Figure 3

Knockout of pDNAJB6 inhibits the formation of autophagosomes and viral replication in PCV2-infected cells. A Schematic chromatogram representation of sgRNA targeting at the pDNAJB6 genomic region. PAM sequences are underlined and highlighted in green. sgRNA targeting sites are underlined and highlighted in red. Red arrows indicate gRNA targeting sites. B Western blot analysis of the pDNAJB6 expression in PK-15 cells infected with CRISPR/Cas9 lentivirus and then selected by puromycin. The lentiviruses contain gRNA-232 and gRNA-845. C Sequencing of pDNAJB6 locus amplified from the gRNA-232 (pDNAJB6²³²) and gRNA-845 (pDNAJB6⁸⁴⁵) generated cell clone. Red arrows indicate insertions or mutations. Red dashes indicate deleted bases or amino acids. Red characters indicate inserted base or mutated amino acids. D Cell viability of cell lines stably knockout for pDNAJB6. E, F Knockout of pDNAJB6 inhibits PCV2 progeny virion production. Wild-type PK-15, 845PK^pDNAJB6+/+ and 232PK^{pDNAJB6−/−} cells were infected with PCV2 (MOI = 5) for 48 h. PCV2 Cap were detected by Western blot (E), and relative fold-change in PCV2 titers were determined by TCID₅₀ assay (F). G, H Knockout of pDNAJB6 inhibits the accumulation of autophagosomes induced by PCV2 infection or PCV2 Cap protein in PK-15 cells. PK-15, 845PK^pDNAJB6+/+ and 232PK^{pDNAJB6−/−} cells stably expressing EGFP-LC3 were infected with PCV2 (MOI = 1) (G) or transfected with pCI-Cap (H) for 24 h. Scale bar = 10 μm. The right panel graph shows the number of autophagosomes by taking the average number of green puncta in 60 cells. Results are shown as the mean ± SD (n = 3). **p < 0.01 versus wild-type PK-15 cells with the same treatment.