Figure 5

SADS-CoV N protein interferes with the interaction between TRIM25 and RIG-I. A RIG-I interacts with SADS-CoV N protein. HEK293T cells were transfected with Flag-RIG-I, Myc-SADS-CoV N, or empty vector. At 24 h post-transfection, cell lysates were subjected to immunoprecipitation with anti-Myc or anti-Flag antibodies, and subsequent immunoblotting with anti-Myc or anti-Flag antibodies. B The domain of SADS-CoV responsible for interaction with RIG-I was identified. C Schematics of RIG-I truncations. D The domain of RIG-I responsible for interaction with SADS-CoV N was identified. E RIG-I 2CARDs interacts with SADS-CoV N protein. HEK293T cells were transfected with GST or GST-RIG-I 2CARDs together with Myc-SADS-CoV N. The cell lysates were collected and then immunoprecipitated with anti-GST antibody. The immunoprecipitates were detected by Western blotting with the indicated antibodies. F Co-localization of RIG-I with TRIM25 and SADS-CoV N. HEK293T cells were co-transfected with Flag-RIG-I and HA-TRIM25 or GFP-SADS-CoV N for 24 h. The cells were incubated with mouse anti-Flag antibody and rabbit anti-HA antibody before being stained with DAPI. G SADS-CoV N protein dose-dependently inhibited RIG-I interaction with TRIM25. HEK293T cells were co-transfected with Flag-RIG-I and HA-TRIM25 or Myc-SADS-CoV N for 24 h. The cell lysates were collected and then immunoprecipitated with anti-Flag antibody. The immunoprecipitates were detected by Western blotting with the indicated antibodies.