FIG 8.

Transient Nsp15 expression did not affect cellular gene expression. (A) IBV Nsp6 and Nsp15 reduced luciferase reporter gene expression. HEK-293T cells were cotransfected with plasmids expressing the IBV Nsp2-16 protein in vector pCMV-Myc and pRL-TK plasmids expressing TK promoter-driven Rluc. SARS-CoV Nsp1 was used as a positive control. At 24 hpt, cells were used for luciferase activity assays. (B) The inhibitory effect of Nsp15 was dose dependent. Different doses of Nsp15 protein (0.5–2.0 μg) and pRL-TK plasmids were cotransfected into 293T cells, and luciferase activity was detected. (C) Endoribonuclease activity affected inhibition of reporter genes. Nsp15 and EndoU-mutants (Nsp15-H223A, Nsp15-H238A, Nsp15-K278A, and Nsp15-Y334A) were transfected with pRL-TK plasmids. (D) Cells were transfected with IBV Nsp6 and Nsp15 plasmid for 24 h. The cells were pulsed with 3 μm puromycin for 1 h at 37°C and then subjected to Western blot analysis (left). The grayscale values of the protein bands were analyzed using ImageJ (right). ***, P < 0.001.