Figure 3

TRIM25 interacts with SADS-CoV N protein. A Co-IP analysis of the interaction between SADS-CoV N protein and TRIM25 in HEK293T cells. HEK293T cells were transfected with HA-TRIM25, Myc-SADS-CoV N, or empty vector. At 24 h post-transfection, cell lysates were subjected to immunoprecipitation with anti-Myc or anti-HA antibodies, and subsequent immunoblotting with anti-Myc or anti-HA antibodies. B The interaction of endogenous TRIM25 and SADS-CoV N protein was detected with Co-IP assays. IPI-2I cells were infected with SADS-CoV at an MOI of 0.1 for 24 h. Cell lysates were collected and then incubated with anti-mouse IgG or anti-SADS-CoV N mAb (3E9), and detected with the indicated antibodies by Western blotting. C SADS-CoV N protein binds TRIM25 in a manner independent of RNA. HA-TRIM25, Myc-SADS-CoV N, or empty vector were co-expressed in HEK293T cells. At 24 h post-transfection, the cell lysates were either immunoprecipitated with antibody against the HA-tag or treated with 100 mg/mL RNase A on ice for 2 h before IP. SADS-CoV N and TRIM25 were detected by Western blotting with anti-Myc or anti-HA antibodies, respectively. D and E Co-localization of TRIM25 and SADS-CoV N. D HEK293T cells were co-transfected with HA-TRIM25, GFP-SADS-CoV N, or GFP empty vector plasmids. The GFP and GFP-SADS-CoV N were in green, and the TRIM25 fusion protein was in red. Merged images were also presented, and the positions of the nuclei were indicated by DAPI (blue) staining in the merged images. E Vero E6 cells were mock infected or infected with SADS-CoV at an MOI of 0.1 for 24 h. Cells were fixed and co-stained with antibodies directed against SADS-CoV N (green) and TRIM25 (red). The cells were then counterstained with DAPI (blue) and observed with a confocal laser scanning microscope.