Porcine DNAJB6 promotes PCV2 replication via enhancing the formation of autophagy in host cells

Figures

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  Figure 1. PCV2 infection upregulates pDNAJB6 expression in PK-15 cells. A–C PK-15 cells were infected with 1 MOI of PCV2 or mock infection for indicated time, the mRNA level of pDNAJB6 (A), the protein level of pDNAJB6 (B) and the relative viral titers of different infected time were measured by TCID50 (C). D, E qPCR and Western blot analysis of pDNAJB6 mRNA and protein levels in PK-15 cells infected with different doses of PCV2 (MOI = 0, 0.5, 1, or 5) for 36 h. β-actin served as an internal control. Results are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01 versus mock infected cells at same time points; #p < 0.05, ##p < 0.01 versus cells infected with PCV2 for 24 h.

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  Figure 2. PCV2 Cap interacts with pDNAJB6 and co-localizes in both cytoplasm and nucleus. A, B PCV2 Cap interacts with pDNAJB6 in transfected cells. HEK293T cells were co-transfected with GFP-Cap and Flag-pDNAJB6 expression plasmids or co-transfected with the pEGFP-N1 control vector and the Flag-pDNAJB6 expression plasmid. The cells transfected with pEGFP-N1 control vector, GFP-Cap expression plasmid or Flag-pDNAJB6 expression plasmid alone served as controls. The interaction of GFP-Cap with Flag-pDNAJB6 was identified through immunoprecipitation using anti-Flag antibodies or anti-GFP antibodies. C pDNAJB6 interacts with PCV2 Cap protein. PK-15 cells were infected with PCV2 (MOI = 1) for 36 h, and cell lysates were immunoprecipitated using Cap antibodies followed by immunoblots using pDNAJB6 antibodies. D Endogenous pDNAJB6 interacts with GFP-Cap. PK-15 cells were transfected with GFP-Cap, and the cells were lysed for immunoprecipitation with GFP antibodies; pDNAJB6 antibodies were used for immunoblotting. In these co-IP assay, the corresponding whole cell lysate (Lysate) were detected the targeted protein before immunoprecipitation. E Direct interaction of pDNAJB6 with PCV2 Cap.…

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  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, 845PKpDNAJB6+/+ and 232PKpDNAJB6−/− 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…

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  Figure 4. Overexpression of pDNAJB6 promotes the formation of autophagosomes and progeny virion production in PCV2-infected cells. A, B Western blot analyzes of pDNAJB6 protein levels in PK-15 cells with pDNAJB6 overexpression using anti-Flag antibodies (A) or anti-DNAJB6 antibodies (B). PK-15 are the cells without transfection, PKV the cells transfected with blank vector, PKpDNAJB6 the cells transfected with the vector expressing Flag-pDNAJB6. C Cell viability of cell lines stably overexpressing pDNAJB6. D, E Overexpression of pDNAJB6 promotes PCV2 progeny virion production. Wild-type PK-15, PKV and PKpDNAJB6 cells were infected with PCV2 (MOI = 5) for 48 h. PCV2 Cap were detected by Western blot (D). PCV2 titers were determined by TCID50 assay (E). F, G Overexpression of pDNAJB6 promotes the accumulation of autophagosomes induced by PCV2 Cap protein or PCV2 infection in PK-15 cells. PK-15 and PKpDNAJB6 cells that stably express EGFP-LC3 were infected with PCV2 (F) or transfected with pCI-Cap plasmid (G) 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).…

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  Figure 5. The J domain of pDNAJB6 is essential for its interaction with Cap. A, B Schematic representation of mutants of Cap and pDNAJB6. C, D GST pull-down assay. GST or GST fusion proteins were incubated with the indicated proteins and the bound proteins were analyzed by SDS-PAGE. GFP-tagged proteins and GST-tagged proteins were detected by Western blot. E, F The J domain of pDNAJB6 and the fragment (162-234 aa) of Cap are required for the interaction of pDNAJB6 with Cap. HEK-293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with anti-Flag or anti-GFP antibodies followed by immunoblots using anti-GFP or anti-Flag antibodies.

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  Figure 6. The interaction between pDNAJB6 and Cap promotes the production of autophagosomes induced by PCV2 infection. A Wild-type PK-15 cells stably expressing EGFP-LC3 were transfected with pCI-neo, pCI-Cap or pCI-CapΔ162-234 (CapΔ162-234) plasmid for 24 h, respectively. The average number of green puncta was counted and calculated in 60 cells for each group. Results are shown as the mean ± SD (n = 3). **p < 0.01 versus pCI-Cap vector transfected cells. B, C PK-15 and PKpDNAJB6−/− cells stably expressing EGFP-LC3 were transfected with pCI-pDNAJB6, pCI-pDNAJB6ΔJ or blank vector (control) for 24 h, then cells were infected with PCV2 (B) or transfected with pCI-Cap plasmid (C) for 24 h. The average number of green puncta was counted and calculated in 60 cells in each group. Results are shown as the mean ± SD (n = 3). **p < 0.01 versus wild-type PK-15 cells. ##p < 0.01 versus PKpDNAJB6−/− cells that transfected with blank vector (PKpDNAJB6−/−/control).

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  Figure 7. pDNAJB6 interaction with Cap promotes PCV2 replication through enhancing of autophagy. A Disruption of pDNAJB6/Cap interaction impedes PCV2 replication. PKpDNAJB6−/− cells (transfected pCI-pDNAJB6 or pCI-pDNAJB6ΔJ for 12 h), wild-type PK-15, and PKpDNAJB6−/− cells (transfected with pCI-neo (control)) were infected with PCV2 for 48 h, respectively. The relative production of progeny viruses was measured by TCID50 and calculated. **p < 0.01 versus wild-type PK-15 cells. ##p < 0.05 versus PKpDNAJB6−/− cells transfected with full-length pDNAJB6 expression plasmid. B, C The effects of an autophagy inhibitor on the production of progeny virions. After pretreatment with or without 3-MA (5 mM), cells were respectively transfected with the indicated plasmids, and then the cells were infected with PCV2 for 48 h. Relative viral production was measured and calculated. **p < 0.01 versus DMSO treated cells that transfected with same plasmid; ##p < 0.01 versus PKpDNAJB6−/− cells rescued wild-type pDNAJB6 with same treatment. &p < 0.05 versus wild-type PK-15 cells with same treatment. D, E Effect of atg5 silencing on the number of autophagosomes induced by PCV2 infection and progeny PCV2…