PDCoV nsp5 cleaves POLDIP3 at residue glutamine 176 (Q176).

(A) Logo analysis of the cleavage site predicted by WebLogo (http://weblogo.threeplusone.com/), based on the polyprotein and other substrates cleavage of PDCoV nsp5. (B) Schematic representation of POLDIP3 and its mutant sites. (C) Identification of the recognition site in PDCoV nsp5-mediated cleavage of POLDIP3. IPEC-J2 cells were transfected with nsp5-Flag and POLDIP3-HA or POLDIP3 mutants, including POLDIP3Q150A, POLDIP3Q153A, POLDIP3Q154A, POLDIP3Q176A, POLDIP3Q179A and POLDIP3Q197A. At 30 h post-transfection, cell lysates were prepared and analyzed by Western blotting. (D) Q176A is required for POLDIP3 cleavage in the context of PDCoV infection. Wild-type POLDIP3-HA or POLDIP3-HAQ176A mutant was transfected into IPEC-J2 cells for 24 h, followed by PDCoV infection for another 24 h to analyze the POLDIP3 cleavage by Western blotting. (E and F) POLDIP3 cleavage abolishes its antiviral activity. IPEC-J2 cells were transfected with plasmids encoding wild-type POLDIP3, POLDIP3Q176A, POLDIP31–176, POLDIP3177–420 or empty vector for 24 h and then infected with PDCoV for another 24 h. The PDCoV N mRNA and viral titer were determined by RT-qPCR (E) or TCID50 assay (F). The results represent three independent experiments (the means ± SD). *, PPPP value was calculated using Student’s t-tests.