Mechanism Of Newcastle Disease Virus Induces Mitophagy Promotes Virus Replication

Viruses are non-living entities and as such do not inherently have their own metabolism.Viruses have evolved to induce metabolic pathways for multiple ends.Newcastle disease virus(NDV)causes serious economic losses in modern poultry industry.Recently,how NDV alters cellular metabolism is still far from fully understood,and how to supply energy and biological matrix after mitochondrial damage has not reported.In this study,NDV was used as a pattern virus,and the metabolic pattern was analyzed through transcriptome,metabolomics and metabolic flux.Seahorse XF,transmission electron microscopy,immunofluorescence,Western blotting(WB)and gene knockout were used to explore the mechanisms of mitophagy and metabolic reprogramming.It will provide a new perspective for studying the parasitic pattern between virus and host.The main research contents are as follows:1.NDV infection caused mitochondrial dysfunction and stress damage.Combined with flow cytometry,immunofluorescence,Seahorse XF and other methods,our results confirmed that NDV infection induced mitochondrial membrane potential depolarization,reduced oxygen consumption rate(OCR),disruption of mitochondrial respiratory chain homeostasis and accumulation of mitochondrial reactive oxygen species(mROS).Finally,leading to mitochondrial dysfunction.2.NDV infection promotes glucose uptake and activates glycolysis metabolism through mROSHIF1α axis.By measuring glucose uptake,it was found that NDV infection promoted cell uptake of glucose in the medium.Subsequently,targeted metabolomics analysis showed that intermediate products of mitochondrial tricarboxylic acid cycle were significantly reduced,while intermediate products of glycolysis pathway were significantly increased by NDV infection,and a large amount of lactic acid was secreted extracellular,leading to significantly up-regulated extracellular acidification rate(ECAR)of infected cells.Mechanistically,NDV infection induces mitochondrial oxidative damage to release a large number of mROS,promotes the stable expression of HIF1α,and activates glycolysis gene expression.Real-time Seahorse ATP assay further confirmed that glycolysis is the primary energy supply pathway after NDV infection.3.SIRT3 regulates cellular metabolic reprogramming as a "transfer switch" of energy metabolism.It was found that NDV infection induced mitochondrial damage,resulting in reduced energy supply and activation of AMPK-mTORC1 interaction to induce autophagy.With the increase of viral replication,mitochondria gradually changed from a fused state to a separate state.Subsequently,PINK1/ Parkin-dependent mitochondrial autophagy was initiated to degrade damaged mitochondria.Along with the degradation of damaged mitochondria,the deacetylase SIRT3,which locates on the outer membrane of mitochondria,is also degraded.The loss of SIRT3 leads to the activation of HIF1α,which then up-regulates the transcription and expression of glycolysis genes and activates glycolysis metabolism,forcing infected cells to undergo Warburg effect to support viral replication.4.NDV induces degradation of damaged mitochondria,promotes free amino acid recycling and supports viral replication.Amino acid metabolomics analysis showed that NDV infection resulted in a large amount of amino acid consumption in infected cells and induced amino acid starvation.Subsequently,intracellular amino acid sensor GCN2 was activated and inhibition mTORC1 activity to promotes mitophagy.By blocking the amino acid replenishment pathway with different drugs,the results showed that blocking mitophagy severely impaired the replication of NDV.These results suggest that the free amino acids released by mitophagy can be an important raw material for the synthesis of viral proteins.In summary,NDV infection induces mitochondrial damage,which is degraded by PINK1/ Parkindependent mitophagy.On the one hand,degrading metabolic “switch” SIRT3 promotes metabolic reprogramming in infected cells;On the other hand,free amino acids released by mitophagy serve as materials for viral protein synthesis to support progeny viral replication.These results are helpful to understand the regulation of specific metabolic pathways during virus replication and to identify the requirements of specific nutrients for virus replication.