Porcine circovirus 3 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin

Abstract

Porcine circovirus 3 (PCV3) has been detected in major pig-producing countries around the world since its first report in the US in 2016. Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs. We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid (Cap) protein expression on autophagic response in human embryonic kidney cell line 293T (HEK293T). We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin (mTOR) in HEK293T cells. The ubiquitin-proteasome pathway is also involved in the autophagy process. These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

Keywords: Porcine circovirus 3 (PCV3); Capsid (Cap) protein; Autophagy.

Fig. 1

Formation of autophagosomes induced by PCV3 capsid protein shown as EGFP-LC3B punctae and autophagosome-like vesicles (a) Cells were co-transfected with pEGFP-LC3B and pCMV-capsid (Cap) or the control plasmid pCMV, or transfected only with pEGFP-LC3B (mock), or treated with 2 μmol/L rapamycin (Rapa) after 5 h of transfection with pEGFP-LC3B (positive control). After 36 h of transfection, cells were immuno-stained with the rabbit anti-PCV3 Cap polyclonal antibody and counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for nuclei, and then examined for formation of EGFP-LC3B punctae under the confocal microscope. (b) Cells were transfected with pCMV-Cap for 36 h or mock-treated (negative control) or treated with 2 μmol/L Rapa (positive control). Autophagosome-like vesicles were observed by transmission electron microscope. Black arrows indicate the characteristic structures of autophagosomes. Scale bar=1 μm

Fig. 2

Increased conversion of LC3-I to LC3-II and downregulated ratio of p62 to β-actin caused by PCV3 capsid protein Cells were first transfected with pCMV-capsid (Cap) or the control plasmid pCMV. Rapamycin (Rapa) was used as positive control. After 36 h of transfection, the whole cell lysates were subjected to western blotting for β-actin, LC3, p62, and PCV3 Cap. (a) A representative blot showing the effects of PCV3 Cap on target proteins. (b, c) Ratios of LC3-II to LC3-I and p62 to β-actin normalized to mock transfection set at 1.0. Data were expressed as mean±standard error of the mean (SEM), n=3. ^* P<0.05; ^** P<0.01

Fig. 3

Complete autophagy flux induced by PCV3 capsid protein and involvement of ubiquitin–proteasome pathway in the autophagy process Cells were first transfected with pCMV-capsid (Cap) for 5 h and then treated either with 5 μmol/L chloroquine (CQ) or MG132, or left untreated as control. Rapamycin (Rapa) was used as a positive control. After 36 h of transfection, the whole cell lysates were subjected to western blotting for β-actin, LC3, p62, and PCV3 Cap. (a, d) Representative blots showing the effects of CQ or MG132 on target proteins. (b, e) Ratio of LC3-II to LC3-I normalized to mock transfection set at 1.0. (c, f) Ratio of p62 to β-actin normalized to mock transfection set at 1.0. Data were expressed as mean±standard error of the mean (SEM), n=3. ^* P<0.05; ^** P<0.01

Fig. 4

Decreased phosphorylation of mTOR by PCV3 capsid protein Cells were transfected with pCMV-capsid (Cap) or the control plasmid pCMV. Rapamycin (Rapa) was used as a positive control. After 36 h of transfection, the whole cell lysates were subjected to western blotting for mammalian target of rapamycin (mTOR), phosphorylated-mTOR (p-mTOR) (S2448), β-actin, LC3, p62, and PCV3 Cap. (a) A representative blot showing the effects of PCV3 Cap expression on target proteins. (b) Ratio of p-mTOR (S2448) to mTOR normalized to mock transfection set at 1.0. Data were expressed as mean±standard error of the mean (SEM), n=3. ^** P<0.01