Figure 6. ORF48 interacts with STING and blocks STING-dependent innate immunity.

(A) Co-immunoprecipitation of HA-STING and STREP-ORF48. HEK293T cells were transfected with HA-STING, an empty backbone, STREP-ORF37, or STREP48 as shown. Forty-eight hours later, cell lysates were immunoprecipitated with STREP antibody and protein A/G beads. HA or STREP antibodies were used for band detection. (B) Co-immunoprecipitation of endogenous STING and STREP-ORF48. HEK293 cells were transfected with an empty backbone or STREP-ORF48 as shown. Forty-eight hours later, cell lysates were immunoprecipitated with STREP antibody and protein A/G beads. STING or STREP antibodies were used for band detection. (C-F) HUVEC-EV or HUVEC-ORF48 stable cells were transfected with ISD or diABZI for 0, 3, and 6 hours. RT-PCR of IFNβ in each group at six hours was performed as shown in (C) ISD or (E) diABZI. Western blot assays of each group at three and six hours were shown in (D) ISD or (F) diABZI. (G) iSLK.219 cells were treated as described in Figure 5A, and IFNβ levels were detected using RT-PCR. (H) Western blot assays evaluating p-TBK1, TBK1, p-IRF3, and IRF3 levels in the above samples. Beta-actin serves as a loading control. (I-J) HUVEC-ORF48 stable cell lines were transfected with two siRNAs targeting ORF48 for forty-eight hours. Samples were then subjected to 4uM of diABZI for 6 hours, and detected with either (I) RT-PCR for IFNβ production or (J) Western blot assays for p-TBK1, TBK1, p-IRF3, IRF3 and ORF48 evaluation. Data are presented as mean ± s.d. from at least three independent experiments. *indicates p<0.05. ** indicates p<0.01 *** indicates p<0.001 **** indicates p<0.0001 by Student’s t-test.