Coronavirus replication-transcription complex: yakakosha uye yakasarudzika NMPylation yeNiRAN-RdRp subunits kunzvimbo dzakachengetwa munsp9

Yakagadziriswa naPeter Sarnow, Stanford University Chikoro cheMishonga, Stanford University, California, yakabvumidzwa muna Zvita 25, 2020 (yakaongororwa muna Gumiguru 25, 2020)

Isu tinoshuma kudyidzana pakati pema subunits mukudzokorodza kwekoronaviruses-transcription complexes, izvo zvakakosha pakudzokorora uye nekuchengetedza shanduko.Isu takapa humbowo hwekuti iyo NiRAN domain yakabatana nensp12 ine nucleoside monophosphate (NMP) transferase chiitiko mu trans, uye yakaratidza nsp9 (iyo RNA inosunga protein) sechinangwa chayo.NiRAN inokonzeresa iyo covalent yakanamatira yeNMP moiety kune yakachengetedzwa nsp9 amino terminus mukuita kunovimba neMn2+ ions uye padyo neakachengetedzwa Asn zvisaririra.Zvakaonekwa kuti NiRAN chiitiko uye nsp9 NMPylation yakakosha pakudzokorora kwe coronavirus.Iyo data inotibvumira kubatanidza chiitiko ichi cheiyo nested virus enzyme marker kune zvakamboonekwa mukufungidzira kuti kutangwa kweRNA synthesis mukirasi yeRNA mavhairasi kunoshanda uye nekushanduka-shanduka kunoenderana.

Iyo RNA-inotsamira RNA polymerase (RdRps) yeNidovirales (Coronaviridae, Arterioviridae, nedzimwe mhuri gumi nembiri) yakabatana neamino-terminal (N-terminal) domain mune isiri-structural protein (nsp) yakaburitswa kubva mupolyprotein, inonzi NiRAN. 1ab inoumbwa nehutachiona hweprotease (Mpro).Kare, hutachiona hwe arterial NiRAN-RdRp nsp's own GMPylation/UMPylation chiitiko chakashumwa, uye zvakakurudzirwa kuti zvigadzire kanguva kekutamisa nucleoside monophosphate (NMP) kune (isingazivikanwe parizvino) hutachiona uye/kana cell biopolymerization Zvinhu.Pano, tinoratidza kuti iyo coronavirus (Human Coronavirus [HCoV] -229E uye Severe Acute Respiratory Syndrome Coronavirus 2) nsp12 (NiRAN-RdRp) ine Mn2+-inotsamira NMPylation chiitiko, icho chinobva ku nsp9 kuburikidza nekuumbwa kweMpro-mediated nsp9 Mushure. iyo N-terminal flanking nsps inoburitswa proteolytically, iyo phosphoramidate inosungirirwa kune yekutanga amine (N3825) paN-terminal ye nsp9.Uridine triphosphate ndiyo inofarirwa nucleotide mukuita uku, asi adenosine triphosphate, guanosine triphosphate, uye cytidine triphosphate zvakare akakodzera co-substrates.Zvidzidzo zvekuchinja uchishandisa recombinant coronavirus nsp9 uye nsp12 mapuroteni uye genetically engineered HCoV-229E mutants akatemesa zvisaririra zvinodikanwa kuNiRAN-mediated nsp9 NMPylation uye kudzokororwa kwehutachiona mune cell tsika.Iyo data yakasimbisa kufanotaura kweNiRAN inoshanda saiti zvakasara uye yakasarudza basa rakakosha re nsp9 N3826 zvakasara mu nsp9 NMPylation uye kudzokorora kwehutachiona mu vitro.Izvi zvakasara chikamu cheN-terminal NNE tripeptide yakachengetedzwa uye yakaratidza kuti ndiyo yega yakasara yensp9 nemahomologs ayo mumhuri yekoronavirus.Chidzidzo ichi chinopa hwaro hwakasimba hwechidzidzo chinoshanda cheNMPylation chiitiko chemamwe mavhairasi akavharirwa uye chinopa pfungwa dzinobvira dzekugadzirwa kwemishonga inorwisa mavhairasi.

Nidovirales positive-stranded RNA virus inokanganisa akasiyana vertebrates uye invertebrates (1, 2).Odha parizvino inosanganisira mhuri gumi neina (3), idzo mhuri yeCoronavirus yakadzidzwa zvakanyanya mumakore makumi maviri apfuura.Panguva iyoyo, matatu zoonotic coronaviruses akabuda kubva kumhuka uye akakonzera kubuda kukuru kwehutachiona hwekufema muvanhu.Kusanganisira zvirwere zvinotapukira zvinokonzerwa nezvirwere zvakanyanya zvinotapukira.Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (4âââ7).Nidoviruses inogovera yakajairika genome sangano, uye subunit ye ​​membrane-yakasungwa replication-transcription complex (RTC) yakavharirwa mu5-?²-terminal two-thirds uye mainstructural subunit yehutachiona particle, pamwe nezvimwe zvinhu. .Mapuroteni, akavharidzirwa mu3??² magumo echitatu ejenome (1).Kunze kwemhuri imwe yemavhairasi emupurani (Monoviridae) (8), mavhairasi ese anested akavharira maRTC madiki mumafuremu makuru akavhurika ekuverenga (ORF) ORF1a neORF1b, ayo akaturikirwa kubva kugenomic RNA ye.ORF1a inoisa polyprotein (pp) 1a, uye ORF1a uye ORF1b pamwe chete encode pp1ab.Nekubatanidzwa kwakawanda kweiyo main protease (Mpro) yakavharidzirwa neORF1a, zvese pp1a uye pp1ab zvinogadzirwa zvine proteolytically mumhando dzakasiyana-siyana dzisiri dzemapuroteni (nsps), anozivikanwawo se3CLpro, nekuti ine homology ne3Cpro yepicornavirus ( 9).Aya ma nsps anofungidzirwa kuve akaunganidzwa kuita yakakura ine simba RTC, inokonzeresa iyo synthesis ye genomic RNA (replication) uye seti ye subgenomic RNA (kunyorwa), uye inoshandiswa kurongedza kutaura kweORF iri zasi kweORF1b (10? ? ?12).

Iyo yakakosha RTC inosanganisira RNA-inotsamira RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) uye akati wandei RNA kugadzirisa enzymes, ayo anonyanya kuvharirwa muORF1b uye mumhuri yekoronavirus Iine nsp12-nsp16 uye nsp9-nsp12 mumhuri yeArterioviridae (ona chinyorwa 10ââ 12).RdRp neHEL1 inomiririra maviri (chimwe-chishanu) akachengetedzwa madomasi ehutachiwana hweshiri uye ane homology pakati pemamwe mavhairasi eRNA.Core replicase inofungidzirwa kuti inobatsirwa nemamwe ma subunits, kusanganisira akati wandei nsps akaburitswa kubva kucarboxy-terminal (C-terminal) dunhu repp1a, zasi kweMpro (coronavirus nsp5 uye arterial virus nsp4, zvichiteerana).Ivo vane dziviriro yakanangana nemhuri uye zviitiko zvakasiyana-siyana (yakaongororwa mureferensi 10ââ12).

Zvishoma nguva pfupi yadarika, nzvimbo ine yakasarudzika kutevedzana motif maitiro yakawanikwa paamino terminus (N-terminus) padyo neRdRp mumavhairasi ese anested, asi hapana humwe hutachiona hweRNA (16).Zvichienderana nenzvimbo yayo uye nucleotide transferase (nucleoside monophosphate [NMP] transferase) chiitiko, iyi domain inonzi NiRAN (Nestvirus RdRp-inoenderana nucleotide transferase).Musanganiswa wembiri-domain weNiRAN-RdRp unoumba nsp12 mumhuri yeCoronaviridae uye nsp9 mumhuri yeArterioviridae, uye mune imwe nestoviridae, NiRAN-RdRp inotarisirwa kuburitswa seyakazvimirira nsp kubva kuhutachiona hwepolyprotein.Muiyo coronavirus, iyo NiRAN domain ine ??1/450 zvakasara uye yakabatana neiyo C-terminal RdRp domain kuburikidza neiyo linker dunhu (16?19).MuEquine Arteritis Virus (EAV) (Arteriviridae), recombinant nsp9 inoratidza Mn2+ ion-inotsamira (yega) UMPylation uye GMPylation zviitiko, zvinoenderana nemabhesi matatu akachengetedzwa akatevedzana mu nestovirus, AN, BN uye CN Izvo zvakasara mukutevedzana.Apo N inomirira NiRAN) (16).Iyo N-terminal flanking yeiyi motif ishoma kuchengetedza motif preAN.Zvimwe zvezvisaririra izvi zvakare zvakachengetwa muprotein kinases yakabatana kure, kwavakaratidzirwa kuve vanobatanidzwa mune nucleoside triphosphate (NTP) inosunga uye inokonzeresa chiitiko (20, 21).Zvinoenderana nekutarisa uku, akati wandei akakosha anoshanda saiti zvakasara mupseudokinase SelO kubva kuPseudomonas syringae inogona kuunganidzwa neiyo ichangoburwa SARS-CoV-2 nsp7/8/12/13 supercomplex.Conserved Coronavirus NiRAN zvisaririra zvakasimudzwa muelectron microstructure.Recombinant protein (17).Zvinofungidzirwa kuti iyo yakanyorwa (yega) U/GMPylation ichaburitsa mamiriro enguva pfupi kuendesa NMP kune (ikozvino isingazivikanwe) substrate (16), uye kufanana kwechimiro pakati peNiRAN neprotein kinase (17, 19) ) Ndiyo pfungwa yekuti NiRAN inoshandura mamwe mapuroteni.

Mazhinji maficha, kusanganisira yakasarudzika uye yakasarudzika kurongeka kwakarongeka nemavhairasi akaiswa uye kupatsanurwa kwemajini kubva kuRdRp, inoita kuti NiRAN ive yakakosha yekudzora enzyme yemavhairasi akavharirwa, ayo akakosha pakubuda kwavo uye kuzivikanwa kwavo.Kare, mabasa matatu aibvira anosanganisira NiRAN kudzora genome/subgenomic dudziro kana kudzokorodza/kunyorwa kwakadaidzwa.Kana uchifunga nezve data shoma uye isina kukwana iripo panguva iyoyo, basa rega rega rine zvarinobatsira uye nekuipa (16).Mutsvagiridzo iyi, isu tinovavarira kusanganisa biochemical uye kudzosera kumashure genetic zvidzidzo zvekoronavirus inomiririra maviri genera, uye nekuisa zvatakawana kumashure kwekushanduka kwechisikigo kushanduka kwemhuri yekoronavirus, kuti tiwane nzwisiso mune ino Isinganzwisisike nzvimbo.Isu tinoshuma kufambira mberi kukuru mukunzwisisa kweNiRAN kuburikidza nekuzivikanwa kwezvinangwa zvechisikigo muRTC, iyo (pakati pezvitatu zviripo zvinofungidzira) inobatsira pakuita basa redunhu iri mukutanga kuumbwa kweiyo nested virus RNA.Tsvagiridzo iyi zvakare inovhura mikana yemamwe mabasa eNiRAN pane hutachiona host interface.

Kuti tiratidze maitiro enzymatic eiyo corona virus nsp12-inoenderana neNiRAN domain, takagadzira recombinant fomu yemunhu coronavirus 229E (HCoV-229E) nsp12 muE. coli, ine His6 tag paC-terminus, uye takabatanidza iyo mapuroteni ane [α32-P] Isa pamwe chete neNTP pamberi peMnCl2 sezvinotsanangurwa muZvishandiso uye Nzira.Ongororo yechigadzirwa chemaitiro yakaratidza kuvepo kweiyo radiolabeled protein inofamba ne nsp12 (106 kDa), zvichiratidza kuti coronavirus nsp12 inokonzeresa kuumbwa kwecovalent protein-NMP adducts, inonyanya kuumbwa neuridine monophosphate (UMP) (Mufananidzo 1A) Uye B).Quantitative analysis yakaratidza kuti kana ichienzaniswa nedzimwe nucleotides, simba rechiratidzo reUMP rinowedzera ne2 kusvika ku3 nguva (Mufananidzo 1C).Iyi data inoenderana neyakafanotaurwa yeNMP transferase chiitiko cheiyo NiRAN domain yekoronavirus (16), asi inoratidza kuti nucleotide zvido zveNiRAN domain yekoronavirus uye hutachiona hwehutachiona hwakasiyana.

Self-NMPylation chiitiko cheHCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) yakaiswa mukati meiyo yakatarwa [α-32P] NTP pamberi pe6 mM MnCl2 kwemaminetsi makumi matatu (ona Zvishandiso uye Nzira kuti uwane ruzivo).Zvigadzirwa zvemaitiro zvakaparadzaniswa neSDS-PAGE uye zvakasvibiswa neCoomassie brilliant blue.(B) Iprotein yeradiolabeled inoonekwa nekufungidzira kwephosphorous.Nzvimbo dze nsp12-His6 uye mapuroteni molecular mass markers (mu kilodaltons) anoratidzwa muA uye B. (C) Kusimba kwechiratidzo cheradioactive (kureva ± SEM) kwakatemwa kubva mukuedza katatu kwakazvimirira.*P≤0.05.Simba rechiratidzo (muzana) rine hukama neUTP.

Kunyangwe zviitwa zvine chekuita neNiRAN-enzayemu zvakaratidzwa kuve zvakakosha pakudzokorora kweEAV neSARS-CoV musero tsika (16), iyo chaiyo NiRAN basa uye zvinokwanisika zvinangwa hazvisati zvatarwa.Iyo ichangobva kutaurwa yemaitiro akafanana pakati peNiRAN nemhuri yemapuroteni ane protein kinase-senge folds (17, 22) yakatikurudzira kuti tiedze fungidziro yekuti NiRAN inokonzeresa iyo NMPylation yemamwe mapuroteni.Isu takagadzira seti yezvingangoita homologous zvinangwa, zvinosanganisira asiri-akaumbwa mapuroteni akaiswa neHCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), imwe neimwe iine C-terminal His6 tag (SI appendix, Tafura S1) , Uye pinza mapuroteni aya ne [α32-P] uridine triphosphate ([α32-P]UTP) kana paine nsp12.Bovine serum albumin uye MBP-LacZα fusion protein inogadzirwa muE. coli yakashanda sekutonga (Mufananidzo 2A, nzira 1 kusvika ku7).Protein yeradiolabeled yakaongororwa ne sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) uye autoradiography, uye yakawanikwa kuti paiva nechiratidzo chakasimba cheradioactive mumhinduro ine nsp12 uye nsp9.Nzvimbo yechiratidzo inoenderana ne molecular mass ye nsp9, inoratidza nsp12-mediated UMPylation ye nsp9 (Mufananidzo 2B, track 7).Hapana mamwe mapuroteni ekuyedza akawanikwa ari UMPylated, izvo zvakaita kuti tipedzisire kuti nsp9 inzvimbo chaiyo yensp12.Zvichienderana neiyo self-NMPylation data inoratidzwa muFigure 1, nsp12 inokwanisa kuendesa ese mana maNMP kunsp9, kunyangwe kugona kwakasiyana, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Mufananidzo).3 A uye B).Pasi pemamiriro ezvinhu anoshandiswa muyedzo iyi (kupfupisa maitiro uye nguva yekuratidzira, kuderedza kuwanda kwe nsp12; zvinhu uye nzira), iyo self-NMPylation ye nsp12 haina kukwanisa kuonekwa (enzanisa Mufananidzo 2B, mugwagwa 7, uye Mufananidzo 1B), iyo yakaratidza kushanda (Uye akawanda marounds) UMP yakatama kubva ku nsp12 kuenda ku nsp9.UMP transferase chiitiko chinoda kuvepo kweMn2 + ions, sezvakaratidzwa mumufananidzo 3C, ukuwo kushoma kweUMP kutamisa chiitiko kwakaonekwa pamberi peMg2 +, uye hapana chiitiko pamberi pemamwe maviri divalent cations akaedzwa.Data yakafanana yakawanikwa muNMPylation assays ine cytidine triphosphate (CTP), guanosine triphosphate (GTP), uye adenosine triphosphate (ATP) (SI appendix, Mufananidzo S1).

HCoV-229E nsp12-mediated UMPylation ye nsp9.Mutsara weprotein substrates (kusanganisira bovine serum albumin, MBP-lacZα, uye nhevedzano yeHCoV-229E nsps yakanyorwa neC-terminal His6 yakavharidzirwa neORF1a) yakashandiswa kuongorora UMPylation chiitiko cheHCoV-229E nsp12-His6⁺-mediated. protein.Isa puroteni ne [α-32P] UTP kwemaminetsi e10 pasina (A) kana kuvapo (B) ye nsp12 sezvinotsanangurwa muzvinhu uye nzira.Pamusoro peA neB, SDS-polyacrylamide gel yakasvibiswa neCoomassie Brilliant Blue inoratidzwa, uye pazasi peA neB, iwo anoenderana autoradiograms anoratidzwa.Nzvimbo yeprotein molecular mass marker (mu kilodaltons) inopiwa kuruboshwe.Nzvimbo ye nsp12-His6 (B, kumusoro) uye chiratidzo cheradioactive chakaonekwa panguva yekuiswa kwe nsp12-His6 ne nsp9-His6 (B, lane 7) inoratidzwawo, iyo inoratidza kuti [α-32P] UMP kune nsp9-His6. (12.9 kDa), iyo yakanga isina kucherechedzwa kune mamwe mapuroteni akaedzwa.

HCoV-229E NiRAN-mediated biochemical uye virological characterization ye nsp9 NMPylation.(A uye B) Basa reiyo nucleotide co-substrate inoshandiswa mukuita.Nsp12-His6 uye nsp9-His6 yakavhenganiswa uye incubated pamberi peakasiyana [α-32P] NTPs muyero NMPylation assay.(A, pamusoro) Coomassie-stained nsp9-His6 yakapatsanurwa neSDS-PEJI.(A, pasi) Autoradiograph yenzvimbo imwechete yegel.(B) Chiitiko chehukama (chinoreva ± SEM) pamberi peiyo yakasarudzwa nucleotide cofactor inotarwa kubva kune matatu akazvimiririra kuedza.*P≤0.05.(C) Basa resimbi ions.Kuratidzwa ndiyo yakajairwa NMPylation bvunzo pamberi pe [α-32P] UTP uye akasiyana simbi ions, imwe neimwe ine concentration ye1 mM.MuC, kumusoro, Coomassie yakasvibiswa nsp9-His6 inoratidzwa, uye muC, pasi, iyo inoenderana autoradiography inoratidzwa.Hukuru hweprotein yakanyorwa (mu kilodaltons) inoratidzwa kuruboshwe kweA neC. (D) Mutant fomu yeHCoV-229E nsp12-His6 inotakura inotsiva amino acid yakataurwa iri mu [α-32P] UTP, sekutsanangurwa kwazvinoitwa. muZvishandiso uye Nzira.Iyo radiolabeled nsp9-His6 yakagadzirwa muNMPylation reaction inowonekwa nephosphorylation imaging (D, kumusoro).Chiitiko chehukama chichienzaniswa neprotein-yemusango (wt) inoratidzwa muD, uye pasi inotorwa seavhareji (±SEM) kubva kune matatu Akazvimiririra kuedza.Nyeredzi dzinoratidza kutsiva zvisaririra zvisina kuchengetedzwa.(E) The virus titer mutsika supernatant yep1 masero akawanikwa maawa makumi maviri nemana mushure mekutapukirwa kwakatemwa neplaque assay.Makodon anotsiva muNiRAN domain yeinjiniya HCoV-229E mutant anoratidzwa (nhamba dzakasara dzinoenderana nenzvimbo yavo mupp1ab).Iyo yekudzokorora-inoshaya RdRp inoshanda saiti mutant nsp12_DD4823/4AA yakashandiswa sekutonga.

Kuti tiwane kunzwisisa kwakadzama kwenzvimbo inoshanda yeNiRAN uye kuona zvakasara zvine chekuita nebasa rensp9-chaiyo NMP transferase, takaita mutation analysis, umo takatsiva zvakasara zvakasara muNiRAN AN, BN uye CN motifs ( 16) Ndiyo Ala (SI appendix, Mufananidzo S2).Pamusoro pezvo, kukanganisa kweConservative Arg-to-Ls kana Lys-to-Arg kutsiva kwakaongororwa muzviitiko zviviri.Se (negative) kudzora, zvisaririra zvisina kuchengetedzwa kana kushoma munzvimbo yeNiRAN yekoronavirus uye mamwe mavhairasi akaiswa dendere anotsiviwa neAla Kutsiva K4116A (in motif preAN), K4135A (AN), R4178A (BN), D4188A (motif). BN) uye D4280A (CN) inoderedza zvakanyanya kana kutobvisa nsp9 NMPylation kuburikidza nensp12, nepo mapuroteni ane conservative substitutions (R4178K), K4116R) anochengeta 60% uye 80% yebasa ravo, izvo zvinoratidza kuti kuzorora kwezvirambidzo padivi ravo. maketani ari physicochemically sensitive (Mufananidzo 3D).Kutsiva mamwe akati wandei akachengetedzwa E4145A, D4273A, F4281A uye D4283A haina kunyanya kukuvadza, uye nsp9 UMPylation inongoderedzwa zvine mwero.Migumisiro yakafanana yakawanikwa mu nsp9 NMPylation reactions inosanganisira dzimwe NTPs (Mufananidzo 3D uye SI appendix, Figure S3), ichisimbisa kuti migumisiro yakaonekwa pane chaiyo amino acid inotsiva yakasununguka kubva kune rudzi rwe nucleotide co-substrate inoshandiswa.Tevere, isu takayedza maitiro anogona kuitika eiyi nsp12 inotsiva pakudzokorora kwekoronavirus musero tsika.Kuti izvi zviitike, takashandisa matemplate eDNA (cDNA) akakodzera akagadzirwa nemajini akaumbwa muhutachiona hwevaccinia (23, 24) kunyora maseru mashanu -7.Titration yehutachiona hwehutachiona hwakagadzirwa mumaseru aya yakaratidza kuti akawanda HCoV-229E NiRAN mutants aisagoneka (Mufananidzo 3E).Boka revasina-viable viral mutants rinosanganisira dzimwe nzira dzakaratidzwa kubvisa kana kuderedza zvakanyanya NMP transferase chiitiko mu vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), asi kune dzimwe nzira mbiri (K4116R, E4145A) % reserved?Yavo in vitro NMPylation chiitiko inoratidza kuti zvimwe zvirambidzo zvinobatanidzwa.Saizvozvo, mamwe maviri shanduko (R4178K, F4281A) iyo yakakonzera kudzikira pakati nepakati muNiRAN's in vitro NMPylation chiitiko chakaburitsa mavhairasi mhenyu, zvisinei, mavhairasi aya akadzikisa zvakanyanya titers kuburikidza nekudzokorora.Zvinoenderana neiyo in vitro chiitiko data inoratidzwa muFigure 3D, ichitsiva mamwe mana masara asina kuchengetedzwa muconavirus uye/kana mamwe mavhairasi anedendere (K4113A, D4180A, D4197A, D4273A) (8, 16) yakagadzira mavhairasi anoshanda Vana, kunyangwe vaine ine mwero yakaderedzwa titer kana ichienzaniswa nehutachiona hwemhando yemusango (Mufananidzo 3E).

Kuti udzidze kana NiRAN-mediated NMP transferase chiitiko chinoenderana neanoshanda RdRp domain, maviri akachengetedzwa eAsp masara akabatanidzwa mukurongeka kwedivalent metal ions (11) muRdRp motif C akatsiviwa neAla. yayo nsp9 NMPylation chiitiko, zvichiratidza kuti nsp12-mediated in vitro nsp9 NMPylation basa haidi polymerase chiitiko (SI Appendix, Figure S4).

Mushure mekutanga iyo nsp9-chaiyo NMP yekuchinjisa chiitiko che nsp12, takaedza kuratidza iyo NMP-nsp9 adduct ne mass spectrometry (MS).Iyo yakazara protein mass spectrum ye recombinant HCoV-229E nsp9 yakaratidza peak pa12,045 Da (Mufananidzo 4A).Kuwedzerwa kwe nsp12 hakuna kuchinja hutano hwe nsp9, zvichiratidza kuti nsp12 uye nsp9 haizogadziri kugadzikana kwakagadzikana pasi pemamiriro akashandiswa (denaturation) (Mufananidzo 4A).Muhupo hweUTP neGTP, kuyerwa kwehuwandu hwemaitiro ane nsp9 uye nsp12 zvakateerana zvakaratidza kuti protein yakawanda yeUTP yakafambisa 306 Da, uye protein mass yeGTP yakafambisa 345 Da, zvichiratidza kuti imwe neimwe nsp9 molecule inosunga UMP kana GMP. (Mufananidzo 4) C naD).Zvinofungidzirwa kuti simba rinodiwa kuNiRAN-mediated nsp9 NMPylation rinobva kuNTP hydrolysis uye pyrophosphate kuburitswa.Kunyange zvazvo 10-fold molar yakawandisa ye nsp9 (chinangwa) kupfuura nsp12 (enzyme) yakashandiswa mukuita uku, inenge yakakwana NMPylation ye nsp9 yakaonekwa, zvichiratidza kuti kuwirirana pakati pe nsp12 ne nsp9 ndekwenguva pfupi, uye nsp12 inogona NMPylate yakawanda nsp9 in vitro molecule.

Imwe NMPylation ye nsp9 pamberi pe nsp12 uye UTP kana GTP.Inoratidzwa ideconvoluted yakazara protein mass spectrum yeHCoV-229E nsp9 (SI appendix, Tafura S1) (AD).(A) nsp9 chete, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 pamberi peUTP, (D) nsp9 + nsp12-His6 pamberi peGTP.

Kuti uone izvo nsp9 zvakasara UMPylated ne nsp12, nsp9-UMP yakatsemurwa netrypsin.Mapeptide akakonzeresa akapatsanurwa nenano-yakakwirira performance liquid chromatography (HPLC) uye yakaongororwa netandem mass spectrometry (MS/MS) online.Ongororo yedata uchishandisa Byonic software package (Protein Metrics) yakaratidza UMPylation yeN-terminal amino acid.Izvi zvinosimbiswa nemaoko.Iyo tandem mass spectrum ye precursor peptide [UMP]NNEIMPGK (SI appendix, Figure S5A) yakaratidza chidimbu pa421 m/z, zvichiratidza kuti UMP inosunga kune yasara 1 yensp9.

PaN-terminus yensp9, Asn inochengetedzwa pakati penhengo dzeOrthocoronavirinae (SI appendix, Mufananidzo S6).Kunyangwe tichitenda kuti N-terminal primary amine nitrogen ndiyo inonyanya kugamuchira UMP, takasarudza kuwana humwe humbowo hweNMP inosunga paN-terminal.Nechikonzero ichi, iyo isiri-NMPylated uye NMPylated N-terminal peptide nsp9 yakacheneswa neHPLC yakatorwa pamberi peacetone uye sodium cyanoborohydride.Pasi pemamiriro ezvinhu aya, chete maamine ekutanga emahara anogona kugadziridzwa nepropyl (25).Iyo N-terminal nsp9-inotorwa peptide ine nhevedzano NNEIMPGK ine maviri ekutanga maamines, imwe paN-terminus yeAsn uye imwe iri padivi cheni yeLys paC-terminus.Nokudaro, mapoka epropyl anogona kuunzwa kumativi ose maviri.Iyo yakabviswa ion chromatograms yeasiri-NMPylated peptides inoratidzwa muSI appendix, Mufananidzo S5B.Sezvinotarisirwa, N-terminal uye C-terminal (mono) propylated (SI appendix, Figure S5B, upper lane) uye dipropylated peptides (SI appendix, Figure S5B, yakaderera lane) inogona kuonekwa.Iyi pateni inoshanduka nekushandiswa kweNMPylated N-terminal peptide ye nsp9.Muchiitiko ichi, chete C-terminal propylated peptides inogona kuonekwa, asi N-terminal propylated peptides uye dipropylated peptides hazvizivikanwi (SI Appendix, Figure S5C), zvichiratidza kuti UMP yakaendeswa kuN-terminal primary amine Kudzivirira izvi. boka kubva pakuchinja.

Tevere, tinotsiva (neAla kana Ser) kana kudzima zvakasara zvakachengetwa paN-terminus ye nsp9 kutsanangura zvinotemerwa-zvakanangana.Kubva pane yedu MS data inoratidza kuti NiRAN inoumba nsp9-NMP adduct ine yekutanga amine yeN-terminal yakasara ye nsp9, takafungidzira kuti nsp9 NMPylation inoda iyo viral master protease (Mpro, nsp5) kuburitsa iyo nsp9 N-terminal kubva. yayo polyprotein precursor.Kuti tiedze iyi hypothesis, takagadzira precursor protein nsp7-11 ine nsp9 muE. coli uye takaita chiyero cheNMPylation bvunzo pamberi pe [α-32P] UTP (materials uye nzira).Sezvinoratidzwa muFigure 5A (lane 3), iyo uncut nsp7-11 precursor haina radiolabeled ne nsp12.Kusiyana neizvi, kana nsp7-11 yakatsemurwa nerecombinant nsp5 kuburitsa nsp9 (uye imwe nsps) kubva kune inotangira, puroteni ine radiolabeled inotama ne nsp9 inowonekwa, ichisimbisa mhedzisiro yedu kuti NiRAN neN-Selective formation ye covalent nsp9-NMP adducts. .Iyo terminal primary amine yeN-terminal Asn (chinzvimbo 3825 mupp1a/pp1ab).Mhedziso iyi inotsigirwawo nemiedzo uchishandisa nsp9 construct, iyo ine imwe kana maviri mamwe masara paN-terminus.Muzviitiko zvese izvi, NiRAN-mediated UMPylation ye nsp9 yakabviswa (SI Appendix, Mufananidzo S7).Zvadaro, takagadzira puroteni ine imwe kana maviri masara eAsn akabviswa kubva ku3825-NNEIMPK-3832 peptide sequence paN-terminal ye nsp9.Muzviitiko zvese izvi, nsp9 UMPylation yakavharwa zvachose (Mufananidzo 5B), ichipa humwe humbowo hwekuti iyo chaiyo nsp9 N-terminus inoshanda seNMP receptor.

Iyo proteolytic process ye nsp9 uye basa reN-terminal residues mu nsp12-mediated UMPylation.(A) nsp9 UMPylation inoda yemahara nsp9 N-terminal.Nsp7-11-His6 isati yaiswa pa30 °C muNMPylation yekuona buffer ine UTP paine kana kusavapo kwerecombinant Mpro (nsp5-His6).Mushure memaawa matatu, tanga iyo NMPylation assay nekuwedzera nsp12-His6 sezvinotsanangurwa muZvishandiso uye Nzira.Maitiro ane nsp5-His6 (lane 1) uye nsp9-His6 (mukoto 2) yakashandiswa sekutonga.Mushure memaminitsi e10, mhinduro yacho yakaguma uye musanganiswa wekuita wakaparadzaniswa neSDS-PAGE.Protein yakasvibiswa neCoomassie Brilliant Blue (A, pamusoro).Iyo Nsp7-11-His6 precursor uye chigadzirwa chakagadziriswa chinobva ku nsp5-His6 mediated cleavage inoratidzwa kurudyi.Ndapota cherechedza (nekuda kwehukuru hwavo hudiki) kuti nsp7 uye nsp11-His6 haioneki mune iyi gel, uye maitiro anowedzerwa ne nsp5-His6 (nzira 1 uye 4; nzvimbo ye nsp5-His6 inoratidzwa nedenderedzwa rakasimba) kana nsp9-His6 (Lane 2) ine shoma shoma yeMBP (inoratidzwa nematenderedzwa akazaruka) semarara akasara nokuti anoratidzwa seMPP fusion mapuroteni (SI appendix, Tafura S1).(B) Musiyano weNsp9-His6 unoshaya imwe kana maviri N-terminal Asn zvisaririra (zvisaririra zviverengero zvinoenderana nenzvimbo iri mupp1a/pp1ab) uye inocheneswa nekuiswa nsp12-His6 uye [α-32P] UTP.B, SDS-PEJI yakasvibiswa neCoomassie inoratidzwa kumusoro, B, iyo inoenderana otoradiograph inoratidzwa pazasi.Nzvimbo ye molecular weight marker (mu kilodaltons) inoratidzwa kuruboshwe.(C) HCoV-229E nsp9-His6 N-terminal yakachengetedzwa zvakasara zvakatsiviwa neAla kana Ser, uye huwandu hwakafanana hweprotein hwakashandiswa mu nsp12-His6 mediated UMPylation reaction.Zvigadzirwa zvemaitiro zvakaparadzaniswa neSDS-PEJI uye yakasvibiswa neCoomassie Brilliant Blue (C, pamusoro), uye radiolabeled nsp9-His6 yakaonekwa ne phosphorescence imaging (C, pakati).Uchishandisa mhuka yemusango (wt) protein sereferensi (yakaiswa ku100%), iyo yakabatana NMPylation chiitiko (kureva ± SEM) yakaverengerwa kubva kune matatu akazvimiririra kuedza.(D) Virus titers mu p1 cell tsika supernatant yeHuh-7 masero akatapukirwa neHCoV-229E wild-type Huh-7 masero, uye mutants akatakura akatemerwa amino acid inotsiva mu nsp9 akatemerwa plaque assay.Iyo yekudzokorora-yakashomeka RdRp motif C kaviri mutant DD4823/4AA yakashandiswa seyakaipa kutonga.

Iyo N-terminus yensp9 (kunyanya zvinzvimbo 1, 2, 3, uye 6) inochengetedzwa zvakanyanya pakati penhengo dzeOrthocoronavirinae subfamily (SI appendix, Figure S6).Kuti udzidze basa rinobvira rezvisaririra izvi mu nsp12-mediated nsp9 NMPylation, maviri akateedzana masara eAsn paN-terminus ye nsp9 akatsiviwa neAla kana Ser (yega kana musanganiswa).Kuenzaniswa nemhando-yemusango nsp9, kutsiva N3825 neAla kana Ser yakakonzera kuderera kunopfuura kaviri mu nsp12-mediated UMPylation (Mufananidzo 5C).Zvichienderana nemhedziso yedu yekuti NMPylation inoitika paN-terminal primary amine pachinzvimbo chengetani yeparutivi yeN-terminal yasara, takaona yakakosha NMPylation nekutsiviwa kweN3825A neN3825S.Sezvineiwo, kana yechipiri Asn inotsiviwa neAla kana Ser, nsp9 UMPylation yakaderedzwa zvakanyanya (kupfuura 10 nguva), nepo kutsiviwa kweAla pazvinzvimbo 3, 4, uye 6 kunongova nepakati pekuita pa nsp9 UMPylation (Mufananidzo 2). ) .5C).Migumisiro yakafanana yakawanikwa uchishandisa ATP, CTP kana GTP (SI appendix, Figure S8).Pamwe chete, idzi data dzinoratidza basa rakakosha reN2826 (nzvimbo 2 mu nsp9) mu nsp9 NMPylation.

Kuti tiwane humwe humbowo hwekubatana kwekushanda pakati peN-terminus yensp9 neNMPylation, takaita kutevedzana kwakawanda (MSA) kwensp9 kutevedzana kwemhuri yeCoronavirus (yakasiyana pakati pe104 ne113 zvakasara) (SI Appendix, Mufananidzo. S6).Pakazara, mumhando makumi mana nenomwe (dzinozivikanwa uye dzinoisa) emarudzi mashanu emhuri yeOrthocoronavirinae inotapurira mhuka dzakasiyana, shiri, nezvinokambaira, zvisere chete zvasara zvakawanikwa zvisingachinji.Shanduko dzakakura kwazvo, kusanganisira kudzima uye kuiswa, dzakaonekwa mumatenderedzwa pakati pechipiri chimiro cheNsp9, sezvakatsanangurwa neyakapfuura zvimiro zvidzidzo (26 ?? 28).Zvishanu zvisingagumi zvakasara zvakawanikwa mu β strand uye α helix yeC-terminal chikamu che nsp9.Zvisaririra zvitatu zvisingachinji zvinoumba NNE motif yeN terminus ye nsp9.Zvinoratidzwa kuti yechipiri Asn yeiyi motif ndiyo yega yakasara yakasara, iyo zvakare yakagovaniswa neiyo hypothetical nsp9 yeiyo iri kure yakabatana frog coronavirus, uye inomiririra Microhyla letovirus 1 mhando mune subfamily Letovirinae yeAlphaletovirus.Kuchengetedzwa kwezvisaririra mu nsp9 chechipiri chimiro chezvimiro zvinogona kugadziridzwa nekufunga kwechimiro kuchengetedza kupeta kana inozivikanwa RNA inosunga zvivakwa.Nekudaro, kufunga uku hakuite sekunge kunoshanda pakuchengetedza kweNNE, uye chidzidzo ichi chisati chaitwa, chimiro chezvipingamupinyi zvinomisa kusiyanisa kwekutevedzana kwetripeptide zvakavharika zvachose.

Kuti uone kukosha kwensp9-NMPylation uye kuchengetedza kweNNE mukudzokororwa kwekoronavirus, takagadzira HCoV-229E mutants, iyo inotakura imwe kana kaviri inotsiva ye nsp9 N-terminal masara, zvichiratidza kuti nsp9 NMPylation inokuvadza in vitro.Tisati tatanga, tinoedza kupindura mubvunzo kuti izvi zvinotsiva (padyo nensp8|9 cleavage site) zvinokanganisa kugadzirwa kweproteolytic yedunhu reC-terminal pp1a.Seti ye nsp7-11 polyprotein inovaka ine inopindirana inotsiva paN-terminus ye nsp9 yakagadzirwa muE. coli uye yakachekwa nerecombinant Mpro.Iyo proteolytic cleavage yemasaiti ina (kusanganisira iyo nsp9 flanking saiti) haina kukanganiswa zvakanyanya nechero yakaunzwa inotsiva (SI appendix, Figure S9), isingabatanidzi shanduko yezvimiro mumapuroteni aya anokanganisa Mpro-mediated nsp8|9 cleavage (Kana zvimwe) website.

Huh-7 maseru akachinjirwa negenome-urefu HCoV-229E RNA, encoding Ala kana Ser inotsiva mune yakachengetedzwa NNE tripeptides (N3825, N3826, uye E3827) pa nsp9 N terminus, zvichiratidza kuti mazhinji ekuchinja anouraya.Isu takakwanisa kununura hutachiona nekutsiva Ser kana Ala yeN-terminal Asn (N2835A kana N2835S), asi takatadza kudzoreredza hutachiona nehumwe humwe uye kaviri shanduko muNNE sequence (N3826A, N3826S, NN3825 / 6AA, NN3825/6SS) , E3827A) (Mufananidzo 5D).

Mhedzisiro iyi inoratidza kuti kudzokororwa kwemakoronavirus mutsika yematishu inorambidzwa (yakafanana kana yakafanana), ichidzikamisa shanduko yechisikigo yensp9 NMPylation nzvimbo mumuviri, uye kutsigira basa rakakosha remhinduro iyi muhupenyu hwekoronaviruses.

Muchikamu chekupedzisira chekuedza, takagadzira C-terminal His6 yakanyorwa kuti SARS-CoV-2 nsp12 uye nsp9, uye maviri anochinja maitiro e nsp12 muE. coli.Iyo inoshanda saiti zvakasara munzvimbo dzeNiRAN neRdRp zvaive zvakateerana Shandisa Ala pachinzvimbo (Mufananidzo 6A uye SI appendix, Tafura S2).K4465 muSARS-CoV-2 nsp12 inoenderana neK4135 muHCoV-229E (SI Appendix, Figure S2), iyo yakaratidza kuve inodiwa pakuita kweNiRAN uye HCoV-229E kudzokorora (Mufananidzo 3D uye E).Izvi zvakasara zvinoenderanawo neiyo arterial virus EAV nsp9 K94 yakasara, iyo yakamboratidzwa kuti yakakosha kuNiRAN self-UMPylation/self-GMPylation (16).Sezvinoratidzwa mumufananidzo 6B, SARS-CoV-2 nsp12 ine UMP yekufambisa chiitiko uchishandisa nsp9 se substrate, nepo nsp12_K4465A inoshanda saiti mutant isingaite.Iko kutsiva kaviri muSDD hunhu kutevedzana kweRdRp motif C haikanganisi UMP transferase chiitiko (Figure 6B), zvichiratidza kuti RdRp chiitiko hachina mhedzisiro yakananga mu nsp9 UMPylation.Data yakafanana yakawanikwa uchishandisa CTP, GTP uye ATP (SI appendix, Figure S10).Muchidimbu, data iri rinoratidza kuti NiRAN-mediated nsp9 NMPylation ine chiitiko chekuchengetedza mumakoronavirus anomiririra genera rakasiyana reorthocoronavirus subfamily.

SARS-CoV-2 nsp12-mediated NMPylation ye nsp9.(A) Coomassie yakasvibiswa SDS-polyacrylamide gel inoratidza recombinant protein inoshandiswa muNMPylation test.Sekutonga, puroteni inoshanduka ine inoshanda saiti inotsiva muNiRAN domain (K4465A) uye RdRp domain (DD5152/3AA) yeSARS-CoV-2 nsp12 yakashandiswa.Kuverengera kwasara kunoenderana nechinzvimbo mupp1ab.(B) Autoradiograph yeUMPylation yekuongorora uchishandisa nsp9-His6 uye [α-32P] UTP se substrate ye nsp12-His6 (wild type [wt] uye mutant).Iyo molecular mass (mu kilodaltons) yeprotein yakanyorwa inoratidzwa kuruboshwe.

Nzvimbo dzeNiRAN dzinowanzo chengetedzwa muNidovirales (16), zvichiratidza kuti zvinokonzeresa maitiro enzymatic akakosha pakudzokorora kweNidovirus.Muchidzidzo ichi, takakwanisa kuratidza kuti iyo NiRAN domain yekoronavirus inotamisa NMP (yakagadzirwa kubva kuNTP) kuenda kunsp9, isinganzwisisike RNA inosunga protein inobatanidzwa mukudzokorodza kwehutachiona (26 ?? 29), kuti ione sechisikigo chinangwa shamwari ye coronavirus RTC.

Iyo NiRAN domain inogovera matatu sequence motifs (AN, BN, uye CN), iyo ine nhamba shoma kwazvo yezvisaririra izvo zvakachengetwa mumhuri dzese mune monophyletic asi yakanyanya kusiyanisa Nidovirales order (8, 16).Zvidzidzo zvenguva pfupi yapfuura zvakaratidza kuti zvine hukama kumhuri isina kurongeka yeprotein kinase-senge mapuroteni, ayo akambonzi mhuri yeSelO (17, 19, 22, 30, 31).Mapuroteni ane hukama neSelO ane kinase folds, asi anoshaya akati wandei akachengetedzwa saiti masara mune classical kinases (22, 32).Zvichienderana nereverse orientation yeATP mamorekuru akasungirirwa kunzvimbo inoshanda uye yakagadziriswa nekudyidzana kwakananga, SelO yakafungidzirwa uye yakazosimbiswa kuendesa AMP (panzvimbo yephosphate) kune protein substrate (22), nepo imwe bhakitiriya SelO-yakafanana neprotein YdiU ine. nguva pfupi yadarika yakaratidzwa kukonzeresa kubatanidzwa kweUMP kuTyr uye masara ake emaprotein substrates akasiyana (33).

Kuti tisimbise uye tiwedzere kufanotaura kweiyo putative inoshanda saiti masara eiyo coronavirus NiRAN domain, takashandisa biochemical uye reverse genetics nzira kuita shanduko yekuongorora pane coronavirus nsp12 (Mufananidzo 3D uye E uye SI appendix, Mufananidzo S3 uye tafura) S1â S4).Iyo data inoratidza kuti kutsiva kweHCoV-229E K4135, R4178 uye D4280 neAla kunobvisa mu vitro NMP kutamisa chiitiko uye kudzokorora hutachiona musero tsika (Mufananidzo 3D uye E uye SI appendices, Mufananidzo S3), inotsigira kuvepo kwavo muNTP γ-phosphate. (K4135, R4178) uye kurongeka kweiyo inoshanda saiti simbi ions (D4280).Iyo E4145A inotsiva Glu yakachengetedzwa muhuwandu hwehutachiwana hweshiri yakafanotaurwa kudzikamisa nzvimbo yeK4135 (17) yakaratidzwa kubvisa kudzokororwa kwehutachiona, asi zvinoshamisa, chiitiko chakachengetwa mu in vitro NMPylation assay (Mufananidzo 3D uye E uye SI appendix, Mufananidzo S3 Uye Matafura S1–S4).Kucherechedzwa kwakafanana kwakaitwa apo iyo inomiririra inotsiva yakaunzwa muYdiU homolog yeSalmonella typhimurium (E130A) (33).Zvakatorwa pamwechete, idzi data dzinotsigira basa rekutonga reiyi yakachengetedzwa yakasara kwete iyo inokonzeresa basa.

Kutsiva iyo yakachengetedzwa Phe residue (F4281A) mukati meiyo nestovirus muHCoV-229E NiRAN domain (8) yakakonzera kudzikira kweNMPylation chiitiko mu vitro uye kuderera kukuru kwehutachiona kudzokororwa musero tsika (Mufananidzo 3D, E uye SI) appendikisi, Mufananidzo S3).Iyo data inoenderana nebasa rakakosha rekutonga reiyi yasara, senge homologous DFG motif Phe zvakasara zvakaratidzwa kare.Mune classical protein kinases, iyo chikamu cheMg2 + inosunga loop uye inobatsira kuunganidza uye kugadzirisa musana???Inodiwa kuti iite basa rekuita rinoshanda (32, 34).Kutsiva Ala neArg yeK4116 zvisaririra (mune preAN motif), zvichiteerana, yakabvisa kudzokororwa kwehutachiona uye, sezvaitarisirwa, yakave nemhedzisiro yakasiyana paNMP transferase chiitiko mu vitro, zvichienderana neamino acid parutivi cheni yakaunzwa (Mufananidzo 3D Uye E uye SI appendices. , Mufananidzo S3).Iyo data inoshanda inopindirana neruzivo rwekuumbwa, zvichiratidza kuti iyi yakasara yakagadzira kudyidzana neATP phosphate (17).Munzvimbo yeNiRAN yedzimwe mhuri dzine hutachiona, nzvimbo yeHCoV-229E pp1a/pp1ab K4116 inogarwa naLys, Arg kana Yake (8), zvichiratidza kuti kurambidzwa kushanda kweiyi zvakasara kwadzoreredzwa.Kutsiviwa kweD4188A uye D4283A kunobvisa kana kuderedza zvakasimba basa re enzyme uye kubvisa kudzokorora kwehutachiona (Mufananidzo 3).Zvisaririra zviviri izvi zvinochengetedzwa muzhinji (asi kwete ese) mavhairasi akaiswa mudendere (8), zvichiratidza basa rakakosha remhuri asi ringangove risingaite.Kutsiva kweAla kwemamwe akati wandei maLys neAsp asara (K4113A, D4180A, D4197A uye D4273A) asina kuchengetedzwa muCoronaviridae kana dzimwe mhuri dzeNestioviridae (8) dzakashandiswa sekutonga.Sezvinotarisirwa, izvi zvinotsiviwa zvakanyanya kushivirira, nekuderera kudiki kwekuita kwe enzyme uye kudzokorora kwehutachiona mune dzimwe nguva (Mufananidzo 3 uye SI appendix, Mufananidzo S3).Pakazere, iyo coronavirus mutagenesis data inopindirana neiyo yega-GMP uye inodzosera genetics data yeEAV NiRAN-RdRp (16), umo EAV nsp9 (coronavirus nsp12 ortholog) yakasara K94 (inoenderana neHCoV- 229E K4135) mabasa akakosha), R124 (inoenderana neR4178), D132 (inoenderana neD4188), D165 (inoenderana neD4280), F166 (inoenderana neF4281).Uye zvakare, iyo HCoV-229E mutagenesis data inowirirana uye yakawedzerwa kubva kune yakambotaurwa SARS-CoV reverse genetics data (16), yakafanana chaizvo neiyo inocherechedzwa inoenderana CN motif Phe-to-Ala mutant SARS-CoV_nsp12 phenotype inotsanangurwa -F219A uye HCoV-229E_F4281A (Mufananidzo 3 D uye E uye SI appendix, Mufananidzo S3 uye Tafura S1-S4).

Kuenzaniswa neEAV orthologs (16), ine yakajeka yakasarudzika yeUTP neGTP (mune-self-NMPylation reaction), kudzidza kwedu kunoratidza kuti iyo coronavirus NiRAN domain (inomiririrwa neHCoV-229E uye SARS-CoV-2) inogona kunyatsoshanda. akatamisa Ese mana maNMP, kunyangwe paine kakuda kweUMP (Figure 1 ne3).Iyo yakaderera yakaderera yeiyo chaiyo NTP co-substrate inopindirana neichangobva kutaurwa SARS-CoV-2 nsp7/8/12/13 supercomposite chimiro, umo ADP-Mg2+ inosungira kune inoshanda saiti yeNiRAN, asi kwete neadenine Chikamu. yekuumbwa kwekubatana chaiko (17).Muchidzidzo chedu, rudzi rwe nucleotide inoshandiswa muNMPylation reaction haina mutsauko pane basa remutant protein (SI Appendix, Figure S3), zvichiratidza kuti hapana chimwe chezvisara izvi chakanyatsobatana nekusungwa kweiyo nucleobase chaiyo.Hwaro hwemagadzirirwo uye hunogona kuita biological kukosha kweakasiyana NTP co-substrate zvido zvinoonekwa munzvimbo dzeNiRAN dzekoronavirus uye arterial virus zvinoramba zvichidzidzwa;dzingave dzechokwadi kana kuti dzinogona kunge dziri nekuda kwekuganhurirwa kwezvidzidzo zvavo.Parizvino, hazvigone kutongerwa kunze kuti ingangoita NMPylator chiitiko cheiyo arterial virus NiRAN domain (ichienzaniswa neyaimbozivikanwa yekuzvimiririra-NMPylation chiitiko) ine akasiyana co-substrate yaunofarira, tichifunga kuti kufanana pakati peiyo arterial uye coronavirus. NiRAN domain iri pamuganho wayo.Sequence-based Enzanisa (16).Kuenzaniswa nepseudokinase SelO, inoshandisa Mg2 + se cofactor, basa rekoronavirus uye arterial virus NiRAN inotsamira paMn2 + (16) (Mufananidzo 3C uye SI appendix, Mufananidzo S1).Kutsamira kweMn2 + uye kudiwa kuri pachena kweUTP chinhu chisina kujairika cheprotein NMPylators, uye ichangobva kusimbiswa muYdiU protein yeSalmonella typhimurium, iyo inokonzeresa iyo yakasimba Mn2 + -inotsamira protein chaperone UMPylation kuchengetedza masero kubva mukushushikana induction Cell ATP dziva. 33).

Iyo ichangobva kutsanangurwa kufanana kwechimiro pakati peiyo coronavirus NiRAN domain uye cellular protein kinases (17, 19) inopa imwe tsigiro yekugona kweNiRAN kubatanidza NMP kune mamwe mapuroteni atakataura muchidzidzo ichi.Takatarisana nekutsvaga kwedu kwezvinobvira NiRAN tarisiro pamapuroteni akavharidzirwa neHCoV-229E ORF1a, anozivikanwa kubatsira zvakananga kana zvisina kunanga RTC's ORF1b-encoded replicase (12, 35).Zviyedzo zvedu zvinopa humbowo hwakakwana hweiyo inoshanda uye yakananga NMPylation ye nsp9 (Mufananidzo 2).Kana chinangwa cheprotein chikashandiswa mune imwe molar yakawandisa iyo 8 kusvika ku10 nguva yakakwirira kudarika iyo enzyme (nsp12), inosimbiswa kuti nsp9 yakakwana (mono) NMPized (Mufananidzo 4).Takagumisa kuti kuwirirana pakati pe nsp12 ne nsp9 inguva pfupi uye haizogadziri yakagadzikana yakaoma ne nsp9 (pasipo mamwe maRTC subunits).Mhedziso iyi inotsigirwa neprotein yekudyidzana zvidzidzo paSARS-CoV proteome (35).Ongororo yeMS yakaratidza iyo yekutanga amine yeN-terminal yakasara yensp9 seNMPylation saiti (SI appendix, Mufananidzo S5).Kuumbwa kwephosphoramidate bond uye N-terminal amino boka rinosiyanisa basa reNiRAN-mediated NMPylation kubva kuPseudomonas syringae SelO-mediated AMPylation reaction, iyo inokonzera kuumbwa kweO-linked AMP kuSer, Thr, kana Tyr zvakasara Peptide adduct ( 22), uye S. typhimurium YdiU inoumba O-yakabatana (neTyr) uye N-yakabatana (neYake) peptide-UMP adducts.Ruzivo rushoma rwunowanikwa paSelO mhuri yemapuroteni inoratidza kuti nhengo dzeiyi yakakura mapuroteni mhuri inosiyana zvakanyanya mukuumbwa kwepeptide-NMP adducts.Ichi chicherechedzo chinonakidza chinokodzera kumwe kufunda.

Iyo data yakawanikwa muchidzidzo ichi yakaita kuti tifunge kuti iyo NMPylation ye nsp9 inoda yemahara N-terminus.Muchirevo chekudzokorora kwehutachiona, izvi zvichapihwa neproteolytic cleavage ye nsp8|nsp9 yekugadzirisa saiti mune replicase polyprotein pp1a inopindirana neMpro uye pp1ab.Mumakoronavirus mazhinji, mutsauko uripo pakati peiyi saiti chaiyo (VKLQ|NNEI muHCoV-229E) uye mamwe ese maconavirus Mpro cleavage saiti iAsn (panzvimbo peimwe diki yasara, senge Ala, Ser Kana Gly) inogara P1â???Nzvimbo (36).Iyo peptide cleavage data yakawanikwa muzvidzidzo zvekutanga yakaratidza kuti cleavage inobudirira ye nsp8|nsp9 saiti yaive yakaderera pane yemamwe masayiti, zvichiratidza kuti 1) iyi saiti chaiyo inogona kuve nebasa rekutonga mukurongeka kwakarongeka kweC-terminal. pp1a dunhu, kana 2) a Basa reiyo yakakosha kuchengetedzwa nsp9 N-terminus mukudzokororwa kwehutachiona (37).Yedu data (Mufananidzo 5A) yakaratidza kuti iyo recombinant fomu ye nsp9 inotakura iyo chaiyo N-terminal sequence yakabudirira NMPized ne nsp12.Iyo N-terminal flanking sequence yakabviswa ne factor Xa (nsp9-His6; SI appendix, Tafura S1) kana Mpro-mediated cleavage (nsp7-11-His6; Mufananidzo 5A uye SI appendix, Tafura S1).Zvakakosha, iyo isina kuchekwa nsp9-ine precursor nsp7-11-His6 yakaratidza kuramba NMPylation ye nsp12, inowirirana nedata redu, zvichiratidza kuti nsp9-NMP adduct inoumbwa kuburikidza neN-terminal primary amine (SI appendix, Figure S5) .Kuti tiwane kunzwisisa kwakadzama kweNiRAN substrate chaiyo, isu takabva tatarisa kune iri padyo neN-terminal masara e nsp9.Mukushaikwa kwemamwe mapuroteni, anoshanduka-shanduka, achivadzivirira kuti vasaonekwe muchimiro chisina kunyorwa che nsp9 (26 28, 38), zvichiratidza kusiyana kwavo kwechisikigo kushoma Izvi zvinokonzerwa nekutevedzana kwakakosha-chaiyo (kwete chechipiri chimiro chine chekuita) basa re nsp9 N-terminal fragment.Ala kutsiva kwezvakachengetwa zvakasara munzvimbo ino (Figure 5C uye D uye SI appendix, Mufananidzo S8) inoratidza kuti N3826 yakakosha kune nsp9 NMPylation in vitro, nepo N3825A uye E3827A inotsiva inotungamira mukudzikira kweNMPylation, nepo M3829A uye P38 inotsiva .Zviripachena zvinokanganisa nsp9 NMPylation.Kunyangwe kutsiva kweN-terminal Asn (N3825A, N3825S) kunongova nemhedzisiro ine mwero pa nsp9 NMPylation uye kudzokororwa kwehutachiona mu cell tsika (Mufananidzo 5C uye D), kudzima kutevedzana kweAsn kubva kuN-terminal 3825-NN dipeptide. yakaratidza kuva Inouraya kune mavhairasi, zvichiratidza kuti imwe yeAsn inosara inodiwa pamberi peimwe yakasara paN-terminus, zvichida Asn, kunyange zvazvo zvichiita sekuti kutsiva kwezvakasara zvakafanana kunogona kushivirirwa zvishoma (Mufananidzo 5B, C, uye D).Isu tinogumisa kuti iyo 3825-NN dipeptide, kunyanya yakachengetedzwa uye yakakosha N3826 yakasara mukati meiyo coronavirus renji (SI appendix, Figure S6), inovimbisa kusungirirwa kwakaringana uye kutaridzika kwensp9 N-terminus munzvimbo inoshanda yeNiRAN.

Kutsiva Ala (E3827A) yeGlu yakachengetwa yeese madiki mhuri inochengeta nsp9 NMPylation in vitro asi inouraya mavhairasi musero tsika (Mufananidzo 5C uye D), zvichiratidza basa rekuwedzera reiyi inosara, semuenzaniso, mukudyidzana kwakakosha (NMPylated kana isina kuchinjwa. ) nsp9 N-terminus nezvimwe zvinhu zvinobatanidzwa mukudzokorora kwehutachiona.Nsp9 mutations haina kukanganisa proteolytic process ye nsp9 kana chero yakatarisana nsps (39) (SI Appendix, Figure S9), zvichiratidza kuti inouraya phenotypes akati wandei nsp9 mutations akaonekwa haana kukonzerwa nekusagadzikana kweC proteolytic process-terminal pp1a nzvimbo. .

Iyo data iri pamusoro apa inopa humbowo hwekuti mushure meMpro-mediated kurapwa kwensp8|9 cleavage saiti mupp1a/pp1ab, iyo N-terminus yensp9 inogona kuve UMPylated (kana kugadziridzwa zvishoma neimwe NMP).Pamusoro pezvo, kuchengetedzwa kwakanaka kweN-terminus yensp9 (kusanganisira imwe uye isingachinjiki masara eAsn mumhuri yekoronavirus) uye reverse genetics data yakawanikwa muchidzidzo ichi (Figure 3E ne5D) yatitungamira kuti tipedzise kuti iyo yakatsanangurwa nsp9 NMPylation. ine hukama nebiological uye yakakosha pakudzokorora kwekoronavirus.Migumisiro inoshanda yeiyi shanduko inoramba ichidzidzwa, semuenzaniso, maererano neyakatsanangurwa kare (isina-yakananga) nsp9 (isina kugadziridzwa fomu) RNA inosunga chiitiko (2628).N-terminal NMPylation inogonawo kukanganisa kupindirana kwensp9 neprotein kana RNA substrates kana kuumbwa kwemagungano akasiyana-siyana.Izvi zvakaonekwa muzvidzidzo zvezvimiro uye zvakasimbiswa kuve zvinoshanda zvine hukama nekoronavirus kudzokororwa, kunyangwe kunyanya pakushaikwa kweMunyaya yekushandurwa uku (26- ââ29, 40).

Kunyangwe iyo yakanangana neiyo coronavirus NiRAN domain ichiri kuda kuratidzwa zvakadzama, data redu rinoratidza kuti puroteni yakanangana neiyo coronavirus NiRAN domain yakatetepa.Kunyange zvazvo kuchengetedzwa kwezvinyorwa zvakakosha zvesaiti zvakasara (8, 16) munharaunda yeNiRAN yemhuri dzose dze nidovirus inotsigira zvakasimba basa reNMPylator yakachengetedzwa mapuroteni aya, kuzivikanwa kweiyo substrate inosunga zvimedu zvehomwe yedunhu iri Kuchengetedza uye kuchengetedza zvinoramba zvichionekwa. , uye inogona kusiyana pakati pemhuri dzakasiyana dzeNidovirales zvinangwa.Saizvozvo, zvakanangwa zvemamwe mavhairasi akaiswa nested hazvisati zvatemwa.Anogona kunge ari kure orthologs e nsp9 kana mamwe mapuroteni, nekuti iwo anoteedzana ari kunze kwemashanu ekudzokorora madomasi ayo anowanzo chengetedzwa mumavhairasi akavharirwa haana kuchengetedzwa zvishoma (8), kusanganisira genome array pakati peMpro neNiRAN, Pakati pavo, nsp9 iri munzvimbo. coronavirus.

Pamusoro pezvo, isu hatigone kutonga kunze kwekugona kuti iyo NiRAN domain ine zvimwe (kusanganisira cellular) zvinangwa.Muchiitiko ichi, zvakakodzera kutaura kuti mabhakitiriya homologues muprotein iri kubuda NMPylators (NMPylators) (30, 31) inoita seine "master regulators"?NMP inogadzirisa akasiyana-siyana mapuroteni emasero kudzora kana kubvisa mabasa avo epasi, nekudaro achitamba chikamu mumhando dzakasiyana dzezvipenyu, senge cellular stress mhinduro uye redox homeostasis (22, 33).

Muchidzidzo ichi (Mifananidzo 2 uye 4 uye SI Appendix, Figures S3 uye S5), takakwanisa kuratidza kuti nsp12 yakaendesa UMP (NMP) chikamu kune imwe chete (yakachengetwa) nzvimbo mu nsp9, nepo mamwe mapuroteni asina kuchinjwa mu inoshandiswa Pasi pemamiriro ezvinhu, yakanyatsotsanangurwa (pane kusununguka) substrate chaiyo inotsigirwa.Zvichienderana neizvi, zvichienzaniswa neN-terminal nsp9 NMPylation, nsp12's pachake NMPylation basa rakaderera zvikuru, kuonekwa kwayo kunoda nguva yakareba yeautoradiography kuratidzwa, uye kuwedzera kwe10-fold mu nsp12 concentration inoshandiswa.Pamusoro pezvo, kuongorora kwedu kweMS kwakatadza kupa humbowo hweNMPylation yensp12, iyo inoratidza kuti NiRAN domain self-NMPylation ndeye (papamusoro) chiitiko chechipiri.Nekudaro, zvinofanirwa kucherechedzwa kuti zvimwe zvidzidzo zvakapa humbowo hwekutanga kuti iyo yega-AMPylation mamiriro ebhakitiriya NMPylator inogona kudzora chiitiko chavo cheNMPylation pane mamwe mapuroteni substrates (22, 33).Naizvozvo, kumwe kutsvagurudza kunodiwa kuti uongorore zvinogona kuita basa rekuita-yega-NMPylation zviitiko zvakashumwa zveEAV nsp9 (16) uye coronavirus nsp12 (chidzidzo ichi), kusanganisira yakadero chaperone-semhedzisiro pakupetwa kweC-terminal RdRp domain ( 16)).

Pakutanga, fungidziro dzakati wandei maererano neanogona kudzika mabasa eiyo nidoviral NiRAN domain akatariswa, kusanganisira RNA ligase, RNA -capped guanylate transferase uye protein priming chiitiko (16), asi hapana kana chimwe chazvo chinowirirana neanowanikwa pasi pemvura mabasa.Ruzivo rwakawanikwa munzvimbo dzinotevera rwakafanana panguva imwechete pasina kuita mamwe fungidziro.Iyo data yakawanikwa muchidzidzo ichi inonyanya kuenderana (asi haigone kuratidza) kuti NiRAN domain inobatanidzwa mukutanga kweprotein-induced RNA synthesis.Zvaimbotendwa kuti basa reNiRAN domain mu5??²-RNA capping kana RNA ligation reactions haina kukanganiswa neizvi uye Tsigiro yeimwe data.Naizvozvo, semuenzaniso, iyo inoshanda saiti yeNiRAN inoonekwa seine yakachengetwa Asp seyakajairika base (D252 muPseudomonas syringae SelO; D4271 muHCoV-229E pp1ab; D208 muSARS-CoV-2 nsp12) (SI Appendix, mufananidzo 2 )S2) (17, 22, 33), nepo catalysis muATP-inotsamira RNA ligase uye RNA capping enzyme inoitwa neiyo covalent enzyme-(lysyl-N)-NMP yepakati, iyo inosanganisira isina- Yakashandurwa Lys yakasara. 41).Uye zvakare, iyo inoshamisa inoteedzana-yakavakirwa chaiyoiyo yekoronavirus NiRAN yeakachengetwa mapuroteni tarisiro uye iyo yakasununguka chaiyo yeNTP co-substrates (inofarira UTP) inopikisa NiRAN-mediated capping enzyme kana RNA ligase-senge mabasa.

Zviripachena, basa rakawedzerwa rakawanda rinodiwa kuratidza uye, kana zvichiratidzwa, kutsanangura pamusoro pebasa rinobvira rensp9-UMP (nsp9-NMP) muprotein-induced RNA synthesis, iyo ichabatanidza akati wandei anonakidza asi (kusvika parizvino) mishumo yakambotaurwa. .Kucherekedza kwakazvimirira.Semuenzaniso, zvakatemwa kuti kupera kweiyo negative-strand RNA yekoronavirus inotanga neoligo (U) tambo (42, 43).Kucherekedza uku kunopindirana nepfungwa yekuti kuumbwa kwe-negative-strand RNA kunotanga nekusunga iyo UMPylated fomu ye nsp9 kune poly(A) muswe (zvinokonzeresa), izvo zvinogona kusimudzirwa neiyo RNA inosunga Chiitiko uye/kana kudyidzana nayo. imwe RTC protein.Iyo UMP chikamu chakapihwa nensp9 chinogona kushandiswa se "primer" ye nsp7/8/nsp12-mediated oligouridylation, uchishandisa iyo 3??²-poly(A) muswe mune genomic RNA kana Imwe oligo (A) -ine kutevedzana. inoshanda se template, yakafanana neyo nzira yakagadzirirwa picornavirus VPg protein (44).Ko kana chikumbiro "chisiri chetsika"????Kutanga kwe (protein-induced) negative-strand RNA synthesis inopa chinongedzo kune zvakaonekwa, zvichiratidza kuti iyo coronavirus negative-strand RNA ine UMP (panzvimbo yeUTP) pakupera kwayo (42), iyo inofungidzirwa kuti inoratidza kuti nucleic acid Dicer inotsemura magumo phosphorylated neisingazivikanwe uridine-chaiyo endonuclease.Kana yakasimbiswa, iyi nucleic acid hydrolytic chiitiko inogona kubatsira kuburitsa iyo oligomeric UMPylated fomu ye nsp9 kubva ku5 ² kumagumo kweiyo nascent negative strand.Basa rinogoneka re nsp9 muprotein priming zvakare rinopindirana neyakapfuura reverse genetics zvidzidzo, izvo zvakaratidza kuti nsp9 (uye nsp8) inodyidzana zvine mutsindo uye zvakanyanya neyakachengeterwa cis-inobata RNA chinhu padyo nemagumo matatu eiyo coronavirus genome.45).Maererano neshumo iri, izvi zvakamboonekwa zvino zvinogona kuongororwa zvakare nekuwedzerwa kuburikidza nekumwe tsvagiridzo.

Muchidimbu, data redu rakasarudza chiitiko cheiyo proprietary nested virus enzyme tag yakabatana neRdRp paN-terminus.Mukoronavirus, iyi ichangobva kuwanikwa yeNiRAN-mediated UMPylator/NMPylator chiitiko chinoshandiswa kuvimba neMn2+ uye padyo neAsn zvisaririra uye kukonzera kuumbwa kwe (pasi-simba) phosphoramidate zvisungo neN-terminal primary amine.Kuburikidza neMpro-mediated cleavage pa nsp8|9 cleavage saiti, iyo nsp9 tarisiro inogona kushandiswa kuNMPylation, ichiratidza kubatana kunoshanda pakati peprotease neiyo NiRAN domain, inoenda kuRdRp.Kuchengetedzwa kwemasara akakosha munsp12 NiRAN inoshanda saiti uye nsp9 chinangwa, chakasanganiswa nedata rakatorwa kubva kumakona maviri anosanganisira SARS-CoV-2, inopa humbowo hwakasimba hwekuti nsp9 NMPylation iconavirus Conservative maficha zvakare idanho rakakosha mukudzokorodza hutachiona.Iyo data iripo inotitungamira kugumisa kuti iro chairo basa reNMPylated fomu ye nsp9 muprotein-induced RNA synthesis inyaya inonzwisisika yekoronavirus uye mamwe mavhairasi akavharirwa, uye NiRAN inogonawo kunanga mamwe mapuroteni asingazivikanwe.Dzora hutachiona.Kudyidzana kwevaenzi.Kana ikasimbiswa, kubatanidzwa kweprotein primers muviral RNA synthesis kuchawedzera kutevedzana kweMpro/3CLpro neRdRp madomasi pakati peyakamboonekwa coronavirus uye picornavirus-senge supergroup (9), iyo ikozvino yakabatanidzwa muPisonivirites ichangoburwa. 46) muchikwata.

Yedu data inoratidzawo kuti zvakakosha, zvekusarudza uye zvekuchengetedza enzyme zviitiko zvakaonekwa muchidzidzo ichi zvinogona kushandiswa sezvinangwa zvemishonga inorwisa mabhakitiriya.Makomisheni anovhiringa kusungirirwa (uye kunotevera kugadziridzwa) kweiyo yakachengetedzwa nsp9 N-terminus munzvimbo inoshanda yeNiRAN inogona kuvandudzwa kuita inoshanda uye inoshanda zvakasiyana-siyana antiviral mishonga, yakakodzera kurapwa kwemhuka uye yemunhu coronaviruses kubva akasiyana (sub) genus Utachiona. , kusanganisira SARS-CoV-2 uye Middle East Respiratory Syndrome Coronavirus.

Kutevedzana kwecoding yeprotein yekoronavirus yakagadzirwa muchidzidzo ichi yakakwidziridzwa neRT-PCR ichishandisa RNA yakaparadzaniswa kubva kuHuh-7 ine HCoV-229E kana Vero E6 ine hutachiona neSARS-CoV-2, uye yakaiswa uchishandisa akajairwa cloning maitiro.pMAL-c2 (New England Biological Laboratory) kana pASK3-Ub-CHis6 (47) kutaura vector (SI Appendix, Tables S1 uye S2).Single codon substitutions yakaunzwa nePCR-based site-directed mutagenesis (48).Kuti ibudise iyo MBP fusion protein, E. coli TB1 masero akashandurwa neakakodzera pMAL-c2 plasmid construct (SI appendix, Tafura S1).Protein yefusion yakacheneswa neamylose affinity chromatography uye yakatsemurwa nefactor Xa.Zvadaro, iyo C-terminal His6-tagged protein yakacheneswa neNi-immobilized metal affinity chromatography (Ni-IMAC) sezvakambotsanangurwa (49).Kuti ibudise ubiquitin fusion protein, E. coli TB1 masero akashandisa zvakakodzera pASK3-Ub-CHIs6 plasmid construct (SI Appendix, Tables S1 uye S2) uye pCGI plasmid DNA encoding ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Shanduko (47).Iyo C-terminal His6-tagged coronavirus protein yakacheneswa sezvakatsanangurwa kare (50).

Iyo self-NMPylation bvunzo yeHCoV-229E nsp12-His6 yakaitwa sezvakatsanangurwa muEAV nsp9 (16).Muchidimbu, nsp12-His6 (0.5 µM) ine 50 mM 4-(2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES)-KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 µM buffer iyo NTP yakatsanangurwa uye 0.17 µM yakaenzana [α32-P]NTP (3,000 Ci/mmol; Hartmann Analytic) pa30 °C kwemaminitsi makumi matatu.Mune mamwe ese (standard) NMPylation assays ye nsp12-mediated nsp9 NMPylation, maitiro ekuita anogadziriswa seanotevera: nsp12-His6 (0.05 µM) uye nsp9-His6 (4 µM) pamberi pe50 mM HEPES-KOH (pH 8.0). ), 5 mM DTT, 1 mM MnCl2, 25 µM yakaratidza NTP, uye 0.17 µM inoenderana [α32-P]NTP.Mushure mekuvhara kwemaminetsi gumi pa30 ° C, mhinduro yemuenzaniso yakasanganiswa neSDS-PAGE sampuli buffer: 62.5 mM tris (hydroxymethyl) aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol uye 0.005% bromophenol bhuruu.Iyo puroteni yakashongedzwa nekudziya pa90 ° C kwemaminetsi mashanu uye yakaparadzaniswa ne12% SDS-PAGE.Iyo gel yakagadziriswa uye yakasvibiswa neCoomassie Brilliant Blue mhinduro (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), yakasvibiswa, uye yakafumurwa kune phosphorescent imaging screen kwemaawa makumi maviri (kuona nsp12 kubva kuNMPylation) kana (maximum) 2 Maawa (kuongorora nsp9 NMPylation).Mufananidzo weTyphoon 9200 (GE Healthcare) wakashandiswa kuongorora skrini uye ImageJ yakashandiswa kuongorora kusimba kwechiratidzo.

Kuongororwa kweMS, 1 µM nsp12-His6 uye 10 µM nsp9 (isina hexahistidine tag) yakashandiswa muNMPylation analysis (SI appendix, Tafura S1) uye kuwedzera kwe500 µM UTP uye GTP yakashandiswa.Zvichienderana nekutarisisa kwavo uye inotarisirwa kunaka kweprotein, iyo Mvura ACQUITY H-Kirasi HPLC sisitimu ine MassPrep column (Mvura) yakashandiswa desalt 1 kusvika 10 µL yebuffered protein mhinduro online.Protein yakabviswa munyu inoburitswa muelectrospray ion sosi yeSynapt G2Si mass spectrometer (Mvura) kuburikidza neinotevera gradient yebuffer A (mvura/0.05% formic acid) nebuffer B (acetonitrile/0.045% formic acid), uye tembiricha yekoramu ndiyo 60 ° C uye kuyerera kwe 0.1 mL/min: elution isocratically ne 5% A kwemaminetsi maviri, uye mutsara gradient kusvika 95% B mukati me8 maminetsi, uye chengetedza 95% B kwemamwe maminetsi mana.

Positive ions ine huremu hwe500 kusvika 5000 m/z dzinoonekwa.Glu-fibrinopeptide B inoyerwa ese masekonzi makumi mana neshanu kune otomatiki mass drift kugadzirisa.Shandisa MassLynx chiridzwa software neMaxEnt1 yekuwedzera kuburitsa avhareji spectrum mushure mekubvisa yekutanga uye kutsetseka.

UMPylated HCoV-229E nsp9 yakagayiwa nekuwedzera sequencing-giredhi yakagadziridzwa trypsin (Serva) uye yakavharirwa husiku pa37 °C.A Chromabond C18WP spin column (chikamu nhamba 730522; Macherey-Nagel) yakashandiswa kubvisa uye kuisa pfungwa pamapeptides.Pakupedzisira, peptide yakanyungudutswa mu25 µL yemvura, yaive ne5% acetonitrile uye 0.1% formic acid.

Iwo masampuli akaongororwa neMS vachishandisa Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Iyo yekupedzisira nanoâ HPLC system (Dionex), yakashongedzerwa netsika yekupedzisira-yakaiswa 50 cm??75 μm C18 RP column yakarongedzwa ne 2.4 μm magnetic beads (Dr. Albin Maisch High Performance LC GmbH) Batanidza kune mass spectrometer online kuburikidza neProxeon nanospray source;jekiseni 6 µL ye trypsin digestion solution mu300 µm mukati dhayamita ×??1 cm C18 PepMap pre-concentration column (Thermo Scientific).Ichishandisa mvura/0.05% formic acid seyakanyungudika, sampu yacho yakangobatwa nega uye yakabviswa munyu pachiyero chekuyerera che6 µL/min.

Aya anotevera magiraidhi emvura/0.05% formic acid (solvent A) uye 80% acetonitrile/0.045% formic acid (solvent B) akashandiswa kuwana kupatsanurwa kwe tryptic peptides pakuyerera kwe300 nL/min: 4% B ye 5 maminetsi, ipapo 30 A linear gradient kusvika 45% B mukati memaminitsi, uye mutsara wekuwedzera kusvika 95% solvent B mukati me5 maminetsi.Batanidza chromatographic column kune Stainless steel nano-emitter (Proxeon), uye pfapfaidza iyo yakananga kune inopisa capillary ye mass spectrometer uchishandisa mukana we2,300 V. Ongororo yekuongorora ine resolution ye60,000 muOrbitrap mass analyzer inobatanidzwa. ine data inokwana katatu MS/MS scans, dynamically excluded for 30 seconds, using linear ion trap collision induced dissociation kana yakakwirira simba collision dissociation yakasanganiswa nekuonekwa kwe orbitrap, Chisarudzo chacho 7,500.


Nguva yekutumira: Aug-03-2021