Fig. 4.

Coronavirus nsp1 is a major IFN antagonist. (A) Degradation of CREB-binding protein (CBP) by PEDV nsp1. Cells were seeded on coverslips and transfected with PEDV nsp1 gene. Porcine reproductive and respiratory syndrome virus (PRRSV) nsp1α is known to degrade CBP in the nucleus and thus included as a control. Cells were stained at 24 h post-transfection for indirect immunofluorescence assay (IFA) using rabbit anti-FLAG polyclonal antibody (red) and mouse anti-CBP monoclonal antibody (green). Nuclei were stained with DAPI (blue). White arrows indicate nsp1-expressing cells. Yellow arrows indicate control cells. (B) PEDV nsp1 does not alter the expression of p300. Cells were grown to 90% confluence and HA-tagged p300 gene was co-transfected with PEDV nsp1 gene or empty vector pXJ41. Cells were stained at 24 h post-transfection for detection of nsp1 (red) using rabbit anti-FLAG polyclonal antibody and for p300 (green) using mouse anti-HA monoclonal antibody. Nuclei were stained with DAPI (blue). (C) Suppression of type I IFN induction by nsp1 of bovine coronavirus (BCoV) and transmissible gastroenteritis coronavirus (TGEV). HeLa cells were seeded in 24-well plates and co-transfected with pIFN-β-luc along with individual nsp1 gene and pRL-TK at a ratio of 1:1:0.1. PRRSV nsp1α is a known IFN suppressor and was included as a control. At 24 h post-transfection, cells were stimulated with poly(I:C) (0.5 μg/ml) for 12 h and the luciferase activities were measured. The reporter experiments were repeated three time, each in triplicate. Asterisks indicate the statistical significance. Statistical analysis was performed by Student’s t-test. *, P < 0.05, **, P < 0.01, and ***, P < 0.001.