The Regulation And Molecular Mechanism Of MERS-CoV Nsp1 On Autophagy In Host Cells

Object:Middle East respiratory syndrome coronavirus(MERS-CoV)is a highly pathogenic coronavirus that was first discovered in Saudi Arabia in 2012.MERS-CoV was seemingly transferred from infected dromedaries to humans,with a mortality rate of 36%.MERS-CoV,SARS-CoV and SARS-CoV-2 are all β-coronaviruses(β-CoVs).In addition to causing fever,headache,and fatigue,they can cause lung and kidney failure,seriously threatening human life.Therefore,elucidating how coronavirus-encoded proteins interact with host factors is crucial on understanding the infectivity and pathogenicity of these viruses,benefiting the development of strategies to prevent and treat coronaviruses.MERS-CoV is enveloped RNA viruses that contain a single 5’-capped and 3’polyadenylated RNA genome(～30 kb)that codes for two large overlapping open reading frames(ORFla and ORF1b)as well as a variety of structural and accessory proteins.Upon entry into host cells,ORF1a and ORF1b encode two large polyproteins,pp1a and pp1ab,which are then cleaved into 16 functional nonstructural proteins(nsps)that take important part in viral infection and RNA genome replication.nsp1 is the first viral protein encoded by ORF1a.As a potent pathogenicity factor,nsp1 plays an important role in inhibiting host protein expression via degrading mRNA and inhibiting protein translation.On the one hand,MERS-CoV nspl promotes the degradation of host mRNAs by inducing an endonucleolytic cleavage in template mRNAs.On the other hand,nspl mediates translation inhibition in host cells by interacting with the 5’ UTR of host mRNA.In addition,MERS-CoV nsp1 selectively targets the nucleartranscribed host mRNAs for inhibition,whereas mRNAs that are cytoplasmic in origin,escape the inhibitory effects of MERS-CoV nspl.We found previously MERS-CoV nsp1 located in a novel ribonucleosome complex formed via liquid-liquid phase separation(LLPS),resulting in template-dependent cleavage and degradation of ribosome protein genes and oxidative phosphorylation(OXPHOS)protein genes in infected cells,thereby impairing the cellular metabolic processes.Autophagy is a highly conserved degradation pathway in eukaryotes that maintains cell survival,material recycling,and homeostasis.Normally,low autophagy levels are beneficial for maintaining a stable intracellular environment.When cells are subjected to various stress conditions,such as energy deprivation or viral infection,autophagy can be activated to promote the initiation and nucleation of a crescent-shaped phagophore,which subsequently expands until it approaches the autophagosome.Upon closure,autophagosomes fuse with lysosomes/late endosomes to form autolysosomes,which degrades its contents to meet the metabolic needs and renew some organelles.Coronavirus infection can regulate autophagy.For highly pathogenic coronaviruses,ORF3a,ORF8b,ORF9b and nsp6 of SARS-CoV can induce autophagy,whereas ORF3a,ORF7a,M,and nsp6 of SARS-CoV-2 can promote LC3 punctate aggregation.Furthermore,nsp3,nsp4,and nsp6 of MERS-CoV,as well as the accessory proteins ORF4b and ORF5a,can promote LC3 lipidation.The fusion of autophagosomes and lysosomes is mainly mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE)complexes,tether complexes,Rab molecules,and cytoskeleton components.Viral infection can also affect autophagy by affecting lysosomal biogenesis and function.Our previous study demonstrated that MERS-CoV nspl disturbs mitochondrial function by selectively downregulating the mRNA of OXPHOS.However,whether MERS-CoV nsp1 affects autophagy and its related mechanisms have not been elucidated.In this study,we aimed to clarify the mechanism by which MERS-CoV nspl-mediated autophagy regulation,so as to evaluate the effect of MERS-CoV nsp1-mediated autophagy on cell death and tissue damage.Furthermore,to provide new insights for the prevention and treatment of the coronavirus.Methods:1.To determine the effect of MERS-CoV nsp1 on autophagosome accumulation(1)Ctrl,nsp1,nspl-CD and nsp1-mt were transfected into HEK293T and A549 cells,respectively.The effects of MERS-CoV nspl on the expression of LC3 and p62 were detected by western blotting.(2)Ctrl,nsp1,nsp1-CD and nsp1-mt and EGFP-LC3 were co-transfected into HEK293T and A549 cells,respectively.Confocal microscopy was used to detect the influence of MERSCoV nsp1 on the number of EGFP-LC3 labeled autophagosomes.(3)Ctrl,nsp1,nspl-CD and nsp1-mt were transfected into HEK293T and A549 cells,respectively.The morphology and the number of autophagosomes induced by MERS-CoV nspl was detected by transmission electron microscopy(TEM).2.To explore the influence of MERS-CoV nsp1 on autophagic flux(1)Ctrl or nsp1 were transfected into HEK293T cells,and treated with autophagy inhibitors bafilomycin A1(BafA1)and hydroxychloroquine(HCQ).The effects of MERS-CoV nsp1 on the expression of LC3 and p62 were detected by western blotting.(2)Ctrl or nsp1 and mCherry-EGFP-LC3 or mCherry-LC3 were co-transferred into HEK293T cells,respectively.The influence of MERS-CoV nsp1 on autophagosome formation and the colocalization of LC3 and LAMP1 were detected by confocal microscopy.3.To elucidate the mechanism of MERS-CoV nsp1-induced autophagy(1)Ctrl and nsp1 were transfected into HEK293T cell silencing Beclinl and ATG5,respectively.The effects of MERS-CoV nsp1 on the expression of LC3 and p62 were detected by western blotting.(2)Ctrl,nsp1 and nsp1-CD were transfected into HEK293T cells,respectively.Western blotting was used to detect the activation of mTOR affected by MERS-CoV nspl.(3)Ctrl,nsp1 and nsp1-CD were transfected into HEK293T cells,respectively.Western blotting and confocal microscopy were used to detect the influence of MERS-CoV nspl on the expression of mTOR,p-mTOR,AMPK,p-AMPK,P70S6K,p-P70S6K,LC3,p62 and the number of EGFP-LC3 labeled autophagosomes pretreated with CC or NAC.(4)Ctrl,nspl and nspl-CD were transfected into HEK293T cells,respectively.The effect of MERS-CoV nsp1 on cell mitochondrial stress test was detected by seahorse analyser.(5)Ctrl,nspl and nspl-CD were transfected into HEK293T cells,respectively.The influence of MERS-CoV nspl on ATP and reactive oxygen species(ROS)production was detected by chemiluminescence and flow cytometry.4.To explore the mechanism of MERS-CoV nspl inhibits autophagic flux(1)Ctrl and nsp1 were transfected into HEK293T cells,and the proteins interacting with MERS-CoV nsp1 were detected by Co-IP/MS assay.Co-IP was used to verify whether the interaction between MERS-CoV nsp1 and Myosin Ⅵ can affect the interaction between Myosin Ⅵ and Tom 1.(2)Ctrl,nsp1 and nsp1-CD were transfected into HEK293T cells,respectively.RNA-sequence(RNA-seq)and RT-qPCR were used to detect the influence of MERS-CoV nspl on the mRNA of lysosome-related genes.(3)Ctrl,nsp1 and nsp1-CD were transfected into HEK293T cells,respectively.Confocal microscopy was used to detect the influence of MERS-CoV nsp1 on lysosomal biogenesis,acidification,cathepsin B and the colocalization of lysosomes and acid lysosomes.(4)Ctrl,nspl and nspl-CD were transfected into HEK293T cells,respectively.Western blotting was used to detect the influence of MERS-CoV nspl on the expression of LAMP 1,LAMP2,CTSD,ATP6V1B2 and epidermal growth factor receptor(EGFR)degradation.5.To clarify the mechanism by which MERS-CoV nspl impair lysosomal biogenesis and acidification(1)Ctrl,nspl and nspl-CD were transfected into HEK293T cells,respectively.Subcellular fractionation and confocal microscopy were used to detect whether MERS-CoV nspl can affect the nuclear translocation of transcription factor EB(TFEB).(2)Ctrl,nspl and nsp1-CD were transfected into HEK293T cells and treated with actinomycin D(Act D),or HEK293T cells silencing XRN1.The effect of MERS-CoV nspl on the expression of lysosome-related genes was detected by RT-qPCR.(3)The nsp1-located granules were sorted according to the nsp1-GFP granules sorting method,and then two different components of the pre-sorted and sorted were analyzed by mass spectrometry(MS).(4)Ctrl,nspl and nspl-CD were transfected into HEK293T cells,respectively.RIP-qPCR was used to detect the binding of MERS-CoV nspl and the mRNA of lysosome-related genes.(5)Ctrl-GFP,nspl-GFP and nspl-CD-GFP were transfected into HEK293T and A549 cells,respectively.The colocalization of MERS-CoV nspl and LAMP1 mRNA was determined by RNA-fluorescence in situ hybridization(RNA-FISH).6.To explore the biological significance of MERS-CoV nspl-induced autophagy(1)Ctrl and nsp1 were transfected into HEK293T cells,and then treated with 3-MA,Z-VAD,Nec-1 and Fer-1.CCK8 was used to detect whether the inhibition of MERS-CoV nspl on the cell viability depended on nsp1-induced autophagy.(2)Ctrl and nspl were transfected into HEK293T cell silencing Beclinl and ATG5,respectively.Annexin V/7-AAD staining was used to detect whether the effect of MERS-CoV nsp1 on the apoptosis depended on nsp1-induced autophagy.7.To determine whether MERS-CoV nspl can be used as an autophagy receptor(1)The protein secondary structure of MERS-CoV nspl was predicted by JPRED database todetermine whether it contained the LIR motif.(2)Co-IP was used to detect whether MERS-CoV nspl can interact with LC3 by LIR motif.8.To explore whether nspl induces autophagy in transgenic mouse models(1)MERS-CoV nspl transgenic mice were commissioned by Southern Model Biotechnology and nsp1 positive mice were selected by genotype identification of F3 generation mice.RTqPCR was used to detect the expression of nspl in lungs,kidneys,liver,spleen and heart after doxycycline(DOX)induction for 21 days.(2)Mouse embryonic fibroblasts(MEFs)were extracted,and the influence of MERS-CoV nsp1 on autophagy was detected by western blotting and confocal microscopy after DOX induction.(3)Hematoxylin-eosin(HE)staining was used to detect the influence of MERS-CoV nspl on the tissue damage of lung,kidney and liver after DOX induction.(4)Western blotting and immunohistochemistry(IHC)were used to detect the effect of MERSCoV nspl on the expressions of p62 and LC3 lipidation in the lungs and kidneys after DOX induction.Results:1.MERS-CoV nspl triggers the accumulation of autophagosomesMERS-CoV nspl promoted the conversion of endogenous LC3-I to LC3-II in a timedependent and dose-dependent manner in HEK293T and A549 cells.Confocal microscopy showed that nspl promotes the formation of EGFP-LC3-labelled dots in HEK293T and A549 cells.Further studies suggested that the mutation and deletion of nspl endonuclease activity site inhibited the accumulation of autophagosomes induced by nspl,while the translation inhibition site had no significant influence.These data demonstrated that MERS-CoV nspl triggers the accumulation of autophagosomes depending on its endonuclease activity.2.MERS-CoV nspl induces autophagy but inhibits autophagic fluxNext,we analyzed the influence of MERS-CoV nspl on autophagic flux.Higher LC3II:LC3-I ratios were detected in nspl-expressing cells than Ctrl upon BafA1/HCQ treatment,indicating that MERS-CoV nspl induced autophagy.In addition,LC3-II:LC3-I levels in nsploverexpressing cells were not significantly influenced by treatment with or without HCQ/BafA1,indicating that nsp1 inhibited autophagic flux.Next,we used mCherry-EGFP-LC3 to monitor autophagic flux.Many yellow autophagic vacuoles were observed in nspl-overexpressing cells and the co-localization of lysosomes and autophagosomes was significantly reduced by nspl.These results demonstrated that MERS-CoV nspl induces autophagy but inhibits autophagic flux.3.MERS-CoV nspl induces autophagy by AMPK-mTOR signalingMERS-CoV nspl-induced autophagy depended on ATG5 and Beclinl,suggesting that MERS-CoV nsp1 induces autophagy via the canonical autophagy pathway.Next,we found that MERS-CoV nspl promotes AMPK activation not ERK1/2 and Akt,accompanied with the inhibition of mTOR signaling,depending on the endonuclease activity of nspl.Furthermore,we found that MERS-CoV nspl leads to a decrease in ATP synthesis and an increase in ROS production by interfering with mitochondrial respiratory function,ultimately inducing autophagy by activating AMPK and inhibiting mTOR.4.MERS-CoV nspl impairs lysosomal biogenesis and acidificationMERS-CoV nspl decreased the mRNA levels of lysosome-related proteins and this process depending on its endonuclease activity by using RNA-seq and RT-qPCR analysis.Next,we found that MERS-CoV nspl inhibits the number of lysosomes and the expression of lysosome proteins,suggesting that MERS-CoV nspl inhibits lysosomal biogenesis.Meanwhile,MERS-CoV nspl inhibited the degradation of EGFR,the number of acidic lysosomes,the activity of cathepsin B,the co-localization of lysosomes and acid lysosomes,suggesting that MERS-CoV nsp1 inhibits lysosomal acidification.Taken together,we found nsp1 inhibits autophagic flux by impairing lysosomal biogenesis and acidification.5.MERS-CoV nspl impairs lysosomal biogenesis and acidification via its endonuclease activityBy using Act D to interfere with transcription processes,it was demonstrated that MERSCoV nspl downregulates the lysosome-related genes independent of the transcription of upstream transcription factors.Meanwhile,we found that the MERS-CoV nsp1 inhibits the mRNA levels of lysosome-related genes independent of host ribonucleases XRN1.Furthermore,we found that the MERS-CoV nspl can bind to the mRNA of lysosome-related genes that are enriched in nspl-located granules,then cleave these mRNAs via its endonuclease activity,resulting in the downregulation of lysosome-related genes,and ultimately impairing lysosomal biogenesis and acidification.6.MERS-CoV nspl-induced autophagy impairs cell viabilityTo clarify the significance of MERS-CoV nspl-induced autophagy,we analyzed the influence of nspl on cell viability.Compared with Ctrl,nspl inhibited cell viability,which could be recovered by sh-ATG5/BECN 1.Simultaneously,3-MA or sh-ATG5/BECN 1 treatment significantly decreased the nsp1-induced apoptotic rate.The results suggested that MERS-CoV nspl-induced autophagy promotes apoptosis,inhibiting cell viability.7.MERS-CoV nspl interacts with LC3 via the LIR motif.MERS-CoV nspl can interact with LC3 by Co-IP analysis.Next,we predicted the protein secondary structure of MERS-CoV nsp1 using the JPRED database and found a LC3interacting region(LIR)motif at the β-sheet of 69-76.Furthermore,we found that the LIR motif of MERS-CoV nspl is crucial for the interaction of MERS-CoV nsp1 with LC3,while the LIR motif have no influence on MERS-CoV nspl-induced autophagy.8.MERS-CoV nspl induces autophagy in transgenic mouse modelFinally,we used MERS-CoV nspl-transgenic murine model to verify the effect of nspl on autophagy.First of all,MEFs were derived from nspl-transgenic pregnant mice and cultured in vitro.The results showed that MERS-CoV nspl induced autophagy but inhibited autophagic flux by impairing lysosomal acidification in MEFs treated with DOX.Furthermore,we isolated the lungs and kidneys of MERS-CoV nspl transgenic mice,and found that nspl also leads to autophagy induction in these tissues treated with DOX,which was related to tissue damage.Conclusions:Our current study illustrated that MERS-CoV nspl-located granules were rich in RNAbinding proteins and the mRNAs of OXPHOS protein genes and lysosome-related genes.As an endonuclease,nspl cleaved the mRNA of OXPHOS protein genes,which disturbed mitochondrial function.The increased ROS and decreased ATP led to the activation of AMPK and the inhibition of mTOR activity,thereby triggering autophagy.On the other hand,nspl directly bound to and downregulated the mRNA of lysosome-related genes,blocking the autophagic flux by impairing lysosomal biogenesis and acidification.In turn,nspl-induced autophagy suppressed cell viability,causing cell death.Our study clarifies systematically the specific mechanism of MERS-Co V nspl-induced autophagy,providing new ideas for coronavirus drug development and clinical therapy.Innovation and significance:1.This study clarified systematically the specific mechanism of MERS-CoV nspl regulate autophagic flux via mTOR signaling and dysfunctional lysosomes.MERS-CoV nspl increased ROS and reduced ATP levels in cells,which activated AMPK and inhibited the mTOR signaling pathway,resulting in autophagy induction.Meanwhile,MERS-CoV nspl bound and downregulated the mRNA of lysosome-related genes,which diminished lysosomal biogenesis and acidification,inhibiting autophagic flux.2.This study proved that mRNA of lysosome-related genes is located in nsp1-located granules,and MERS-CoV nspl directly binds and degrades it,impairing lysosomal biogenesis and acidification.3.This study found MERS-CoV nspl-induced autophagy promotes cell death,proving that autophagy is an important reason of tissue injury caused by MERS-CoV infection.4.This study revealed that MERS-CoV nspl can be used as an important cytotoxic factor.nspl and autophagy can be used as potential targets for treatment and prevention of coronavirus infection.