Figure 4

SADS-CoV N protein inhibits CCD-dependent TRIM25 oligomerization. A and C Schematics of SADS-CoV N and the SADS-CoV N2 domain truncations. B and D The domain of SADS-CoV N responsible for interaction with TRIM25 was identified. HA-TRIM25 together with GFP empty vector, GFP-SADS-CoV N, or SADS-CoV N truncations were co-expressed in HEK293T cells for 24 h. Whole cell lysates were collected and subjected to IP with anti-HA antibody, followed by immunoblotting with anti-GFP or anti-HA antibodies. E Schematics of TRIM25 truncations. F The domain of TRIM25 responsible for interaction with SADS-CoV N was identified. Myc-SADS-CoV N, GFP empty vector, or TRIM25 truncations with GFP were co-expressed in HEK293T cells. At 24 h post-transfection, the cell lysates were immunoprecipitated with antibody against GFP. SADS-CoV N and TRIM25 truncations were detected by Western blotting with anti-Myc or anti-GFP antibodies, respectively. G SADS-CoV N did not interact with mutants of TRIM25 lacking the CCD domain. HEK293T cells were co-transfected with Myc-SADS-CoV N together with empty vector, HA-TRIM25, or HA-TRIM25 DelCCD. At 24 h post-transfection, whole cell lysates were collected and used for IP with anti-HA antibody, followed by immunoblotting with anti-Myc or anti-HA antibodies. H SADS-CoV N protein inhibited the interaction of differently labeled TRIM25. HEK293T cells were transfected with Myc-SADS-CoV N together with empty vector, Flag-TRIM25, or HA-TRIM25. At 24 h post-transfection, whole cell lysates were collected and used for IP with anti-Flag antibody, followed by immunoblotting with anti-Myc, anti-HA, and anti-Flag antibodies. I SADS-CoV N protein expression interferes with TRIM25 multimerization. HEK293T cells were transfected with HA-TRIM25 in the presence or in the absence of Myc-SADS-CoV N for 24 h. The cell lysates were separated with Native PAGE followed by Western blotting to detect the expression of individual proteins.