Porcine parvovirus triggers autophagy through the AMPK/Raptor/mTOR pathway to promote viral replication in porcine placental trophoblasts.

Autophagy has been demonstrated to play important roles in the infection and pathogenesis of many viruses. We previously found that porcine parvovirus (PPV) infection can induce autophagy in porcine placental trophoblast cells (PTCs), but its underlying mechanism has not yet been fully revealed. In this study, we showed that PPV infection inhibited the activation of mTORC1 and promoted the expression of Beclin 1 and LC3II in PTCs. Treatment with a mTOR activator inhibited the expression of Beclin 1 and LC3II, as well as autophagy formation, and reduced viral replication in PPV-infected PTCs. Furthermore, we found that inhibition of AMPK expression, but not the inhibition of PI3K/Akt, p53, or MAPK/ERK1/2 pathway activation, can significantly increase mTOR phosphorylation in PPV-infected PTCs. Then, we found that the regulation of mTOR phosphorylation by AMPK was mediated by Raptor. AMPK expression knockout inhibited the activation of Raptor, decreased the expression of Beclin 1 and LC3II, suppressed the formation of autophagosomes, and reduced viral replication during PPV infection. Together, our results showed that PPV infection induces autophagy to promote viral replication by inhibiting the activation of mTORC1 through activation of the AMPK/Raptor pathway. These findings provide information to understand the molecular mechanisms of PPV-induced autophagy.

Autophagy Promotes Porcine Parvovirus Replication and Induces Non-Apoptotic Cell Death in Porcine Placental Trophoblasts.

Autophagy plays important roles in the infection and pathogenesis of many viruses, yet the regulatory roles of autophagy in the process of porcine parvovirus (PPV) infection remain unclear. Herein, we show that PPV infection induces autophagy in porcine placental trophoblasts (PTCs). Induction of autophagy by rapamycin (RAPA) inhibited the occurrence of apoptotic cell death, yet promoted viral replication in PPV-infected cells; inhibition of autophagy by 3-MA or ATG5 knockdown increased cellular apoptosis and reduced PPV replication. Interestingly, we found that in the presence of caspase-inhibitor zVAD-fmk, PPV induces non-apoptotic cell death that was characterized by lysosomal damage and associated with autophagy. Induction of complete autophagy flux by RAPA markedly promoted PPV replication compared with incomplete autophagy induced by RAPA plus bafilomycin (RAPA/BAF) in the early phase of PPV infection (24 h.p.i.). Meanwhile, induction of complete autophagy with RAPA increased lysosomal damage and non-apoptotic cell death in the later phase of PPV infection. Therefore, our data suggest that autophagy can enhance PPV replication and promote the occurrence of lysosomal-damage-associated non-apoptotic cell death in PPV-infected porcine placental trophoblasts.

Construction and characterization of the infectious clone of porcine parvovirus carrying genetic marker.

Porcine parvovirus (PPV) is one of the major pathogens that bring about reproductive failure of pregnant sows. However, the study of the pathogenesis mechanism is circumscribed due to the lack of efficient genetic manipulation method. Infectious clone is a powerful tool for further studying the genetic mechanisms of PPV. In the present study, the gene fragment (157-4812) of PPV was amplified by PPV China isolate strain as a template, and PPV DNA fragments (1-182) forming Y-structure within in 5' end and (4788-5074) forming U-structure in 3' end were synthesized. And then, the above three fragments were inserted into plasmid pKQLL to congregate a PPV full-length recombinant plasmid by means of In-Fusion cloning technology. After the successful sequencing identification of the recombinant plasmid, the EcoR I restriction site was brought out as a genetic marker by nonsense mutation (A3058 T) to produce plasmid Y-PPV, which was transfected into PK-15 cells for rescue of virus. The rescued viral particles were observed under transmission electron microscopy, and the sequencing analysis showed that Y-PPV could stably carry the genetic marker. It could be seen that Y-PPV has similar replicate capability and pathogenicity as the wild-type parental PPV strain by cellular and animal experiments. These results confirmed that Y-PPV maintain similar biological characteristics with wild-type parental PPV strain. Infectious clone could be a valuable tool for studying the individual genes of PPV and applications in gene deletion or live vector vaccines.