PDCoV nsp5 cleaves POLDIP3 through its protease activity.

(A) PDCoV nsp5 cleaves POLDIP3. IPEC-J2 cells were co-transfected with plasmids of POLDIP3-HA and PDCoV nsp5-Flag or empty vector, and then cell samples were collected at 30 h post-transfection for Western blotting analysis. (B) POLDIP3 cleavage in a PDCoV nsp5 dose-dependent manner. IPEC-J2 cells were co-transfected with POLDIP3-HA and increased quantities of PDCoV nsp5-Flag or empty vector. Cell lysates were prepared and analyzed by Western blotting at 30 h post-transfection. (C) PDCoV nsp5 cleaves POLDIP3 depending on its protease activity. IPEC-J2 cells were transfected with POLDIP3-HA along with the wild-type PDCoV nsp5-Flag (nsp5) or its protease-defective mutant (nsp5 H41A or nsp5 C144A). After 30 h post-transfection, cells were collected and lysed for Western blotting. (D) PDCoV nsp5-mediated cleavage of POLDIP3 independent of proteasome degradation and autophagy. IPEC-J2 cells transfected with POLDIP3-HA and PDCoV nsp5-Flag were treated with MG132 (10 μM) or 3-MA (5 mM), followed by detection of cleavage by Western blotting. (E) PDCoV infection induces POLDIP3 cleavage. IPEC-J2 cells were infected with PDCoV and harvested to detect POLDIP3 expression and cleavage at 24 h post-infection.