points and then were transfected with poly (dA:dT) at a concentration of 1 µg/ml. After 12 h, cells were harvested for RNA extraction and RT-PCR analysis of Ifnb (D) and downstream Isg54 (E) and Isg56 (F) mRNA expression. (G, H and I) PAMs were infected with PCV2d (MOI = 1) for indicated time points and then were transfected with poly (dA:dT) (1 µg/ml) for 12 h. Cells were harvested for for RNA extraction and RT-PCR analysis of Ifnb (G), Isg54 (H) and Isg56 (I) mRNA expression. (J and K) ELISA analysis of the expression of 2’3’-cGAMP (J) and IFN-α (K) in PAMs treated as above. Data are represented as means ± SD from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.

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  Fig 2. PCV2d Rep inhibits IFN-I production. (A-C) iPAMs were transfected with increasing doses of PCV2d Rep for 24 h. Cells were stimulated with poly (dA:dT) at a concentration of 1 ug/mL. After another 12 h, cells were harvested for RNA extraction and RT-PCR detection of Ifnb (A), Isg54 (B) and Isg56 (C). (D) Schematic representation of cGAS-STING signaling pathway. (E, F, G, and H) iPAMs were co-transfected pcGAS and pSTING (E), pcGAS (F), pSTING (G) and pTBK1 (H) plasmids and increasing doses of PCV2d Rep plasmid for 24 h. The cells were harvested for RNA extraction and RT-PCR analysis of Ifnb mRNA expression. Data are represented as means ± SD from three biological replicates. ns, no significance, **p < 0.01, ***p < 0.001, Student’s t-test.

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  Fig 3. Localization of Rep during PCV2d infection. (A and B) Immunoblotting analysis of PCV2d Rep protein expression in ST cells (A) and PAMs (B) infected with PCV2d (MOI = 1) for 0, 24, 36. 48, 60 and 72 h. (C) Confocal microscopy analysis of PCV2d Rep localization in ST cells infected with PCV2d (MOI = 1) for indicated time points, followed by labeling with Rep-specific primary antibodies and secondary antibodies (red). Cell nuclei were stained with DAPI (blue). The fluorescent signals were observed with confocal immunofluorescence microscopy. Scale bar, 50 μm.

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  Fig 4. PCV2d Rep disrupts cGAS oligomerization and phase separation. (A) HEK-293T cells were transfected with increasing doses of FLAG- PCV2d Rep for 24 h. The whole cell lysate for streptavidin pull-down was incubated with biotin-HSV60 (40 ug/mL) and pull-down assay of the binding between PCV2d Rep and dsDNA. (B) Pull-down assay for assessment of the interactions between SUMO-PCV2d Rep protein and biotin-HSV60 (40 ug/mL) DNA. After mixing the purified recombinant proteins in NP-40 buffer at 4°C for 6 h, the mixed buffer was immunoprecipitated with Streptavidin mAb. The immunoprecipitated complex was analyzed by immunoblotting with the indicated antibodies. (C) Pull-down assay for assessment of the interactions between the whole cell lysate in HEK-293T cells transfected with increasing doses of FLAG-tagged PCV2d Rep or pcGAS full length protein and biotin-HSV60 (40 ug/mL) DNA. (D) Pull-down assay for assessment of the interactions between SUMO-PCV2d Rep or pcGAS full length protein and biotin-HSV60 (40 ug/mL) DNA. (E) Immunoblotting assay for cGAS complexes incubated with cross-linker between recombinant pcGAS full length and increasing doses of PCV2d Rep proteins. (F) The confocal…

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  Fig 5. Rep proteins from PCV2a and PCV4 disrupt cGAS oligomerization and phase separation. (A) iPAMs were transfected with Rep of PCV1, PCV2a, PCV2d, PCV3 and PCV4 for 24 h. Cells were stimulated with poly (dA:dT) at a concentration of 1 ug/mL. After another 12 h, cells were harvested for RNA extraction and RT-PCR detection of Ifnb. (B) HEK-293T cells were transfected with increasing doses of FLAG-tagged Rep of PCV2a (left) and PCV4 (right) for 24 h. The whole cell lysate for streptavidin pull-down was incubated with biotin-HSV60 (40 ug/mL) and pull-down assay of the binding between Rep and dsDNA. (C and D) Pull-down assay for assessment of the interactions between the whole cell lysate in HEK-293T cells transfected with increasing doses of FLAG-tagged PCV2a (C) and PCV4 (D) Rep or pcGAS full length protein and biotin-HSV60 (40 ug/mL) DNA. (E and F) Immunoblotting assay for pcGAS complexes incubated with cross-linker between recombinant full-length pcGAS and increasing doses of Rep proteins of PCV2a (E) or PCV4 (F). (G) Confocal microscopic analysis of GFP-pcGAS phase separation, where recombinant full-length EGFP-pcGAS (10 μM) was incubated with PCV2a Rep (10 μM) or PCV4 Rep (10 μM) or…

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  Fig 6. PCV Rep suppresses cGAS activation by self-DNA. (A, B and C) iPAMs were transfected with increasing doses of PCV2d Rep for 24 h. Cells were stimulated with BPTES at a concentration of 10 uM. After another 6 h, cells were harvested for for RNA extraction and RT-PCR detection of Ifnb (A), Isg54 (B) and Isg56 (C). (D, E and F) iPAMs were transfected with Rep proteins of PCV1, PCV2d, PCV2a and PCV4 for 24 h. Cells were stimulated with BPTES (10 uM) for 6h. Cells were harvested for RNA extraction and RT-PCR detection of Ifnb (D), Isg54 (E) and Isg56 (F). (G) The confocal microscopy analysis of cGAS foci in HeLa cells co-transfected with encoding pcGAS-EGFP and FLAG-PCV1, PCV2d, PCV2a or PCV4 Rep plasmids for 24 h, were stimulated with BPTES (10 uM). After another 6 h, cells were labelled with FLAG specific primary antibodies and secondary antibodies (red). Cell nuclei were stained with DAPI (blue). The fluorescent signals were observed with confocal immunofluorescence microscopy. Scale bar, 10 μm. Data are represented as means ± SD from three biological replicates. ns, no significance, **p < 0.01, ***p < 0.001, Student’s t-test.

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  Fig 7. Identification of the key DNA-binding region in PCV2d Rep. (A) Computationally derived binding model of PCV2d Rep with HSV45 dsDNA. (B) Pull-down assay for assessment of the interactions between the whole cell lysate in HEK-293T cells transfected with a plasmid encoding different mutations (wild-type, Q12A, R199-W202A, R199A or Q12A/ R199-W202A) of PCV2d Rep or full-length pcGAS protein and biotin-HSV60 (40 ug/mL) DNA. (C) iPAMs were transfected with a plasmid encoding different mutations (wild-type, Q12A, R199-W202A, R199A or Q12A/ R199-W202A) of PCV2d Rep for 24 h. Cells were stimulated with poly (dA:dT) at a concentration of 1 ug/mL for 12 h. Cells were harvested for RNA extraction and RT-PCR detection of Ifnb. (D) Immunoblotting assay for cGAS complexes incubated with cross-linker between recombinant full-length pcGAS and wild-type, Q12A or R199-W202A of PCV2d Rep mutant proteins. (E) Confocal microscopic analysis of EGFP-pcGAS phase separation, where recombinant full-length EGFP-pcGAS (10 μM) was incubated with wild-type, Q12A or R199-W202A of PCV2d Rep mutant proteins or SUMO (10 μM) proteins, along with Cy3-45 bp DNA (5 μM). The fluorescent signals were observed with…

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  Fig 8. Working model of circovirus Rep-mediated immunosuppression via dsDNA binding and pcGAS inhibition. During pathogenic PCV infection, cGAS binds dsDNA from PCV or released mtDNA, triggering liquid-liquid phase separation (LLPS) condensate formation and subsequent IFN-I production. To establish an immunosuppressive environment, circovirus Rep protein is expressed in the cytoplasm, where it binds dsDNA, blocking recognition and activation, thereby suppressing the antiviral immune responses mediated by IFN-I and ISGs.