Functional And Mechanism Of Mitochondrial Fission And Mitophagy Proteions In Anti-PRRSV Infection

Porcine Reproductive and Respiration Syndrome Virus(PRRSV),the etiology of Porcine Reproductive and Respiration Syndrome(PRRS),is a single positive strand RNA virus,which causes reproduction failure of breeding sow and respiration disease of piglets.It causes severe economic losses to worldwide pig industry because of its highly pathogenic,morbidity and mortality.Because of host immune suppression and subclinical syndrome and accompanied infection of PRRSV infection,it is difficult to prevent and cure the disease.Vaccine and medicine cannot cure the disease because of the highly mutation and antibody dependent of PRRS virus.Besides,large number of dead pig,drug residue causes environmental damage and food safety problem.Taken together,clarify the interaction between PRRSV and host will give us advantage to find new medicine target and breeding a new anti-virus pig strain.Mitochondria are not only essencial energy organelle,but also play important role in innate and adaptive immunity.In order to maintain healthy mitochondria network,Mitochondria undergo constantly fusion,fission and degraded by mitophagy.Recent researches have revealed that mitochondrial fussion and fission play important role in regulate innate immune response.Mitochondrial fusion enhances innate immune response,while mitochondrial fission inhibits it.According to this many viruses have developed strategies to target mitochondria to escape host innate immune responses.However,whether there has interaction between mitochondrial dynamic,mitophagy and PRRSV infection are not clarify,which is the subjects of our research.Mainly results of our research are as follows:1 Drp1 was phosphorylated at Ser616 and mitochondrial translocation induced by PRRSV infection.In this research we utilized Marc145 cells to study the host cell response induced by PRRSV infection.We analyzed mitochondria morphology after PRRSV infection by using transmission electron microscope and confocal microscope.We found that PRRSV infection induced mitochondrial fragment and mitochondrial cristae swell.These results indicated that PRRSV infection induced the change of mitochondrial morphology which became fragmented.In order to clarify the molecular mechanism of mitochondrial morphology changes induced by PRRSV infection,we analyzed the expression of mitochondrial fission factors.By using Western Blotting,confocal microscope and subcellular isolation,we found that PRRSV infection induced the phosphorylation of mitochondrial fission factor Drp1 at Ser616 site which resulted in mitochondrial fission.At the same time,Phosphorylated Drp1 translocated to the surface of mitochondria which directly regulates mitochondrial fission.Taken together,these results indicated that PRRSV infection induces Drp1 dependent mitochondrial fission.2 Inhibiting Drp1 blocks PRRSV infection by enhancing apoptosisMitochondria are close related with apoptosis,innate and adapted immune response and virus infection.In our research,we found that virus-induced mitochondrial fission enhances virus proliferation,which through negative regulate apoptosis.In order to interference Drp1-dependent mitochondrial fission,we used si RNA to inhibit Drp1 expression.We found that virus proliferation was reduced under the condition of interfering Drp1 expression.Moreover,cleaved-Caspase3 and cleaved-PARP were upregulated in Drp1 deficient cells,compared with control cells.Overall,We can concluded that Drp1-dependent mitochondrial fission induced by PRRS infection enhances PRRSV proliferation by inhibiting apoptosis.3 Mitochondrial mass was reduced by PRRSV infection which rescued by Parkin inhibition.Mitochondrial fission is closely related with mitochondrial mass.PRRSV infection changes mitochondrial dynamics,whether it can influences mitochondrial mass and mitochondrial DNA copy number? We analyzed mitochondrial DNA copy number and mitochondrial mass by utilizing q PCR and Western Blotting.We found that PRRSV infection clearly reduced mitochondrial DNA copy number in a time dependent manners.Besides,mitochondria marker protein,Tom20,was also reduced after PRRSV infection.PRRSV induced mt DNA depletion was rescued under the condition of Parkin dependent mitophagy was inhibited,indicating that PRRSV induced the reduction of mt DNA copy number was because of PRRSV induced mitophagy4 Parkin translocated to mitochondrial and mediated mitophagy after PRRSV infection.Mitophagy are utilized by cell to destroy cellular dysfunctional and redundant mitochondria.Recently many researches include our group have clarified that PRRSV infection induces autophagy.Our result from transmission electron microscope has revealed that mitochondrial are swell and crista disappear after PRRSV infection.Whether these abnormal mitochondria are eliminated by mitophagy is not known.So we detected mitophagy proteins expression and cellular location by using different methods.In our results,we found that PINK1 and Parkin are upregulated by PRRSV infection.Besides,we isolated mitochondrial fragment and analyzed Parkin expression,we found Parkin was enrich at mitochondrial fragment.While LC3 II was reduced at mitochondrial fragment under the condition of Parkin restricted.Taken together,we concluded that PRRSV infection induces PINK1/Parkin dependent mitophagy.Whether PRRSV infection can induces completely mitophagy? In order to clarify this question,we constructed a dual fluorescence plasmid which can locate at mitochondrial surface.We found that there are more red fluorescence in PRRSV infected cells,which is clearly different with yellow fluorescennce in control cells.Therefore we concluded that PRRSV infection induces completely mitophagy.5 Inhibiting Parkin blocks PRRSV infection by enhancing apoptosisIt was said that PRRSV infection can utilize autophagy to facilitate its proliferation.Besides,Many RNA viruses can induces mitophagy which can facilitate theirs infection.What's the role of mitophagy in PRRSV infection? In order to understand the role of mitophagy in PRRSV infection,we utilized si RNA to interfere Parkin expression.We found that PRRSV proliferation was greatly reduced by interfering Parkin.What's more,apoptosis proteins cleaved-PARP and cleaved-Caspase3 were significantly accumulated in Parkin inhibited cells.Taken together,we concluded that Parkin dependent mitophagy enhances PRRSV proliferation by inhibiting apoptosis.