| AimsMiddle East Respiratory Syndrome Coronavirus(MERS-CoV)is a highly pathogenic coronavirus,which was first identified in Saudi Arabia in 2012.It is classified into beta coronaviruses with SARS-CoV and SARS-CoV-2.Compared with other coronavirus infections that cause main symptoms such as fever,headache,fatigue,and chills,these three viruses can also result in severe acute respiratory distress syndrome(ARDS)and organ failure such as lung and renal failure,which is a serious threat to human health.The lethality of SARS-CoV and SARS-CoV-2 is less than 10%,while MERS-CoV infection has a fatality rate as high as 36%.Unfortunately,there is still no effective drug and perfect treatment plans for the MERS-CoV infection.Therefore,revealing the pathogenic mechanism of host body damage caused by MERS-CoV infection can provide a theoretical basis for the development of drugs for MERSCoV infection,and certain guiding significance for improving the treatment plan of MERSCoV infection.Recent studies have demonstrated viral infection-induced the immune system disorder,oxidative stress,mitochondrial damage,and abnormal energy metabolism will lead to multiorgan failure,which is a serious threaten to human health.The mitochondria damage and oxidative stress are closely associated with the activation of immune system.Mitochondria are the energy centers of cells,providing the major source of ATP needed for metabolism.However,mitochondrial damage will release DNA and ROS,which act as pathogen-associated molecular patterns(PAMPs)and damage-associated molecular patterns(DAMPs)to active the NF-κB signaling pathway and inflammasome signaling pathway,resultting in a strong inflammatory response.This will induce cell apoptosis and cause organ failure.However,it is still unclear whether MERS-CoV can cause mitochondrial damage.MERS-CoV is a positive-sense RNA virus with a single-stranded genome approximately 30 knt in size.The MERS-CoV genome contains 10 genes and encodes 11 proteins.The orf1ab gene at the 5’-end occupies about two-thirds of the genome encodes two polyproteins,orfl a and orf1ab.These polyproteins were subsequently cleaved into 16 nonstructural proteins(nsp)by papain-like protease nsp3 and 3C-like protease nsp5.The other 9 genes at the 3’-end encode 4 structural proteins,including S protein(spike protein),E protein(envelope protein),M protein(membrane protein)and N protein(nucleocapsid protein),as well as 5 accessory proteins ORF3,ORF4a,ORF4b,ORF5 and ORF8b.However,it is still unclear whether these proteins can damage mitochondria and affect cell viability.Therefore,exploring the mechanism of viral proteins encoded by MERS-CoV on affecting cell function can provide a theoretical basis for the development of drugs for MERS-CoV infection.In this study,we constructed the nonstructural protein overexpression vector encoded by the orf1ab gene of MERS-CoV(nsp1-nsp16).Through the detection of cell viability,cell cycle and cell migration,nsp1 was found to regulate cell viability.Furthermore,the mechanism by which MERS-CoV nsp1 affected cell viability was explored via transcriptome sequencing and bioinformatics analysis,and confirmed by molecular biologic experiments.Finally,MERSCoV nsp1 transgenic mice(nsp1-tg)was constructed to verify the influence of MERS-CoV nspl on cell viability in vivo.This study revealed the mechanism of MERS-CoV affects cell viability,which provides a potential target for the development of therapeutic drugs for MERSCoV infection,and is helpful for optimizing clinical treatment strategy for MERS-CoV infection.Methods1.The nonstructural protein overexpression vectors encoded by MERS-CoV orflab gene(nsp1-nsp 16)were constructed by using the classical vector construction method(digestion by restriction endonuclease and ligase by T4 DNA ligase).After overexpression in HEK293T cells,CCK8 assay was performed to screen out the MERS-CoV nsp1 that influenced cell viability.Furthermore,the MERS-CoV nsp1 was overexpressed in A549 cells to confirm the influence of nsp1 on the cell viability by CCK8 assay.2.Designing the point mutation sites on the PCR primers and performing PCR by using HotStart Pfu DNA Polymerase,the nsp1 endonuclease active site mutant(R146A and K147A)nsp1-CD was constructed.The effect of nsp1-CD on cell viability was further detected by CCK8 assay to explore whether the inhibition of cell viability by nspl depends on its endonuclease activity.3.pcDNA3.1,pcDNA3.1-nsp1 and pcDNA3.1-nsp 1-CD were transfected into HEK293T and A549 cells,respectively.After 36 h,the effect of nsp1 and nsp1-CD on cell cycle was detected by flow cytometry in HEK293T and A549 cells.4.pCDH,pCDH-nsp1 and pCDH-nsp1-CD were transfected into HEK293T and A549 cells,respectively.12 h later,the cells were streaked with a pipette tip,and the position and size of the scratch were recorded by using microscope.24 h later,the size of the scratch was recorded again by using microscope.The migration capacity of cells was assayed by using Image J software.5.Transcriptome sequencing was used to analyze the mechanism of MERS-CoV nsp1/nsplCD on cell viability.pCDH,pCDH-nsp1 and pCDH-nsp1-CD were transfected into HEK293T cells,respectively.36 h later,the cells were collected and washed twice with pre-cooled PBS.After that,1 ml TRIzol reagent was added and frozen in liquid nitrogen.These samples were ubsequently delived in dry ice to Shanghai Personalbio Technology Co.Ltd for mRNA sequencing.6.Bioinformatics analysis of the effect of MERS-CoV nspl/nsp1-CD on cellular process regulation.Differentially changed genes(up-regulated and down-regulated)in transcriptome sequencing and the proteome of nspl-located granules were analyzed by KEGG category enrichment and GO category enrichment,exploring the mechanism of nsp1 and nsp1-CD on cellular process regulation(https://david.ncifcrf.gov/tools.jsp).7.pcDNA3.1,pcDNA3.1-nsp1 and pcDNA3.1-nsp1-CD were transfected into HEK293T cells,respectively.24 h later,these cells were fixed,and the ribosomes and mitochondria were observed by transmission electron microscopy.8.pcDNA3.1,pcDNA3.1-nsp1 and pcDNA3.1-nsp1-CD were transfected into A549 cells,respectively.36 h later,these cells were stained with Mito-tracker-Red,and the effects of nsp1 and nsp1-CD on mitochondria were observed by confocal laser microscopy.9.RT-qPCR was performed to detected the mRNA levels of ribosomal protein genes(RPLP1,RPL19,RPS7,RPL36,RPL18,RPL10,RPS18,COX7C)and oxidative phosphorylation protein genes(NDUFA1,COX7C,COX5B,NDUFB7,ATP5F1E,COX4I1),apoptosis related genes(BAD,BCL2L11,BIRC5)and DPP4,AUP1,CHCHD2,C1QBP.10.Western blot was performed to detect the protein expressions of nsp1 and β-actin in HEK293T and A549 cells,and the protein levels of nspl,LSM14A,G3BP1,RPS18,COX4I1 and LAMP1 in pre-sorted-and sorted-nsp1 granules.11.pCDH,pcDH-nspl and pcDH-nspl-CD were transfected into A549 cells,respectively.36h later,the mitochondria were stained by using Janus green B,and then the mitochondria were then observed by using microscope.12.pCDH,pcDH-nsp1 and pcDH-nsp1-CD were transfected into HEK293T and A549 cells,respectively.The changes of oxidative phosphorylation value(OCR value)in HEK293T and A549 cells were detected by Seahorse XF24 Extracellular Flux Analyzer.13.HEK293T cells were transfected with pcDNA3.1,pcDNA3.1-nsp1 and pcDNA3.1-nsp1CD,respectively.36 h later,HEK293T cells were collected and stained with mitochondrial membrane potential assay kit JC-1.Subsequently,mitochondrial membrane potential changes were detected by flow cytometry.14.The overexpression vectors of pcDNA3.1-CHCHD2 and pcDNA3.1-C1QBP were cotransfected with pcDNA3.1,pcDNA3.1-nsp1 or pcDNA3.1-nsp1-CD,respectively.Then,the mRNA levels of ribosomal protein genes(RPLP1,RPS18)and oxidative phosphorylation protein genes(COX7C,NDUFB7,ATP5F1E)were detected by RT-qPCR assay.15.RT-qPCR was performed to detect the effects of nspl and nspl-CD expression on mRNA levels of ribosomal protein gene RPS18 and oxidative phosphorylation protein gene COX4I1 in the presence or not of Actinomycin D(Act D),confirming the influence of nspl on the tanscription of these genes.16.XRN1 knockdown HEK293T cells was constructed by using XRN1-shRNA.pcDNA3.1,pcDNA3.1-nspl and pcDNA3.1-nspl-CD overexpression vector were transfected into these cells,respectively.36 h later,the mRNA levels of the ribosomal protein gene RPS18 and the oxidative phosphorylation protein gene COX4I1 were detected by RT-qPCR assay,confirming whether nsp1 decrease these gene mRNAs through XRN1.17.The nspl overexpression vector pcDNA3.1-nsp1-GFP was tansfected in HEK293T cells.24 h later,cells were collected and nsp1 granules were sorted according to the nsp1-GFP granules sorting method(materials and methods).Subsequently,two different fractions of nspl granules before(pre-sorted)and after sorting(sorted)were performed by fluorescence microscopy and western blot to determine whether nspl located in the known organelles.18.pcDNA3.1-nspl was transfected into HEK293T cells.24 h later,immunofluorescence assay was performed by using nsp1 monoclonal antibody.The localization of nspl in HEK293T cells was observed by confocal microscope.pcDNA3.1-nsp1-GFP was cotransfected with pcDNA3.1-LSM14A-mCHerry and pcDNA3.1-G3BP1-mCHerry into HEK293T cells.24 h later,the localization of nspl with P-bodies(LSM14A)and stress granules(G3BP1)were observed by confocal microscopy.pcDNA3.1-nsp1-GFP was transfected into HEK293T cells.24 h later,mitochondria and lysosomes were stained with Mito-Tracker-Red and Lyso-Tracker-Red,respectively.The colocalization of nsp1 with mitochondria and lysosomes was observed by confocal microscopy.19.pCDH-nspl vector was transfected into HEK293T cells.24 h later,the cells were collected and lysed.Nsp1 and proteins interacting with nsp1 were co-mmunoprecipitated by nsp1 antibody and magnetic beads,and then protein mass spectrometry was performed.Finally,proteins interacting with nspl were analyzed by GO enrichment.20.pCDH-nspl vector was transfected into HEK293T cells.24 h later,the cells were collected and lysed by lysis buffer containing RNA enzyme inhibitors(to avoid RNA degradation).Nsp1 and RNA interacting with Nsp1 were co-immunoprecipitated by Nsp1 antibody and magnetic beads,and then RNAs were extracted by TRizol reagent,analyzed by RT-qPCR,including the Nsp1 degraded mRNA(COX4I1,RPS18,ATP5F1E,CHCHD2,C1QBP)and non-degraded mRNA(BAD,BCL2L11,BIRC5,nspl)in nspl granules.21.Four to five-week-old sex-and age-matched WT mice and MERS-CoV nspl transgenic mice were selected and fed with either a standard diet or a tetracycline diet(2 mg/mL tetracycline)for 2 weeks.During this period,the body weight of the mice was measured daily.After that,the mice were injected intraperitoneally with poly(I:C)(2 μg/g body weight)plus D-galactosamine(0.25 mg/g body weight).The survival of these mice was monitored every 12 h.22.Total RNA was extracted from the lung tissues of mice by TRizol reagent RNA extraction.After that,RT-qPCR assay was performed to analysis the mRNA of nspl,Cox4i1,Rps18 and Rplp1 in the lung tissues of WT and MERS-CoV nspl transgenic mice fed with either a standard diet or a tetracycline diet.Results1.MERS-CoV nspl significantly inhibits cell viability,cell cycle,and cell migrationMERS-CoV nspl could significantly inhibit cell viability in HEK293T cells,while other nonstructural proteins had no significant effect on cell viability.Further experiments showed that MERS-CoV nspl inhibited the viability of HEK293T and A549 cells in a dose-dependent manner.Meanwhile,MERS-CoV nspl significantly blocked the cell cycle at the G1 phase.Besides,MERS-CoV nspl significantly disturbed cell migration capacity of HEK293T and A549 cells.2.MERS-CoV nspl inhibits cell viability through its endonuclease activityFurther experiments were performed to explore the effects of MERS-CoV nspl endonuclease active site mutant Nsp1-CD on the cell activity,cell cycle and cell migration of HEK293T and A549 cells,which indicated that MERS-CoV nspl inhibits cell viability through its endonuclease activity.3.MERS-CoV nsp1 downregulates the mRNAs of ribosomal protein genes and oxidative phosphorylation protein genesTranscriptome sequencing and bio informatics analysis indicated that MERS-CoV nspl could specifically downregulated the mRNAs of ribosomal protein genes and oxidative phosphorylation protein genes in cells,but had no significant effect on the mRNAs of other biological processes.RT-qPCR results also confirmed that MERS-CoV nspl could downregulated the mRNAs expression of ribosomal protein genes(RPLP1,RPL19,RPS7,RPL36,RPL18,RPL10)and oxidative phosphorylation protein genes(NDUFA1,COX7C,COX5B,NDUFB7)in cells.However,MERS-CoV nspl had no significant effect regulation on the mRN A of MERS-CoV receptor protein gene DPP4 and lipid metabolism related gene AUP1.These results were further confirmed by western blot.Moreover,MERS-CoV nspl downregulated the mRNAs of ribosomal protein genes and oxidative phosphorylation protein genes also depends on its endonuclease activity.4.MERS-CoV nspl decreases ribosome number and disturbs mitochondrial function Transmission electron microscopy revealed that MERS-CoV nspl could decrease ribosome number and cause mitochondrial damage in HEK293T cells and A549 cells.Further investigation showed MERS-CoV nspl could significantly reduce mitochondrial activity and mitochondrial membrane potential,accompanied with the reduction of basal respiration,ATP production and space respriatory capacity.These effects of MERS-CoV nspl also depended on its endonuclease activity.5.MERS-CoV nspl downregulates the mRNAs of ribosomal protein genes and oxidative phosphorylation protein genes by its endonuclease activityMERS-CoV nspl-mediated the downregulation of ribosomal protein genes and oxidative phosphorylation protein genes mRNAs was not associated with C1 QBP and CHCHD2 that are responsible for the transcription of ribosomal and mitochondrial protein genes.Meanwhile,MERS-CoV nspl could still downregulate the mRNAs of these genes in the present of Actinomycin D(Act D)that inhibits mRNA transcription or in cells the exoribonuclease XRN1 knocking down.Therefore,MERS-CoV nspl promoted the degradation of ribosomal protein genes and oxidative phosphorylation protein genes mRNAs depending on its endonuclionase activity.6.MERS-CoV nspl located-granules are rich in nspl downregulated mRNAsBy using laser confocal microscope,we found MERS-CoV nspl formed granules that were distributed in the cells.However,MERS-CoV nspl did not co-localize with P bodies,stress granules(SGs),mitochondria,or lysosomes in the cells.After purified nsp1-located granules and the following mass spectrometry,bioinformatics analysis showed the nsp1-located granules is a novel ribonucleoprotein complex that is similar to P-body and Stress granules(SGs).Through bioinformatics analysis of the transcriptome sequencing data of P-body and Stress granules(SGs),we found there is an unknown ribonucleoprotein complex similar to Pbody and Stress granules(SGs).This novel ribonucleoprotein complex is rich in the mRNAs of ribosomal protein genes and oxidative phosphorylation protein genes.RIP assay also demonstrated that this nspl located ribonucleoprotein complex containing abundant ribosomal protein genes,oxidative phosphorylation protein genes,C1QBP1 and CHCHD2 mRNAs,while the mRNAs not degraded by nspl were almost undetectable.7.MERS-CoV nspl transgenic mice are more sensitive to poly(I:C)-induced deathMERS-CoV nspl could be induced by tetracycline feeding in MERS-CoV nsp1 transgenic(nspl-tg),and the expression of nspl could significantly cause the weight loss of nspl-tg mice compared with WT mice but did not induce mice die.Furthermore,nspl-tg mice exhibited more sensitive to poly(I:C)-induced inflammatory death,accompanied with the decrease of ribosomal protein gene and oxidative phosphorylation protein gene mRNA in the lung tissues such as the mRNA of ribosomal protein gene Rps18 and Rplpl and oxidative phosphorylation protein gene Cox4i1.ConclusionsIn this study,MERS-CoV nspl was found to be able to inhibit cell viability,cell migration and arrest cell cycle at G1 phase,which was dependent on its own endonuclease activity.Further studies demonstrated that MERS-CoV nspl could downregulate the mRNAs of ribosomal protein gene and oxidative phosphorylation protein gene in cells.Meanwhile decreasing ribosome number and inducing mitochondria injury.MERS-CoV nsp1 inhibited the oxidative phosphorylation process of mitochondrial in cells,resulting in reduced mitochondrial membrane potential and the disorder of protein synthesis and energy metabolism.These phenomena all depend on the nspl endonuclease activity.Importantly,we found that MERSCoV nspl localized in a novel ribonucleoprotein complex of cells.And,these novel granules specifically contained mRNAs downregulated by MERS-CoV nspl,but not mRNAs that were not influenced by MERS-CoV nspl.In addition,MERS-CoV nspl expression caused mice body weight loss and made mice more sensitive to polyI:C-induced death.SignificanceThis study demonstrated that MERS-CoV nspl is the major viral virulence factor leading to the decline of cell viability by decreasing the number of ribosomes and disturbing mitochondrial function after MERS-CoV infection.Moreover,the nspl expression can make the mice more sensitive to poly(I:C)-induced death,which may be the reason for the high mortality of MERS-CoV.In addition,this study first discovered nsp1 locates in a novel granules that are specifially rich in the mRNAs of ribosomal protein gene and oxidative phosphorylation protein gene.This phenomenon reveals the molecular mechanism of nspl inhibiting cell vitality.Therefore,this study can provide a new idea and a solid experimental basis for the subsequent development of therapeutic drugs for MERS-CoV,it also provides theoretical guidance for improving the treatment plan. |
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