Journal: Virologica Sinica

Article Title: OGG1 inhibition suppresses African swine fever virus replication

doi: 10.1016/j.virs.2022.11.006

Figure Lengend Snippet: Cytotoxicity of OGG1 inhibitors in PAMs. A Published chemical structures of TH5487 and SU0268. B CC 50 values of TH5487 and SU0268 were determined by using the CCK8 Kit, and data analysis was performed using GraphPad Prism. Data were represented as the average of three independent experiments with standard deviation.

Article Snippet: TH5487 (6749) was purchased from Tocris Bioscience, UK.

Techniques: Standard Deviation

Journal: Virologica Sinica

Article Title: OGG1 inhibition suppresses African swine fever virus replication

doi: 10.1016/j.virs.2022.11.006

Figure Lengend Snippet: OGG1 inhibition suppressed ASFV proliferation. A PAMs were pretreated with TH5487 (5 ​μmol/L) or SU0268 (10 ​μmol/L) for 6 ​h each. Then, PAMs were infected with ASFV (MOI ​= ​1). Samples were harvested at 24 hpi. qRT-PCR was performed to analyse the mRNA level of the early viral gene CP204L and the late gene B646L . GAPDH was served as a reference gene. B, C PAMs were treated with SU0268 (10 ​μmol/L) or TH5487 (5 ​μmol/L) for 6 ​h prior to ASFV (MOI ​= ​1) infection. The samples were collected at 0.5, 1, 3, 6, 9, 12, and 24 hpi. The relative expression levels of CP204L and B646L were detected by qRT-PCR. GAPDH was used as a reference gene. D OGG1 silencing in PAMs was determined by qRT-PCR, and GAPDH served as a reference gene. E, F PAMs were transfected with si-OGG1 or si-NC (scramble) for 24 ​h, then infected with ASFV (MOI ​= ​1) for 24 ​h. The expression of viral protein was tested by Western blotting and the mRNA level of CP204L and B646L were analyzed by qRT-PCR. The control group was only infected with ASFV (MOI ​= ​1) for 24 ​h. GAPDH was used as a reference gene. G MA104 ​cells were transfected with HA-OGG1 (2 ​μg) expression plasmids for 24 ​h and then infected with ASFV (MOI ​= ​1) for 24 ​h. Western blot was performed to detect the protein level of OGG1, P72 and β-actin. Data were shown as mean with standard deviation. Statistical analysis was performed by Student's t -test. ∗∗∗∗ P ​< ​0.0001; ∗∗∗ P ​< ​0.001; ∗∗ P ​< ​0.01; ∗ P ​< ​0.05.

Article Snippet: TH5487 (6749) was purchased from Tocris Bioscience, UK.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Control, Standard Deviation

Journal: Virologica Sinica

Article Title: OGG1 inhibition suppresses African swine fever virus replication

doi: 10.1016/j.virs.2022.11.006

Figure Lengend Snippet: ASFV yield was decreased by OGG1 inhibitors. A, B Titration of infectious ASFV was quantified by using the HAD assay. PAMs were pretreated with TH5487 and SU0268 for 6 ​h, and ASFV was then added to the cells and titrated in triplicate using 10-fold serial dilutions. HAD was determined on day 7 post-infection, and DMSO treatment was used as a negative control. C The viral titer was measured in PAMs after ASFV infection at MOIs of 0.5, 1 or 5 ​at 7 days post-infection. The viral titer was compared with that in the DMSO-treated group. D, E The mRNA levels of the CP204L and B646L genes were verified by qRT-PCR. PAMs were treated with TH5487 and SU0268 for 6 ​h prior to ASFV (MOI ​= ​1) infection, and the samples were then collected at 24 hpi. GAPDH was served as a reference gene. F PAMs were pretreated with TH5487 and SU0268 at the indicated concentrations for 6 ​h, and cells were harvested after ASFV infection for 24 ​h. The protein level of P72 was determined by Western blot. Data were shown as mean with standard deviation. Statistical analysis was performed by Student's t -test. ∗∗∗∗ P ​< ​0.0001; ∗∗∗ P ​< ​0.001; ∗∗ P ​< ​0.01; ∗ P ​< ​0.05; ns, not significant.

Article Snippet: TH5487 (6749) was purchased from Tocris Bioscience, UK.

Techniques: Titration, Infection, Negative Control, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Virologica Sinica

Article Title: OGG1 inhibition suppresses African swine fever virus replication

doi: 10.1016/j.virs.2022.11.006

Figure Lengend Snippet: The expression of ASFV-BER-associated proteins was inhibited by OGG1 inhibitors. A PAMs were pretreated with TH5487 (5 ​μmol/L) or SU0268 (10 ​μmol/L) for 6 ​h. ASFV-BER-associated protein (pO174L, pNP419L and pE296R) mRNA levels were determined by RT-PCR at different time points after ASFV (MOI ​= ​1) infection. The expressed significance was compared with 0.5 hpi. GAPDH was served as a reference gene. B PAMs were pretreated with TH5487 or SU0268 at assigned concentrations for 6 ​h and then harvested after ASFV (MOI ​= ​1) infection at 24 hpi. The mRNA levels were assessed by qRT-PCR. GAPDH was used as a reference gene. C, D PAMs were pretreated with OGG1 inhibitors at the indicated concentrations for 6 ​h. At 24 hpi, mock- and ASFV-infected (MOI ​= ​1) cells were lysed, and ASFV-BER protein levels were assessed by Western blot analysis using specific antibodies. The relative intensity was calibrated by β-tubulin. Data were shown as mean with standard deviation. Statistical analysis was performed by Student's t -test. ∗∗∗∗ P ​< ​0.0001; ∗∗∗ P ​< ​0.001; ∗∗ P ​< ​0.01; ∗ P ​< ​0.05; ns, not significant.

Article Snippet: TH5487 (6749) was purchased from Tocris Bioscience, UK.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Virologica Sinica

Article Title: OGG1 inhibition suppresses African swine fever virus replication

doi: 10.1016/j.virs.2022.11.006

Figure Lengend Snippet: OGG1 interacted with ASFV MGF360-14L and affected IFN-β transcription. A HA-OGG1 (2 ​μg) and Flag-MGF360-14L (2 ​μg) plasmids were cotransfected into MA104 ​cells for 24 ​h. The interaction was analyzed through Co-IP using anti-Flag and anti-HA antibodies. B MA104 ​cells were transfected with HA-OGG1 and Flag-MGF360–14L expression plasmids for 24 ​h. Double immunofluorescent staining revealed colocalization of HA-OGG1 (green) and Flag-MGF360-14 ​L (red) in cells (scale bar ​= ​25 ​μm). C MA104 ​cells were cotransfected with Flag-MGF360-14L (1 ​μg) and HA-OGG1 (0, 1, 2, 3 ​μg) for 24 ​h. Then cells were lysed and tested by Western blotting. D MA104 ​cells were transfected with HA-OGG1 (0, 1, 2, 3 ​μg) for 12 ​h, then infected with ASFV (MOI ​= ​1) for 24 ​h. The mRNA level of ASFV MGF360-14L was analyzed by qRT-PCR. GAPDH was used as a reference gene. E PAMs were transfected with si-OGG1 or si-NC (scramble) for 24 ​h and then infected with or without ASFV (MOI ​= ​1) for 24 ​h. IFN-β mRNA level was detected by qRT-PCR. GAPDH was served as a reference gene . F PAMs were pretreated with 5 ​μmol/L TH5487, or 10 ​μmol/L SU0268 for 6 ​h and then infected with ASFV (MOI ​= ​1) for 24 ​h. The transcription level of IFN-β was determined by qRT-PCR. GAPDH was used as a reference gene. Data were shown as mean with standard deviation. Statistical analysis was performed by Student's t -test. ∗∗∗ P ​< ​0.001; ∗∗ P ​< ​0.01; ∗ P ​< ​0.05; ns, not significant.

Article Snippet: TH5487 (6749) was purchased from Tocris Bioscience, UK.

Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Staining, Western Blot, Infection, Quantitative RT-PCR, Standard Deviation

Journal: Virologica Sinica

Article Title: OGG1 inhibition suppresses African swine fever virus replication

doi: 10.1016/j.virs.2022.11.006

Figure Lengend Snippet: A mechanism of OGG1 inhibitors suppresses ASFV replication. ASFV infection triggers dynamic changes in ROS and 8-oxoG and consistently increases the expression of DNA repair enzyme OGG1. The small-molecule inhibitors TH5487 and SU0268 block OGG1 binding to 8-oxoG which then upregulated IFN-β transcription and thus to suppress ASFV replication. Additionally, the interaction of OGG1 with viral MGF360-14L protein could disturb IFN-β production to further affect ASFV replication.

Article Snippet: TH5487 (6749) was purchased from Tocris Bioscience, UK.

Techniques: Infection, Expressing, Blocking Assay, Binding Assay