The Process Of SARS-CoV-2 Interfering With The Host Cell Autophagy Machinery And Its Potential Therapeutic Target Study

The current outbreak of SARS-CoV-2 was first reported in December 2019 and is the third serious outbreak of?coronavirus since SARS-CoV and MERS-CoV.It spread to more than 200 countries in a terrible speed since February 2020.The World Health Organization announced that coronavirus disease 2019(COVID-19)caused by SARS-CoV-2 has escalated into a global epidemic On March 11,2020.In December,novel SARS-CoV-2 mutants were detected in different countries and regions such as the UK and SouthAfrica,and the mutants were more infectious.As of March 10,2021,more than 100 million cases and 2.6 million deaths have been reported worldwide,with a global death rate of 2.2%,and these figures are still on the rise in most countries.The severe epidemic situation challenges human economy,society and life safety.We still know little about the intracellular life process of SARS-CoV-2,and the mechanism research is in urgent need.Therefore,the discovery of anti-SARS-CoV-2 target based on this mechanism study will undoubtedly become a strong agent for the fight against SARS-CoV-2.Autophagy is an evolutionarily conservative cell process,which forms a double membrane structure named autophagosome.Autophagosome selectively encapsulates defective or damaged intracellular substances,including long-lived proteins and defective organelles,such as mitochondria and lysosomes.The autophagosome fuses with lysosome at the terminal stage of autophagy to form autophagolysosome,and the inclusions will be degraded.Autophagy clears damaged organelles and pathogens,and plays a role of defense and host protection.Some viruses,including measles virus,HIV-1,herpes B virus,coxsackievirus virus and coronavirus,etc.,that have got adaptability could hijack autophagic elements to promote self-replication.So far,the interaction between SARS-CoV-2 and host cell autophagy has not been clarified.This study discusses the interference of SARS-CoV-2 with host autophagy machinery in order to find effective therapeutic targets.1.The study of SARS-CoV-2 interfering with host cell mitophagyMitophagy is a special type of autophagy.The damaged mitochondria selectively become autophagic substrates,which are degraded in autophagolysosomes and provide substrate for cell metabolism and new mitochondria production.At present,no experimental study has been conducted to elucidate the subcellular localization of SARS-CoV-2 genome.In this study,we used immunoelectron microscopy and immunofluorescence co-localization technology to mark ds RNA,an important intermediate product in the replication of SARS-CoV-2 RNA.We demonstrated for the first time that mitochondria are one of the replication sites of SARS-CoV-2 RNA.By using the knockdown technique of Tom20,we found that the mitochondrial outer membrane protein Tom20 may play an important role in the mitochondrial localization and replication of SARS-CoV-2 RNA.To investigate the influence of SARS-CoV-2replication on mitochondrial function,we detected the changes of mitochondrial membrane potential by JC-1 staining,the permeability changes of mitochondria permeability transition pore(MPTP)by TMRM staining,and the release of active oxygen by CM-H2DCFDA probe.The results showed that SARS-CoV-2 infection caused mitochondrial damage such as the depolarization of mitochondrial membrane potential,increased permeability of MPTP and enhanced release of reactive oxygen stress.The mitochondrial damage was treated by Mdivi-1 and Cyclosporin A,which inhibited SARS-CoV-2 replication,suggesting that the abnormal function of mitochondria provided favorable conditions for SARS-CoV-2 replication.Mitochondrial damage is the prerequisite for activating mitophagy.The classical pathway of mitophagy was detected by Western Blotting and Immunofluorescence.The results showed that SARS-CoV-2 increased Pink1 and Parkin expression and caused their mitochondrial aggregation.In addition,by knockdown of Pink1 protein,we found the mitochondrial aggregation of P62 protein which was regulated by Pink1,suggesting that SARS-CoV-2 infection activated the Pink1-Parkin pathway and recruited the P62 to mitochondria.The P62-labeled mitochondria become selective autophagic substrates,which will be further wrapped by autophagosomes,and the damaged mitochondria and virus genomes will be degraded and removed in lysosome.The virus interferes with this process.By Western blotting and immunofluorescence,we found that the damaged mitochondria were not effectively removed,and the mitochondria were not become the substrate of autophagosome and lysosome.We further used Co-immune precipitation to explore the mechanism of SARS-CoV-2 intervention on this process.The results showed that SARS-CoV-2 infection impeded the binding between P62 and LC3,thus preventing autophagosome from wrapping the P62-labeled mitochondria.This study is the first to demonstrate that mitochondria could be one of the replication sites of SARS-CoV-2.SARS-CoV-2 replication causes mitochondrial damage,which induces mitophagy through Pink1-Parkin pathway as a result of host cell defense.The virus inhibits the binding of p62 and LC3 to hinder mitophagy,and thus can resist the defense mechanism of host cell.2.The study of SARS-CoV-2 regulating autophagic processThe nsp6 protein of mouse hepatitis virus(MHV),one of the members of?coronavirus,induced autophagy in Vero cells in an Atg5-dependent manner.In addition,overexpression of Plpro protein of SARS-CoV and MERS-CoV on three human cell lines promoted autophagosome formation,but inhibited the fusion of autophagosome and lysosome,which leads to autophagosome aggregation.Similarly,the infection of MERS-CoV inhibited autophagolysosome formation in Vero B4 cells.So far,there is no published report on the autophagic process and pathway changes induced by SARS-CoV-2 infection.In this part,we confirmed by Western Blotting,as well as immunohistochemistry and transmission electron microscopy,the accumulation of autophagosomes in Vero E6 cells,Huh-7 cells and lung tissues of non-human primates after SARS-CoV-2 infection.Next,we used m Cherry-EGFP-LC3 plasmid,autophagy and lysosome co-labeling,and Western Blotting assays and found that the increase of autophagosome induced by SARS-CoV-2 infection could result from:(1)the increase of autophagosome biosynthesis;(2)the inhibition of autophagosome-lysosome fusion.Finally,Western blotting was performed in Vero E6 cells to examine the signaling cascade in autophagosome formation.It was found that the SARS-CoV-2-induced Akt-mTOR pathway inhibition,VPS34 activation,and increased Atg5,Atg12 and Atg16 abundance,was consistent with the increased biosynthesis of autophagosome.This study elucidates the alterations in autophagic process after SARS-CoV-2 infection,which is manifested by the impairment of autophagosome-lysosome fusion,and the augmented autophagosome formation that is mediated by Akt-mTOR pathway and VPS34 complex.3.The study on VPS34 as a potential anti-SARS-CoV-2 drug targetThe regulation of autophagic process is considered to be one of the potential strategies for anti-SARS-CoV-2.At present,there is no clear drug target in autophagy pathway.In the previous chapter,we observed a series of changes in autophagic process induced by SARS-CoV-2 infection.In order to clarify the function of autophagy on SARS-CoV-2 infection,we regulated the activity of autophagy related proteins through drug intervention or siRNA interference or gene knockout in Vero E6 cells.The results showed that VPS34 inhibitors SAR405 and 3-MA,as well as atg14 knockdown,one of the key subunits of VPS34 complex,had significant inhibition on SARS-CoV-2replication,while Atg5 gene knockout had no significant effect on virus replication.Next,we evaluated the anti-SARS-CoV-2 effect of VPS34 inhibitors in vivo.The results showed that the VPS34 inhibitor 3-MA significantly inhibited the replication of SARS-CoV-2 in the lung tissue of hACE2 transgenic mice and improved the viral pneumonia.In contrast,Rapamycin,the mTOR inhibitor,aggravates SARS-CoV-2infection.Finally,we further evaluated 3-MA on human lung tissues.For the first time,we reported that human lung xenografted mice could be used as an infection model of SARS-CoV-2.The results showed that VPS34 inhibitor 3-MA significantly inhibited the N gene and E gene copies of SARS-CoV-2 in human lung tissues,and reduced the number of infectious virus particles,and had therapeutic effect on SARS-CoV-2 related pneumonia.This study demonstrated that the inhibition of autophagic VPS34 complex exerts anti-SARS-CoV-2 effects,which provides a feasible way for drug design.4.The evaluation of anti-SARS-CoV-2 activities of autophagosome-lysosome fusion inhibitors in vivo and in vitroAutophagosome-lysosome fusion inhibitors inhibits the final stage of autophagy.In the previous chapter,we found that SARS-CoV-2 acted in a similar way as autophagosome-lysosome fusion inhibitors.However,chloroquine(CQ),a typical autophagosome-lysosome fusion inhibitor,has been used as a therapeutic drug in the early stage of COVID-19 outbreak.The mechanisms may be that excessive autophagosome accumulation leads to the death of infected cells and the termination of SARS-CoV-2 replication cycle.At present,few studies have reported the anti-SARS-CoV-2 effect of autophagosome-lysosome fusion inhibitors except for CQ.This study evaluated the anti-SARS-CoV-2 effects of autophagosome-lysosome fusion inhibitors,including CQ,bafilomycin A1 and NH₄Cl on cells and hACE2 transgenic mice.The results showed that CQ,bafilomycin A1 and NH₄Cl inhibited the replication of SARS-CoV-2 in Vero E6,Huh-7 and 293T-hACE2 cells.CQ and bafilomycin A1increased the cell proliferation of Vero E6 cells after SARS-CoV-2 infection.Moreover,in the hACE2 transgenic mice model of SARS-CoV-2 infection,CQ and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues,as well as improved structures of alveolar septum and pulmonary alveoli.This study demonstrated that in addition to CQ,the autophagosome-lysosome fusion inhibitor bafilomycin A1 may be a potential drug candidate for anti-SARS-CoV-2 treatment.