Enterovirus A71 3AB protein facilitates immune evasion by blocking cGAS recognition of mtDNA

Figure 1. EV-A71 infection disrupts mitochondria and results in mtDNA release. (A) Mitochondrial morphology analyzed in RD cells infected with EV-A71. After 12 h of EV-A71 infection (MOI = 1) in RD cells, mitochondrial morphology was examined by transmission electron microscopy. Scale bars, 1 μm. (B) After infecting HeLa or RD cells with EV-A71 (MOI = 1) for 24 or 18 h, respectively, the mitochondrial membrane potential (ΔΨm) was assessed with JC-10, with CCCP serving as a positive control. In healthy mitochondria, JC-10 accumulates in the mitochondrial matrix and forms aggregates that emit red fluorescence (Ex/Em = 540/590 nm). In contrast, in depolarized or damaged mitochondria, JC-10 remains in its monomeric form in the cytoplasm, emitting green fluorescence (Ex/Em = 490/525 nm). The ratio of red to green fluorescence serves as an indicator of mitochondrial membrane potential. Images were examined by Nikon C2 confocal microscopy (left panel). Scale bars, 20 μm. The ratio of red to green fluorescence for JC-10 was analyzed with a multifunctional microplate reader (right panel). Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical…

Figure EV1. EV-A71 2B protein induces mitochondrial damage. (A–D) HeLa, U251, RD, and THP-1 cells were infected with EV-A71 (MOI = 1) for 12, 12, 6, and 24 h, respectively. Cytosolic fractions were isolated, and whole-cell lysates (WCL) or cytosolic fractions (Cyt) were analyzed by Western blotting. (E) HeLa cells were infected with EVA-71-UV for 12 h. Cytosolic fractions were isolated, and qPCR was conducted to measure the cytosolic mtDNA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (F) Mitochondrial morphology analyzed in RD cells transfected with EV-A71 2B plasmid. After 18 h of EV-A71 2B transfection in RD cells, mitochondrial morphology was examined by transmission electron microscopy. Scale bars, 1 μm. (G) After 18 h of EV-A71 2B plasmid transfection in HeLa or RD cells, the mitochondrial membrane potential was assessed with JC-10, with CCCP serving as a positive control. Images were examined with a Leica STELLARIS 5 confocal microscope (left panel). Scale bars, 10 μm. The ratio of red to green fluorescence for JC-10 was analyzed with a multifunctional microplate reader (right panel). Data were presented as mean ±…

Figure 2. EV-A71 infection modulates the mtDNA‒cGAS‒STING pathway. (A–C) HeLa, U251, and THP-1 cells were infected with EV-A71 at an MOI of 1 for 12 or 24 h. The mRNA expression of IFN-β was assessed by qRT‒PCR. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (D, E) HeLa (D) and U251 (E) cells were transfected with 100 ng/mL poly(dA:dT) for 6 h, followed by EV-A71infection (MOI = 1) for 12 h. qRT‒PCR was then performed to measure the expression of IFN-β mRNA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (F–I) EV-A71 infection (MOI = 1) impaired the activation of the IFN-I pathway in TFAM-knockout cells. (F) sgRNA-mediated knockout of TFAM was assessed by Western blotting with an anti-TFAM antibody. (G) Cytosolic mtDNA levels were quantified by qPCR in TFAM knockout and mock-treated cells. (H, I) TFAM knockout and mock-treated cells were infected with EV-A71 for 12 h. IFN-β mRNA expression was measured by qRT‒PCR, and protein levels of cGAS, STING, as well as total and phosphorylated TBK1 and IRF3 were analyzed by western blotting. Data were presented as…

Figure EV2. TFAM knockout enhances IFN-I expression and suppresses EV-A71 infection. (A, B) HeLa and THP-1 cells were infected with PRV for 12 h, followed by EV-A71 infection (MOI = 1) for 12 h and 24 h, respectively. qRT‒PCR was then performed to measure the expression of IFN-β mRNA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (C) IFN-β mRNA expression was detected by qRT‒PCR in TFAM knockout cells. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (D) Intracellular viral load was assessed at 12 h post-infection using qRT‒PCR in TFAM knockout cells. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (E, F) TFAM knockout cells were treated with RU.521 (10 μM) or H151 (1 μM) for 4 h, after which IFN-β mRNA expression was measured by qRT‒PCR (E), and protein levels of total and phosphorylated TBK1 and STING were analyzed by western blotting (F). Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. The experiments were repeated at least…

Figure 3. EV-A71 3AB modulates the cGAS‒STING pathway. (A) After 12 and 24 h of EV-A71 infection (MOI = 1), Western blotting analysis was performed to assess total cGAS, STING, TBK1, and IRF3 levels in HeLa cells. (B) HeLa cells were transfected with 100 ng/mL poly(dA:dT) for 6 h, followed by EV-A71 or EV-A71-UV (MOI = 1) infection for 12 h. Intracellular cGAMP levels were measured by ELISA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (C) HeLa cells were transfected with 100 ng/mL poly(dA:dT) for 6 h, followed by EV-A71 or EV-A71-UV (MOI = 1) infection for 12 h. qRT‒PCR was then performed to measure the expression of IFN-β mRNA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (D) cGAS, STING, poly(dA:dT), and IFN-β luciferase reporter plasmids were transfected into 293 T cells, which were then infected with EV-A71 or EV-A71-UV (MOI = 1) for 12 h. The IFN-β luciferase activity was measured with a dual-luciferase reporter assay. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (E) cGAS,…

Figure EV3. EV-A71 3AB inhibits DNA-mediated IFN-I responses. (A) cGAS, STING, poly(dA:dT), and IFN-β luciferase reporter plasmids were transfected into 293 T cells, which were then infected with EV-A71 or UV-inactivated EV-A71 for 12 h. The overexpression of cGAS and STING was assessed by Western blotting. (B) cGAS, STING, poly(dA:dT), and IFN-β luciferase reporter plasmids were transfected into 293T cells, which were then transfected with EV-A71 nonstructural plasmids for 24 h. The overexpression of cGAS and STING was assessed by Western blotting. (C) HeLa cells were transfected with 100 ng/mL poly(dA:dT) for 6 h, followed by transfection with EV-A71 3AB plasmid for 24 h. qRT‒PCR was then performed to measure the expression of IFN-β mRNA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (D) MG63 cells were transfected with 100 ng/mL poly(dA:dT) for 6 h, followed by transfection with EV-A71 3AB plasmid for 18 h. CXCL10 protein expression in supernatant was determined by ELISA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (E, F) HeLa cells were transfected…

Figure 4. EV-A71 3AB sequesters mtDNA from the cGAS-bound state. (A) HeLa cells were transfected with 100 ng/mL poly(dA:dT) for 6 h, followed by transfection with EV-A71 3AB plasmid for 24 h. Intracellular cGAMP levels were measured by ELISA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (B, C) Investigation of the interaction between 3AB and cGAS by co-IP. 3AB and cGAS plasmids were transfected into 293 T cells, and cell lysates were immunoprecipitated with either anti-HA or anti-Myc antibodies, followed by detection with the respective other antibodies. (D, E) 3AB impaired the binding between cGAS and mtDNA. After 24 h of co-transfection with 3AB, cGAS plasmids and mtDNA, the cytosol was isolated, followed by immunoprecipitation with anti-Myc (D). mtDNA was assessed by dot blotting (D) or qPCR (E). Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (F, G) HeLa cells were co-transfected with cGAS-GFP, mCherry-3AB plasmids and mtDNA for 24 h. (F) Representative images of cGAS-GFP and mCherry-3AB were examined with confocal microscope. Scale bars, 5 μm. (G)…

Figure EV4. EV-A71 3AB does not inhibit STING agonist–induced innate immune responses. (A) After 24 h of 3AB overexpression, Western blotting analysis was performed to assess total cGAS, STING levels in HeLa cells. (B) The EV-A71 3AB plasmid was transfected into HeLa cells for 24 h, followed by treatment with diABZI (5 μM) for 4 h. Protein levels of IRF3, as well as total and phosphorylated TBK1 and STING, were analyzed by Western blotting. (C) HeLa cells were transfected with EV-A71 3AB plasmid for 24 h, followed by transfection with 2'3'-cGAMP (5 μg/mL) for 6 h. Protein levels of IRF3, as well as total and phosphorylated TBK1 and STING were analyzed by Western blotting. (D) The EV-A71 3AB plasmid was transfected into MG63 cells for 18 h, followed by treatment with diABZI (5 μM) for 4 h. Protein levels of IRF3, as well as total and phosphorylated TBK1 and STING, were analyzed by western blotting. (E, F) MG63 cells were transfected with the EV-A71 3AB plasmid for 18 h, followed by treatment with diABZI (5 μM) for 4 h. The protein levels of IFN-β (D) and CXCL10 (E) in supernatant were determined by ELISA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used…

Figure 5. EV-A71 3AB protein binds mtDNA. (A) After 24 h of co-transfection with 3 AB or 3A plasmid and mtDNA, the cytosol was isolated, followed by immunoprecipitation with anti-HA. qPCR was performed to measure the cytosolic mtDNA levels. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (B) After 24 h of co-transfection with 3AB plasmid and mtDNA, the cytosol was isolated, followed by immunoprecipitation with anti-HA. Dot blotting analysis was then used to detect the pulled-down BrdU-mtDNA. (C) Colocalization of mtDNA and 3AB in HeLa cells. Cells were co-transfected with mCherry-3AB plasmid and EdU-mtDNA for 24 h. The nuclei were stained with DAPI. Images were examined with a Nikon C2 confocal microscope. Scale bars, 10 μm. (D) The 3 AB or 3A plasmid was transfected into TFAM knockout and mock-treated cells for 24 h, the cytosol was isolated, followed by immunoprecipitation with anti-HA. qPCR was performed to measure the cytosolic mtDNA levels. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (E, F) After 12 h of EV-A71 infection (MOI = 1), the cytosol was…

Figure 6. Mapping the functional domains of EV-A71 3AB protein responsible for mtDNA binding. (A, B) Schematic representation of 3AB mutants. Three 3AB mutants plasmids were constructed and expressed in HeLa cells. (C) After 24 h of co-transfection with 3AB mutants plasmids and mtDNA, the cytosol was isolated, followed by immunoprecipitation with anti-HA. Dot blotting analysis was then used to detect the pulled-down BrdU-mtDNA. (D) The 3AB mutants plasmids were transfected into TFAM knockout and mock-treated cells for 24 h, the cytosol was isolated, followed by immunoprecipitation with anti-HA. qPCR was performed to measure the cytosolic mtDNA levels. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (E) HeLa cells were transfected with 100 ng/mL poly(dA:dT) for 6 h, followed by transfection with 3AB mutants plasmids for 24 h. qRT‒PCR was performed to measure IFN-β mRNA expression. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (F) Purification of the recombinant GST, GST-3B, and GST-3AB proteins produced in the Escherichia coli BL21 DE3 strain. The proteins…

Figure EV5. EV-A71 3AB protein directly interacts with cytosolic mtDNA. (A) After 24 h of co-transfection with 3AB mutants plasmids and mtDNA, the cytosol was isolated, followed by immunoprecipitation with anti-HA. qPCR was performed to measure the cytosolic mtDNA levels. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (B) cGAS, STING, poly(dA:dT), and IFN-β luciferase reporter plasmids were transfected into 293 T cells, which were then transfected with EV-A71 mutants plasmids for 24 h. The overexpression of cGAS and STING was assessed by Western blotting, and IFN-β luciferase activity was measured with a dual-luciferase reporter assay. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (C) Purified GST-3AB protein was coated onto a 96-well ELISA plate and incubated overnight at 4 °C. BrdU-labeled mtDNA was then added and incubated for 2 h at room temperature. After washing, the bound mtDNA was detected using anti-BrdU antibodies. Data were presented as mean ± SEM, n = 4 biological replicates. Unpaired t test was used for statistical analysis. (D) Purified…

Figure 7. CVA9 and CVA16 3AB inhibit IFN-I signaling by targeting cytosolic mtDNA. (A) HeLa cells were infected with CVA9 (MOI = 1) for 24 h. Cytosolic fractions were isolated, and qPCR was conducted to measure the cytosolic mtDNA. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (B, C) After 24 h of co-transfection with CVA9 or CVA16 3AB plasmid and mtDNA, the cytosol was isolated, followed by immunoprecipitation with anti-HA. qPCR was performed to measure the cytosolic mtDNA levels. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (D) The CVA9 3AB or CVA16 3AB plasmid was transfected into TFAM knockout and mock-treated cells for 24 h, the cytosol was isolated, followed by immunoprecipitation with anti-HA. qPCR was performed to measure the cytosolic mtDNA levels. Data were presented as mean ± SEM, n = 3 biological replicates. Unpaired t test was used for statistical analysis. (E) The CVA9 3AB or CVA16 3AB plasmid was transfected into TFAM knockout and mock-treated cells for 24 h, after which IFN-β mRNA expression was measured by qRT‒PCR. Data were presented…