POLDIP3 is a common substrate of different coronaviruses nsp5.

(A and B) Ectopic expression of POLDIP3 inhibited PEDV and TGEV replication by RT-qPCR. POLDIP3 proteins were overexpressed for 24 h, and then cells were inoculated with PEDV or TGEV. The levels of PEDV (A) and TGEV (B) N mRNA were determined at 24 h post infection by RT-qPCR. (C and D) Overexpression of POLDIP3 decreased PEDV and TGEV infection by TCID50 assay. Cells were subjected to PEDV (C) and TGEV (D) infection for 24 h following POLDIP3 overexpression for 24 h. The virus yield was measured by TCID50. (E and F) PEDV and TGEV N proteins were inhibited by POLDIP3 overexpression by Western blotting analysis. The target cells were treated as previously mentioned in (A), and PEDV (E) and TGEV (F) replications were evaluated by detecting N proteins by Western blotting. (G to I) Nsp5s from PEDV, TGEV and SARS-CoV-2 harbor the conserved function to cleavage porcine and human POLDIP3. The target cells were transfected with specific POLDIP3-HA along with plasmids encoding Flag-tagged nsp5s or their catalytic defective mutants from PEDV (C144A) (G), TGEV (C144A) (H) and SARS-CoV-2 (C145A) (I), respectively. At 24 h post-transfection, cells were lysed and detected by immunoblotting. (J) Nsp5s from PEDV, TGEV and SARS-CoV-2 cleavage POLDIP3 at the same conserved site. The target cells were co-transfected with wild-type POLDIP3-HA or mutant (POLDIP3-HAQ176A) together with nsp5 plasmids from PEDV, TGEV or SARS-CoV-2. After 24 h, cells were lysed and detected by immunoblotting. The results represent three independent experiments (the means ± SD). *, PPPP value was calculated using Student’s t-tests.