| Porcine parvovirus?PPV?mainly causes reproductive disorders in sows,which is characterized by abortion,stillbirth,deformity,mummified fetus and weak offspring in the first pregnant sows.Studies have confirmed that porcine placental trophoblast cells?porcine placental trophoblast cells,PTCs?are the main target cells in the pathogenesis of PPV,and the dysfunction and death of PTCs are closely related to reproductive disorders.In recent years,a large number of studies have confirmed that the infection and pathogenesis of a variety of viruses are closely related to host cell autophagy,However,whether PPV induces host cell autophagy,and the role and mechanism of PPV and its non-structural proteins in host cell autophagy have not been reported so far.In this study,we first detected the level and characteristics of autophagy in PPV-infected PTCs,and further examined the signal transduction pathway of PPV-induced autophagy and the effect of autophagy on PPV replication.Finally,we systematically studied the role and mechanism of PPV non-structural proteins NS1 and NS2 and their host interaction proteins in the process of autophagy.The results are as follows:1. The autophagy level of PTCs,was detected at different time points after PPV infection.The results of western blotting showed that the autophagy marker-LC3?began to increase after PPV infection with PTCs 12h,and was significantly higher than that of Mock group at24 h,36 h and 48 h after infection?p<0 05?.The results of fluorescence microscope showed that there were a large number of GFP LC3 puncta representing autophagosomes in PTCs infected with PPV for 24 h.Transmission electron microscopic observation showed that there were 0.2?1.0?m,double-membraned autophagic vesicles accumulated in PPV-infected PTCs at 24 h.p.i..The cells were pretreated with autophagy flux inhibitors?CQ or BAF?,and then infected with PPV.Western blotting showed that the level of LC3 II in PPV infection group was significantly higher than that in Mock group.The characteristics of autophagy were detected within 48 h after PPV infection.Western blotting showed that with the extension of PPV infection time,the accumulation of p62 in cells increased and began to decrease at 48 h.The results of laser confocal detection showed that there were a large number of RFP-GFP-LC3 yellow fluorescent puncta representing autophagosomes in most of PPV-infected cells at24 h.p.i,while large amount of RFP-LC3 whereas large amount of red fluorescent puncta representing autolysosomes were observed in the positive control group?complete autophagy induced by EBSS?.Moreover,a large number of LC3 in PTCs of could not co-locate with LAMP1in PPV-infected PTCs at 24 h.p.i.,while LC3 in EBSS treatment group could co-locate with LAMP1.The results showed that incomplete autophagy was mainly induced by PPV infection within 48 h.Furthermore,laser confocal microscopy was used to detect the formation of autophagosomes and autophagy lysosomes after PPV infection with PTCs 24 h,48 h and72 h.The results showed that there were a large number of RFP-GFP-LC3 yellow fluorescent puncta representing autophagosomes in PPV infected cells at 24 h and 48 h,but with the extension of infection time,there were a large number of RFP-LC3 red fluorescent puncta representing autolysosomes in PPV infected cells at 72 h.The above results showed that PPV infection induced PTCs autophagy,which mainly induced the formation of autophagosomes in the early stage of infection?24 h and 48 h?,incomplete autophagy occurred in cells,and autophagy flux was further induced in the later stage of infection?72 h?,and complete autophagy occurred in cells.2. With western blotting,the signal molecules related to autophagy were detected in PPV infected PTCs.The results showed that the expression level of p-m TOR at 6 h,12 h and 24 h after PPV infection was significantly lower than that in Mock group?p<0.05?.The expression of Beclin1 began to increase after 6 h.p.i and lasted for 24 h.p.i in PPV infected PTC,suggesting that m TOR may be involved in the regulation of PPV-induced autophagy.Further detection showed that m TOR activating molecule Rheb could significantly inhibit the high expression of Beclin1 and LC3?induced by PPV infection.The specific inhibitors of signal pathways upstream of m TOR were used to treat the cells,and the effects of various inhibitors on LC3 II in PPV infected cells were detected by western blotting.The results showed that the inhibitors of p53,PI3K/Akt and MAPK/Erk1/2 signaling pathways had no significant effect?p>0.05?on the level of LC3 II in PPV infected cells,while Compound C,the specific inhibitor of AMPK,significantly decreased the level of LC3 II in PPV infected cells?p<0.01?.The results of further detection showed that PPV infection could increase the level of phosphorylated Raptor in PTCs,and Compound C treatment,could attenuate the increase of Raptor phosphorylation induced by PPV.The wild type PTCs and AMPK knockout(AMPK-/-)PTCs were separately infected with PPV.Western blotting showed that the phosphorylation level of Raptor and expression levels of Beclin 1 and LC3 II in AMPK-/-cells were decreased compared to that in wild type PTCs.The results show that PPV infection can inhibit the activation of m TOR and induce autophagy by activating AMPK/Raptor signal pathway.3. Western blotting and realtime Q-PCR results showed that in PPV infected PTCs,the level of LC3 II,NS1 and PPV DNA copies in rapamycin?RAPA?pretreated group were higher than those in control DMSO group?p<0 01?.Further western blotting and realtime Q-PCR detection showed that in PPV infected PTCs,the level of LC3 II,NS1 and PPV DNA copie in 3-methyladenine?3-MA?pretreatment group were lower than those of the control DMSO group?p<0.05?.Furthemore,cells pretreated with ATG5 specific interference RNA?si ATG5?decressed the level of LC3 II,NS1 and PPV DNA copies in PPV infected PTCs,compared to in negative interference RNA control group?si NC??p<0.05?.The results show that autophagy induced by autophagy inducer can increase the replication level of PPV in cells,and inhibition of autophagy by autophagy inhibitor or down-regulation of ATG5 can reduce the replication level of PPV in cells.4. PTCs were infected UV-inactivated PPV?UVPPV,lost the ability of replication,but contained PPV structural proteins?,western blotting results showed that LC3?increased with the increase of infection dose and infection time,and the level of p62 protein decreased at 24h after infection,and decreased continuously at 36 h and 48 h.The levels of LC3?and p62in UVPPV infected cells treated with autophagy inhibitor CQ and BAF were significantly higher than those in untreated group.The results of confocal laser scanning showed that there were a large number of red fluorescent puncta representing autolysosomes in the cells infected with UVPPV at 24 h.p.i.,indicating that UVPPV can induce the formation of autolysosomes.PTCs was transfected with NS1 or NS2,and then infected with UVPPV,western blotting results showed that overexpression of NS1 could significantly inhibit the degradation of p62in UVPPV infected cells.After NS1 gradient transfection,PTCs were infected with UVPPV.Western blotting and laser confocal detection showed that as the amount of NS1 transfection increased,intracellular p62 gradually increased,and autophagosomes in the cells?RFP+GFP+yellow fluorescent puncta?increased,while autolysosomal formation decreased.The above results indicate that PPV NS1 can inhibit complete autophagy induced by UVPPV,suggesting that PPV NS1 plays a key role in PPV-induced incomplete autophagy of PTCs.Furthermore,PTCs were quantitatively transfected with NS1,gradient transfected NS2,and then infected with UVPPV.Laser confocal detection showed that in NS1 single transfection cells,UVPPV infection mainly induced the formation of autophagosomes?RFP+GFP+yellow fluorescent puncta?,but with the increase of NS2 transfection,the formation of autolysosomes?RFP+GFP-red fluorescent puncta?gradually increased.The above results suggest that UVPPV infection induces complete autophagy in PTCs,NS1 plays an important role in PPV-induced incomplete autophagy,and NS2 plays a key role in inducing the transformation from incomplete autophagy to complete autophagy.5. The host interaction protein of NS1 was screened by IP tandem mass spectrometry.CO-IP and GST-pulldown identified RAB2A as the direct interaction protein of NS1.Further identification of the interaction mode between RAB2A and NS1,CO-IP and laser confocal identification results showed that in the process of PPV infection,NS1 could interact with RAB2A,NS1 and RAB2A were co-located,and the N-terminal aa1-86 of NS1?the common domain of NS1 and NS2?could be co-located with RAB2A.The results of isothermal titration calorimetry showed that the N-terminal 1 aa?86 aa of NS1 could interact with RAB2A directly.The interaction between NS2 and RAB2A was identified,and the results of laser confocal detection showed that NS2 and RAB2A were co-located.The host interaction protein of NS2was screened by IP tandem mass spectrometry,and VAMP7 was identified as the direct interaction protein of NS2 by CO-IP and GST-pulldown.Further identification of the interaction mode between VAMP7 and NS2,CO-IP and laser confocal identification results showed that in the process of PPV infection,NS2 could interact with VAMP7,NS2 and VAMP7 were co-located,NS2 87 aa?161 aa and VAMP7 were co-located,and isothermal titration calorimetry showed that NS2 87 aa?161 aa could interact with VAMP7 directly.6. The interaction between RAB2A and VAMP7 was identified by CO-IP.The results of CO-IP showed that there was an interaction between RAB2A and VAMP7 in UVPPV infected cells,and the transfection of NS1 inhibited the interaction between RAB2A and VAMP7.Laser confocal detection showed that RAB2A and VAMP7 were co-located in UVPPV infected cells,but RAB2A and VAMP7 could not be co-located after NS1 transfection.The interaction between RAB2A and VMP7 during PPV infection was identified.The results of laser confocal detection showed that RAB2A could not be co-located with VAMP7 in the early stage of PPV infection?24 h?,and RAB2A could co-locate with VAMP7 in the later stage of PPV infection?72 h?.Although transfection of NS1 inhibited the co-localization of RAB2A and VAMP7 in UVPPV infected cells,transfection of NS2 enabled RAB2A and VAMP7 to co-locate.NS2can be co-located with RAB2A and VAMP7.Furthermore,the interaction regions of NS1,NS2and RAB2A were screened and identified by CO-IP and laser confocal technique.The results showed that the 1 aa?43 aa of NS1 and NS2 was the interaction region with RAB2A,and the-10 EVLK14-site was necessary for the interaction.The NSPPV,realtime Q-PCR detection results of the mutant strain with deletion of-10EVLK14-site showed that the copy number of72h.p.i.NSPPV in PTCs was lower than that of PPV wild type virus?p<0.05.The results of?;western blotting and laser confocal detection showed that the level of LC3II in cells increased and a large number of autophagy lysosomes were formed in cells induced by NSPPV infection.These results suggest that PPV NS1 directly interacts with the host protein RAB2A through the common domain of 1 aa?43 aa at the N-terminal,which inhibits the fusion of autophagosomes and lysosomes mediated by the interaction between RAB2A and VAMP7,which plays an important role in inducing incomplete autophagy in the early stage of PPV infection.PPVNS2 interacts with both RAB2A and VAMP7 to promote the fusion of autophagosomes and lysosomes mediated by the interaction of RAB2A and VAMP7,thus inducing the transformation of cells from incomplete autophagy to complete autophagy in the later stage of PPV infection?72 h?.In this study,it was found that incomplete autophagy was mainly induced in PTCs in the early stage of PPV infection?24 h and 48 h?,and complete autophagy was mainly induced in the later stage?72 h?.The molecular mechanism of PTCs autophagy induced by PPV by activating AMPK/m TOR signal pathway was clarified.Further studies have found that PPV infection promotes self-replication by inducing autophagy.This study also found that in the early stage of PPV infection,the non-structural protein NS1 interacts with the host protein RAB2A to prevent the fusion of autophagosomes and lysosomes,resulting in incomplete autophagy.In the later stage of infection,the non-structural protein NS2 mediates the fusion of autophagosome and lysosome by interacting with NS1,host protein RAB2A and VAMP7,which promotes complete autophagy.The key site?10EVLK14?of the interaction between PPV nonstructural protein NS and RAB2A was identified,and the mutant?NSPPV?which did not interact with RAB2A and had lower replication and pathogenicity than wild type PPV was obtained.The above results clarify the role and regulatory mechanism of PPV and its non-structural proteins NS1 and NS2 in the process of inducing autophagy in PTCs,and provide a basis for further revealing the pathogenic mechanism of PPV. |
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