FIG 8.

Recombinant PDCoV with mutation at K46 and/or K58 of nsp8 displays resistance to the antiviral activity of HDAC6. (A and B) IFA of LLC-PK1 cells infected with rPDCoV-WT, rPDCoV-nsp8-K37R, rPDCoV-nsp8-K46R, rPDCoV-nsp8-K58R, and rPDCoV-nsp8-K46/58R at 6 hpi (A) or 12 hpi (B) using an anti-PDCoV N monoclonal antibody. Bar, 100 μm. (C) Multiple-step growth curves of rPDCoVs on LLC-PK cells. Cells were infected with rPDCoVs (MOI = 0.5) and then collected at the different time points postinfection (6, 12, 18, 24 hpi) and subjected to a TCID₅₀ assay. (D to H) LLC-PK1 cells were transfected with pCAGGS-Flag-HDAC6 or empty vector for 12 h and then infected with rPDCoVs (MOI = 0.5) for 12 h. Cell samples were harvested and subjected to RT-qPCR for the detection of viral genomic mRNA (D) and N subgenomic mRNA (E), Western blotting (F), including density analysis of panel F by ImageJ software (G), and a TCID₅₀ assay (H). The presented results represent the means and standard deviations of data from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant difference.