Ihlelwe nguPeter Sarnow, iStanford University School of Medicine, kwiYunivesithi yaseStanford, eCalifornia, evunyiweyo nge-25 kaDisemba, 2020 (ihlaziywe nge-25 ka-Okthobha 2020)
Sinika ingxelo ngentsebenziswano phakathi kweeyunithi ezingaphantsi kophindaphindo lwekhompleksi ezikhutshelweyo, eziyimfuneko kuphindaphindo kunye nolondolozo lwendalo.Sinike ubungqina bokuba i-domain ye-NiRAN ehambelana ne-nsp12 inomsebenzi wokudlulisa i-nucleoside monophosphate (NMP) kwi-trans, kwaye ichonge i-nsp9 (i-RNA ebophezelayo iprotheni) njengento ekujoliswe kuyo.I-NiRAN yenza i-covalent attachment ye-NMP moiety kwindawo egciniweyo ye-nsp9 amino terminus kwimpendulo exhomekeke kwi-Mn2+ ion kunye neentsalela ze-Asn ezikufutshane.Kwafunyaniswa ukuba umsebenzi we-NiRAN kunye ne-nsp9 NMPylation zibalulekile kuphindaphindo lwe-coronavirus.Idatha ivumela ukuba sidibanise lo msebenzi we-nested virus enzyme marker kwimigqaliselo yangaphambili kwi-hypothesis yokuba ukuqaliswa kwe-RNA synthesis kwiklasi yeentsholongwane ze-RNA zisebenza kwaye zihambelana ngokuguquguqukayo.
I-RNA exhomekeke kwi-RNA polymerase (RdRps) ye-Nidovirales (Coronaviridae, Arterioviridae, kunye nezinye iintsapho ezili-12) inxulunyaniswe ne-amino-terminal (N-terminal) domain kwi-non-structural protein (nsp) ekhutshwe kwi-polyprotein, ebizwa ngokuba yi-NiRAN. I-1ab yenziwe nge-viral main protease (Mpro).Ngaphambili, i-virus ye-arterial ye-NiRAN-RdRp ye-nsp ye-GMPylation / UMPylation yomsebenzi yabikwa, kwaye kwacetyiswa ukuba kuveliswe isikhashana sokudluliselwa kwe-nucleoside monophosphate (NMP) kwi-virus (engaziwa ngoku) kunye / okanye i-cell biopolymerization Things.Apha, sibonisa ukuba i-coronavirus (Human Coronavirus [HCoV]-229E kunye neSevere Acute Respiratory Syndrome Coronavirus 2) nsp12 (NiRAN-RdRp) inomsebenzi we-NMPylation oxhomekeke kuMn2+, othatyathwe kwi-nsp9 ngokusekwa kwe-Mpro-mediated nsp9 Emva koko. i-N-terminal flanking nsps ikhutshwe ngeproteolytically, i-phosphoramidate iboshwe kwi-amine yokuqala (N3825) kwi-N-terminal ye-nsp9.I-uridine triphosphate yeyona nucleotide ekhethwayo kule mpendulo, kodwa i-adenosine triphosphate, i-guanosine triphosphate, kunye ne-cytidine triphosphate zikwayi-co-substrates efanelekileyo.Izifundo zoguqulo zisebenzisa i-coronavirus nsp9 kunye ne-nsp12 iiproteni kunye ne-HCoV-229E eguqulweyo eyenziwe ngokwemfuza yagqiba iintsalela eziyimfuneko kwi-NiRAN-mediated nsp9 NMPylation kunye nokuphindaphindwa kwentsholongwane kwinkcubeko yeseli.Idatha iqinisekisile ukuqikelelwa kweendawo zokuhlala ze-NiRAN ezisebenzayo kwaye yagqiba indima ebalulekileyo ye-nsp9 N3826 i-residus kwi-nsp9 NMPylation kunye nokuphindaphinda kwentsholongwane kwi-vitro.Le ntsalela yinxalenye yolandelelwano lwe-N-terminal ye-NNE tripeptide egciniweyo kwaye ingqineke iyintsalela engaguqukiyo ye-nsp9 kunye neehomologs zayo kusapho lwe-coronavirus.Olu phononongo lubonelela ngesiseko esiluqilima sophononongo olusebenzayo lomsebenzi we-NMPylation wezinye iintsholongwane ezihlala kwindlwane kwaye liphakamisa iithagethi ezinokwenzeka kuphuhliso lwamachiza alwa neentsholongwane.
I-Nidovirales intsholongwane ye-RNA ene-positive-stranded ichaphazela iintlobo zezilwanyana ezinomqolo kunye nee-invertebrates (1, 2).Lo myalelo okwangoku ubandakanya iintsapho ezili-14 (3), apho usapho lweCoronavirus lufundwe ngokubanzi kwiminyaka engama-20 edlulileyo.Ngelo xesha, ii-zoonotic coronaviruses ezintathu zavela kwimikhosi yezilwanyana kwaye zabangela ukuqhambuka okukhulu kosulelo olubi lokuphefumla ebantwini.Kubandakanya iindyikityha ezizingisileyo ezibangelwa zizifo ezosulelayo eziqatha.IRespiratory Syndrome iCoronavirus 2 (SARS-CoV-2) (4âââ7).Ii-Nidoviruses zabelana ngombutho oqhelekileyo we-genome, kunye ne-subunit ye-membrane-bound-bound-transcription complex (RTC) ifakwe ngekhowudi kwi-5-?²-terminal yesibini esithathwini kunye ne-subunit engundoqo yolwakhiwo lwentsholongwane, kunye nezinye izincedisi. .Iprotheyini, efakwe kwikhowudi yesi-3??² isiphelo sesithathu segenome (1).Ngaphandle kosapho olunye lweentsholongwane zeplanarian (i-Monoviridae) (8), zonke iintsholongwane ezifakwe kwindlwane zifakela ii-subunits ze-RTC kwiifreyimu ezimbini ezivulekileyo zokufunda (ORF) ORF1a kunye ne-ORF1b, eziguqulelwe kwi-RNA ye-genomic ye.I-ORF1a ifaka iikhowudi ze-polyprotein (pp) 1a, kunye ne-ORF1a kunye ne-ORF1b ngokudibeneyo encode i-pp1ab.Ngokuthatha inxaxheba ngokubanzi kwe-protease ephambili (Mpro) efakwe kwikhowudi yi-ORF1a, zombini i-pp1a kunye ne-pp1ab zicutshungulwa ngokweproteolytically kwiindidi ze-non-structural proteins (nsps), ezaziwa ngokuba yi-3CLpro, kuba ine-homology kunye ne-3Cpro ye-picornavirus. 9).Ezi nsps kucingelwa ukuba zidityaniswe kwi-RTC enkulu eguquguqukayo, i-catalyze synthesis of genomic RNA (replication) kunye neseti ye-subgenomic RNA (transcription), kwaye zisetyenziselwa ukulungelelanisa intetho ye-ORF ebekwe ezantsi komlambo we-ORF1b (10? ? ?12).
I-RTC engundoqo iquka i-RNA exhomekeke kwi-RNA polymerase (RdRp) (13), i-superfamily 1 helicase (HEL1) (14, 15) kunye ne-RNA ezininzi ze-enzymes zokucubungula, ezifakwe kwiikhowudi kwi-ORF1b nakwintsapho ye-coronavirus Iqulethe i-nsp12-nsp16 kunye nsp9-nsp12 kusapho lwe-Arterioviridae (jonga isalathiso 10ââ 12).I-RdRp kunye ne-HEL1 zimele iindawo ezimbini (isinye kwisihlanu) ezigciniweyo zentsholongwane yentaka kwaye zinehomology phakathi kwezinye iintsholongwane zeRNA.I-core replicase kukholelwa ukuba incediswa ngamanye ama-subunits, kubandakanywa ii-nsps ezininzi ezincinci ezikhutshiweyo kwingingqi ye-carboxy-terminal (C-terminal) ye-pp1a, emazantsi e-Mpro (coronavirus nsp5 kunye ne-arterial virus nsp4, ngokulandelelana).Banokhuseleko olukhethekileyo kusapho kunye nemisebenzi eyahlukeneyo (ihlaziywe kwireferensi 10ââ12).
Kutshanje, isizinda esineempawu zemotif zolandelelwano ezizodwa zifunyenwe kwi-amino terminus (N-terminus) ekufuphi ne-RdRp kuzo zonke iintsholongwane ezifakwe kwindlwane, kodwa azikho ezinye iintsholongwane ze-RNA (16).Ngokusekelwe kwindawo yayo kunye ne-nucleotide transferase (nucleoside monophosphate [NMP] transferase) umsebenzi, lo mmandla ubizwa ngokuba yi-NiRAN (Nestvirus RdRp-enxulumene ne-nucleotide transferase).Indibaniselwano yedomeyini embini ye-NiRAN-RdRp ibandakanya i-nsp12 kusapho lwe-Coronaviridae kunye ne-nsp9 kusapho lwe-Arterioviridae, kwaye kwenye i-nestoviridae, i-NiRAN-RdRp kulindeleke ukuba ikhutshwe njenge-nsp ezimeleyo kwi-polyprotein eyintsholongwane.Kwi-coronavirus, i-domain ye-NiRAN iqulethe i-??1/450 iintsalela kwaye iqhagamshelwe kwi-C-terminal RdRp domain ngokusebenzisa ummandla we-linker (16?19).Kwi-Equine Arteritis Virus (EAV) (Arteriviridae), i-recombinant nsp9 ibonisa i-Mn2+ ion-dependent (self) imisebenzi ye-UMPylation kunye ne-GMPylation, exhomekeke kwiziseko ezithathu ezigciniweyo zolandelelwano kwi-nestovirus, AN, BN kunye ne-CN Iintsalela kulandelelwano.Apho i-N imele i-NiRAN) (16).I-N-terminal flanking yezi motif yimotif engaphantsi yogcino preAN.Ezinye zezi ntsalela nazo zigcinwe kwiprotheyini kinases ehambelana ngokude, apho iboniswe ukuba ibandakanyeka kwi-nucleoside triphosphate (NTP) yokubopha kunye nomsebenzi we-catalytic (20, 21).Ngokungqinelana nolu qwalaselo, iintsalela zeendawo ezininzi ezisebenzayo kwi-pseudokinase SelO ukusuka kwi-Pseudomonas syringae zinokudityaniswa ne-SARS-CoV-2 nsp7/8/12/13 supercomplex esanda kupapashwa.I-Coronavirus egciniweyo iintsalela ze-NiRAN ezibekwe ngaphezulu kwi-electron microstructure.Iprotheyini edibeneyo (17).Kucingelwa ukuba i-U / GMPylation ebhaliweyo (self) iya kuvelisa imeko yesikhashana ukuhambisa i-NMP kwi-substrate (okwangoku engaziwa) (16), kunye nokufana kwesakhiwo phakathi kwe-NiRAN kunye neprotein kinase (17, 19) ) Ngaba i-hypothesis yokuba I-NiRAN iguqula ezinye iiprotheni.
Iimpawu ezininzi, kubandakanywa unxulumano lwayo olukhethekileyo kunye nolulodwa olucwangcisiweyo kunye neentsholongwane ezifakwe kwindlwane kunye nokwahlulwa kofuzo kwi-RdRp, yenza i-NiRAN ibe yi-enzyme eyimfuneko yokulawula iintsholongwane ezifakwe kwindlwane, ebaluleke kakhulu ekuveleni kwabo kunye nobunikazi.Ngaphambili, imisebenzi emithathu enokwenzeka ebandakanya i-NiRAN yokulawula i-genome / i-subgenomic translation okanye ukuphindaphinda / ukushicilelwa kwakubizwa.Xa kuqwalaselwa idatha enqongopheleyo nengaphelelanga ekhoyo ngelo xesha, umsebenzi ngamnye uneengenelo kunye nokungalunganga (16).Kolu phando, sijonge ukudibanisa i-biochemical kunye nokubuyisela umva izifundo zemfuzo ze-coronavirus ezimele le mijelo mibini, kwaye sibeke iziphumo zethu kwimvelaphi yenguquko yendalo yosapho lwe-coronavirus, ukuze sifumane ukuqonda kulo mmandla umangalisayo.Sinika ingxelo yenkqubela phambili enkulu ekuqondeni kwe-NiRAN ngokuchongwa kweethagethi zendalo kwi-RTC, leyo (phakathi kweengcamango ezintathu ezikhoyo) igalelo kwindima yalo mmandla ekuqaliseni i-synthesis ye-RNA ene-nested virus.Olu phando lukwavula amathuba kwezinye iindima ze-NiRAN kwi-virus host interface.
Ukuze sibonakalise iimpawu ze-enzymatic zentsholongwane ye-corona i-nsp12-enxulumene nesizinda se-NiRAN, siye savelisa uhlobo oluphinda-phindayo lwe-coronavirus yabantu 229E (HCoV-229E) nsp12 e-E. coli, kunye nethegi ye-His6 kwi-C-terminus, kwaye idibanise i iprotheyini ene- [α32-P] I-Incubate kunye ne-NTP phambi kwe-MnCl2 njengoko ichazwe kwiiMathiriyeli kunye neendlela.Uhlalutyo lwemveliso yokusabela lubonise ubukho beprotheyini ene-radiolabeled ehamba kunye ne-nsp12 (106 kDa), ebonisa ukuba i-coronavirus i-nsp12 ivuselela ukwakheka kwe-covalent protein-NMP adducts, eyenziwe ngokukhethekileyo nge-uridine monophosphate (UMP) (Figure 1A) Kwaye B).Uhlalutyo olulinganisiweyo lubonise ukuba xa kuthelekiswa namanye ama-nucleotides, ukunyanzeliswa kwesignali yokufakwa kwe-UMP kwanda ngama-2 ukuya kwii-3 amaxesha (Umfanekiso 1C).Le datha ihambelana nomsebenzi oqikelelweyo we-NMP wokudluliselwa kwe-domain ye-NiRAN ye-coronavirus (16), kodwa ibonisa ukuba ukhetho lwe-nucleotide ye-NiRAN ye-coronavirus kunye ne-virus ye-arterial iyahluka.
Umsebenzi we-Self-NMPylation we-HCoV-229E nsp12.(A) I-HCoV-229E nsp12-His6 (106 kDa) yafukanyelwa nge- [α-32P] NTP echongiweyo phambi kobukho be-6 mM MnCl2 imizuzu engama-30 (jonga iiMathiriyeli kunye neeNdlela ngeenkcukacha).Iimveliso zokusabela zahlulwe yi-SDS-PAGE kwaye zadyojwa nge-Coomassie eluhlaza okwesibhakabhaka.(B) Iprotheyini ene-radiolabeled ibonwa ngomfanekiso we-phosphorous.Izikhundla ze-nsp12-His6 kunye ne-protein molecular mass markers (kwii-kilodaltons) zibonisiwe ku-A no-B. (C) Ubukhulu besignali ye-radioactive (ithetha ± SEM) yamiselwa kwimifuniselo emithathu ezimeleyo.*P≤0.05.Amandla omqondiso (ipesenti) inxulumene ne-UTP.
Nangona imisebenzi ye-enzyme enxulumene ne-NiRAN ibonakaliswe ibalulekile ekuphindaphindweni kwe-EAV kunye ne-SARS-CoV kwinkcubeko yeeseli (16), umsebenzi othile we-NiRAN kunye neethagethi ezinokubakho azikamiselwa.Ukufana kwesakhiwo esandula kuxelwa phakathi kwe-NiRAN kunye nosapho lweeprotheyini ezineprotein kinase-like folds (17, 22) yasishukumisela ukuba sivavanye i-hypothesis yokuba i-NiRAN yenza i-NMPylation yezinye iiprotheni.Senze iseti yeethagethi ezinokubakho, kubandakanywa iiproteni ezingezizo ezezakhiwo ezifakwe kwikhowudi yiHCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), nganye iqulethe ithegi ye-C-terminal His6 (i-SI appendix, iTheyibhile S1) , Kwaye zifukame ezi proteni nge [α32-P] uridine triphosphate ([α32-P]UTP) phambi okanye ukungabikho kwe-nsp12.I-albumin ye-bovine serum kunye ne-MBP-LacZα iprotheni yokudibanisa eveliswe kwi-E. coli yasebenza njengolawulo (Umfanekiso 2A, imizila ye-1 ukuya kwi-7).Iprotheni ene-radiolabeled yahlalutywa yi-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) kunye ne-autoradiography, kwaye kwafunyanwa ukuba kukho umqondiso onamandla we-radioactive kwi-reaction equkethe i-nsp12 kunye ne-nsp9.Isikhundla somqondiso sihambelana ne-molecular mass ye-nsp9, ebonisa i-nsp12-mediated UMPylation ye-nsp9 (Umfanekiso 2B, umkhondo 7).Azikho ezinye iiprotheyini zovavanyo ezifunyenwe zi-UMPylated, eziye zasikhokelela ekubeni sigqibe kwelokuba i-nsp9 yi-substrate ethile ye-nsp12.Ngokuhambelana nedatha ye-self-NMPylation eboniswe kuMfanekiso 1, i-nsp12 iyakwazi ukudlulisela zonke ii-NMP ezine kwi-nsp9, nangona ukusebenza kakuhle kwahlukile, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Umfanekiso).3 A kunye no-B).Ngaphantsi kweemeko ezisetyenzisiweyo kolu vavanyo (nciphisa ukuphendula kunye nexesha lokuvezwa, ukunciphisa ukuxinwa kwe-nsp12; izixhobo kunye neendlela), i-self-NMPylation ye-nsp12 ayikwazanga ukufunyanwa (thelekisa uMfanekiso 2B, umzila we-7, kunye noMzobo 1B), apho ibonakale isebenzayo (Kwaye imijikelo emininzi) i-UMP isusiwe kwi-nsp12 ukuya kwi-nsp9.Umsebenzi wodluliselo we-UMP ufuna ubukho be-Mn2+ ion, njengoko kubonisiwe kuMfanekiso 3C, ngelixa kuphela umsebenzi omncinci wodluliselo we-UMP wabonwa phambi kobukho be-Mg2+, kwaye akukho msebenzi kubukho bezinye ii-divalent cations ezivavanyiweyo.Idatha efanayo ifunyenwe kwii-assays ze-NMPylation eziqulethe i-cytidine triphosphate (CTP), i-guanosine triphosphate (GTP), kunye ne-adenosine triphosphate (ATP) (i-SI appendix, i-Figure S1).
I-HCoV-229E nsp12-mediated UMPylation ye-nsp9.Uthotho lweprotein substrates (kubandakanya i-albumin ye-bovine serum, i-MBP-lacZα, kunye nothotho lwe-HCoV-229E nsps ebhalwe nge-C-terminal His6 encoded ngu-ORF1a) yasetyenziselwa ukuvavanya umsebenzi we-UMPylation we-HCoV-229E nsp12-His6⁺-mediated iprotheni.Faka iprotheni nge- [α-32P] UTP imizuzu eyi-10 ngokungabikho (A) okanye ubukho (B) be-nsp12 njengoko kuchazwe kwizinto kunye neendlela.Phezulu kwe-A kunye ne-B, i-SDS-polyacrylamide ijeli ene-Coomassie Brilliant Blue iboniswa, kwaye ngaphantsi kwe-A kunye ne-B, i-autoradiograms ehambelanayo ibonisiwe.Indawo yeprotein molecular mass marker (kwi kilodaltons) inikwe ngasekhohlo.Isikhundla se-nsp12-His6 (B, phezulu) kunye nesignali ye-radioactive ebonwe ngexesha lokufukamela i-nsp12-His6 kunye ne-nsp9-His6 (B, umzila we-7) nayo ibonisiwe, ebonisa ukuba [α-32P]UMP ukuya kwi-nsp9-His6 ( 12.9 kDa ), engazange ibonwe kwezinye iiprotheni ezivavanyiweyo.
I-HCoV-229E NiRAN-mediated biochemical and virological characterization of nsp9 NMPylation.(A kunye no-B) Indima ye-nucleotide co-substrate esetyenziswe kwi-reaction.I-Nsp12-His6 kunye ne-nsp9-His6 zixutywe kwaye zifukanywe kubukho bezohlukeneyo [α-32P] NTPs kuvavanyo oluqhelekileyo lwe-NMPylation.(A, phezulu) I-Coomassie-stained nsp9-His6 yahlulwe yi-SDS-PAGE.(A, ezantsi) I-Autoradiograph yendawo efanayo yejeli.(B) Umsebenzi onxulumeneyo (uthetha ± SEM) kubukho be-cofactor ye-nucleotide echongiweyo igqitywe kwimifuniselo emithathu ezimeleyo.*P≤0.05.(C) Indima yeeyoni zetsimbi.Kubonisiwe uvavanyo oluqhelekileyo lwe-NMPylation phambi kwe- [α-32P] UTP kunye ne-ioni zetsimbi ezahlukeneyo, nganye ine-concentration ye-1 mM.Ku-C, phezulu, i-Coomassie stained nsp9-His6 ibonisiwe, kwaye kwi-C, ezantsi, i-autoradiography ehambelanayo ibonisiwe.Ubungakanani beprotheyini ebhalwe (kwi kilodaltons) iboniswa ekhohlo lika A kunye no C. (D) Uhlobo oluguquguqukayo lwe HCoV-229E nsp12-His6 ethwele ukutshintshwa kweamino acid echaziweyo iku [α-32P]UTP, njengoko kuchaziwe. kwiiMathiriyeli kunye neendlela.I-radiolabeled nsp9-His6 eveliswe kwi-NMPylation reaction ifunyenwe nge-phosphorylation imaging (D, phezulu).Umsebenzi ohambelanayo xa kuthelekiswa nohlobo lwasendle (wt) iprotheni iboniswe kwi-D, kwaye i-bottom ithathwa njenge-avareji (±SEM) ukusuka kwiilinge ezintathu ezizimeleyo.Iinkwenkwezi zibonisa ukutshintshwa kweentsalela ezingalondolozwanga.(E) I-virus titer kwi-culture supernatant yeeseli ze-p1 ezifunyenwe kwiiyure ezingama-24 emva kokuba usulelo luchongwe ngovavanyo lwe-plaque.Ukutshintshwa kwekhodon kwidomeyini ye-NiRAN yenguqu eyenziwe ngobunjineli ye-HCoV-229E ibonisiwe (intsalela yeenombolo isekelwe kwindawo yazo kwi-pp1ab).I-replication-deficident RdRp indawo esebenzayo i-mutant nsp12_DD4823/4AA isetyenziswe njengolawulo.
Ukuze ufumane ukuqonda okunzulu kwendawo esebenzayo ye-NiRAN kwaye unqume iintsalela ezinxulumene nomsebenzi we-nsp9-specific NMP transferase, senze uhlalutyo lokuguqulwa kwenguqu, apho sithathe indawo yeendawo ezihlala zigcinwe kwi-NiRAN AN, BN kunye ne-CN motifs ( 16) Ngu-Ala (isihlomelo se-SI, uMzobo S2).Ukongeza, impembelelo ye-Conservative Arg-to-Ls okanye iLys-to-Arg substitutions iye yavavanywa kwiimeko ezimbini.Njengolawulo (negative), iintsalela ezingagcinwanga okanye ezingagcinwanga ngaphantsi kwi-domain ye-NiRAN ye-coronavirus kunye nezinye ii-virus ezifakwe kwindlwane zitshintshwa nge-Ala Ukubuyisela i-K4116A (in motif preAN), K4135A (AN), R4178A (BN), D4188A (motif) I-BN) kunye ne-D4280A (CN) iyanciphisa kakhulu okanye ide isuse i-nsp9 NMPylation ngokusebenzisa i-nsp12, ngelixa iiprotheni ezinokutshintshwa kwe-conservative (R4178K) , K4116R) zigcina i-60% kunye ne-80% yomsebenzi wazo, obonisa ukuba ukukhululeka kwezithintelo kwicala labo. amatyathanga anovelwano ngokwe-physicochemically (Figure 3D).Ukutshintsha iintsalela ezininzi ezigciniweyo E4145A, D4273A, F4281A kunye ne-D4283A ayinabungozi kangako, kwaye i-nsp9 UMPylation icuthwe ngokuphakathi kuphela.Iziphumo ezifanayo zifunyenwe kwi-nsp9 ye-NMPylation reactions ebandakanya ezinye ii-NTPs (Umfanekiso we-3D kunye ne-SI isihlomelo, uMzobo S3), eqinisekisa ukuba iziphumo eziboniweyo kwiindawo ezithile ze-amino acid zizimeleyo kuhlobo lwe-nucleotide co-substrate esetyenzisiweyo.Okulandelayo, siye savavanya impembelelo enokubakho yolu tshintsho lwe-nsp12 ekuphindaphindweni kwee-coronavirus kwinkcubeko yeeseli.Ukuza kuthi ga ngoku, sisebenzise izifanekiso zeDNA (cDNA) ezifanelekileyo ezenziwe ngobunjineli ezidityaniswe kwintsholongwane yokugonya ephinda-phindayo (23, 24) ukukhuphela iiseli ezi-5 -7.Ukwenziwa kwenzala yentsholongwane eyosulelayo eveliswe kwezi seli ibonise ukuba uninzi lweenguquko ze-HCoV-229E NiRAN bezingenzeki (Umfanekiso 3E).Iqela le-non-viable viral mutants libandakanya ezinye iindlela eziye zaboniswa ukuphelisa okanye ukunciphisa kakhulu umsebenzi wokudluliselwa kwe-NMP kwi-vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), kodwa kukho ezinye iindlela ezimbini (K4116R, E4145A) % ebekelwe?Umsebenzi wabo we-in vitro we-NMPylation ucebisa ukuba izithintelo ezongezelelweyo ziyabandakanyeka.Ngokufanayo, ezinye iinguqulelo ezimbini (i-R4178K, i-F4281A) ezibangele ukuhla okuphakathi kwe-NiRAN's in vitro NMPylation umsebenzi wavelisa iintsholongwane eziphilayo, nangona kunjalo, ezi ntsholongwane zanciphisa kakhulu ii-titers ngokuphindaphinda.Ngokuhambelana nedatha ye-in vitro yomsebenzi eboniswe kuMfanekiso we-3D, ukubuyisela ezinye iintsalela ezine ezingagcinwanga kwi-coronavirus kunye/okanye ezinye iintsholongwane ezihlala kwindlwane (K4113A, D4180A, D4197A, D4273A) (8, 16) zivelise iintsholongwane ezisebenzayo Inzala, nangona i-titer encitshisiweyo ngokulinganayo xa kuthelekiswa nentsholongwane yohlobo lwasendle (Umfanekiso 3E).
Ukuze ufunde ukuba ingaba umsebenzi wodluliselo we-NMP we-NiRAN-mediated uxhomekeke kwi-domain ye-RdRp esebenzayo, iintsalela ze-Asp ezimbini ezigciniweyo ezibandakanyekayo kukulungelelaniswa kwee-ion zentsimbi (11) kwi-RdRp motif C zathatyathelwa indawo ngu-Ala. umsebenzi wayo we-NMPylation we-nsp9, ebonisa ukuba i-nsp12-mediated in vitro nsp9 umsebenzi we-NMPylation awufuni umsebenzi we-polymerase (Isihlomelo se-SI, uMzobo S4).
Emva kokuseka umsebenzi wodluliselo we-NMP we-nsp9-specific we-nsp12, sizame ukuphawula i-NMP-nsp9 adduct nge-mass spectrometry (MS).Ubuninzi beprotheyini epheleleyo ye-recombinant HCoV-229E nsp9 ibonise incopho kwi-12,045 Da (Figure 4A).Ukongezwa kwe-nsp12 akuzange kutshintshe umgangatho we-nsp9, ebonisa ukuba i-nsp12 kunye ne-nsp9 ayiyi kwenza i-complex ezinzile phantsi kweemeko ezisetyenzisiweyo (i-denaturation) (Umfanekiso 4A).Phambi kwe-UTP kunye ne-GTP, umlinganiselo we-mass reaction equkethe i-nsp9 kunye ne-nsp12 ngokulandelanayo ibonise ukuba ubunzima beprotheyini ye-UTP yahambisa i-306 Da, kunye nobunzima beprotheni ye-GTP yahambisa i-345 Da, ebonisa ukuba i-molecule nganye ye-nsp9 ibophelela i-UMP okanye i-GMP. (Umfanekiso 4) C no-D).Kucingelwa ukuba amandla afunekayo kwi-NiRAN-mediated nsp9 NMPylation avela kwi-NTP hydrolysis kunye nokukhululwa kwe-pyrophosphate.Nangona i-10-fold molar excess of nsp9 (target) kune-nsp12 (enzyme) yasetyenziswa kule reaction, phantse i-NMPylation epheleleyo ye-nsp9 yabonwa, ebonisa ukuba intsebenziswano phakathi kwe-nsp12 kunye ne-nsp9 yinto emfutshane, kwaye i-nsp12 ingakwazi i-NMPylate ngaphezulu kwe-nsp9 in vitro molekyuli.
I-NMPylation enye ye-nsp9 phambi kwe-nsp12 kunye ne-UTP okanye i-GTP.Kubonisiwe yi-deconvoluted epheleleyo yeprotein mass spectrum ye-HCoV-229E nsp9 (i-SI appendix, iTheyibhile S1) (AD).(A) nsp9 yedwa, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 phambi kwe-UTP, (D) nsp9 + nsp12-His6 phambi kwe-GTP.
Ukumisela iintsalela ze-nsp9 UMPylated ngu-nsp12, i-nsp9-UMP yacandwa nge-trypsin.Iipeptide ezifunyenweyo zahlulwa yi-nano-high performance liquid chromatography (HPLC) kwaye zahlalutywa nge-tandem mass spectrometry (MS/MS) kwi-intanethi.Uhlalutyo lwedatha kusetyenziswa iphakheji ye-software ye-Byonic (i-Protein Metrics) ibonise i-UMPylation ye-N-terminal amino acid.Oku kuqinisekiswa ngesandla.I-tandem mass spectrum ye-precursor peptide [UMP]NNEIMPGK (SI appendix, Figure S5A) ibonise iqhekeza kwi-421 m/z, ebonisa ukuba i-UMP ibophelela kwintsalela ye-1 ye-nsp9.
Kwi-N-terminus ye-nsp9, i-Asn igcinwe phakathi kwamalungu e-Orthocoronavirinae (i-SI appendix, uMfanekiso S6).Nangona sikholelwa ukuba i-N-terminal primary amine nitrogen yeyona inokwenzeka ukuba yamkele i-UMP, sigqibe ekubeni sifumane ubungqina obongezelelweyo be-NMP ebophelelayo kwi-N-terminal.Ngesi sizathu, i-peptide engeyiyo i-NMPylated kunye ne-NMPylated N-terminal nsp9 ehlanjululwe yi-HPLC yafunyanwa kubukho be-acetone kunye ne-sodium cyanoborohydride.Ngaphantsi kwezi meko, kuphela ii-amine eziziiprayimari ezikhululekile zingatshintshwa ngepropyl (25).I-peptide ye-N-terminal ye-nsp9-derived peptide kunye nolandelelwano lwe-NNEIMPGK iqulethe ii-amine eziphambili, enye kwi-N-terminus ye-Asn kunye nenye kwicala lekhonkco le-Lys kwi-C-terminus.Ngoko ke, amaqela epropyl anokwaziswa kuzo zombini iziphelo.I-ion chromatograms ekhutshiweyo yeepeptide ezingekho kwi-NMPylated iboniswe kwisihlomelo se-SI, uMfanekiso S5B.Njengoko kulindelekile, i-N-terminal kunye ne-C-terminal (mono) ipropylated (i-SI appendix, i-Figure S5B, indlela ephezulu) kunye ne-dipropylated peptides (i-SI appendix, i-Figure S5B, indlela esezantsi) inokuchongwa.Le pateni iyatshintsha ngokusetyenziswa kwe-NMPylated N-terminal peptide ye-nsp9.Kule meko, kuphela i-C-terminal propylated peptides inokuchongwa, kodwa i-N-terminal propylated peptides kunye ne-dipropylated peptides ayichongwa (iSihlomelo se-SI, uMzobo we-S5C), ebonisa ukuba i-UMP idluliselwe kwi-N-terminal primary amine Ukuthintela oku. iqela ekwenzeni utshintsho.
Okulandelayo, sitshintsha (nge-Ala okanye i-Ser) okanye sicime iintsalela ezigciniweyo kwi-N-terminus ye-nsp9 ukuchaza imiqobo ekujoliswe kuyo.Ngokusekelwe kwidatha yethu ye-MS ebonisa ukuba i-NiRAN yenza i-nsp9-NMP adduct kunye ne-amine yokuqala ye-N-terminal residu ye-nsp9, siye saqikelela ukuba i-nsp9 NMPylation ifuna i-viral master protease (Mpro, nsp5) ukukhulula i-nsp9 N-terminal ukusuka isandulela sayo sepolyprotein.Ukuvavanya le ngcamango, sivelise iprotheni ye-precursor nsp7-11 equkethe i-nsp9 kwi-E. coli kwaye yenze uvavanyo oluqhelekileyo lwe-NMPylation phambi kwe- [α-32P] UTP (izinto kunye neendlela).Njengoko kubonisiwe kuMfanekiso 5A (umzila 3), isandulela se-nsp7-11 esinganqunyulwanga asibhalwanga nge-radiolabeled nge-nsp12.Ngokwahlukileyo, ukuba i-nsp7-11 iqhekezwe yi-nsp5 ephinda iphinde ikhuphe i-nsp9 (kunye nezinye i-nsps) kwi-precursor, iprotein ene-radiolabeled ehamba nge-nsp9 ifunyenwe, iqinisekisa isigqibo sethu sokuba i-NiRAN kunye ne-N- Ukukhetha ukwakheka kwee-covalent nsp9-NMP adducts. .I-terminal engundoqo ye-amine ye-N-terminal Asn (indawo engu-3825 kwi-pp1a/pp1ab).Esi sigqibo sikwaxhaswa yimifuniselo esebenzisa i-nsp9 construct, equlethe intsalela enye okanye ezimbini ezongezelelweyo kwi-N-terminus.Kuzo zombini iimeko, i-NiRAN-mediated UMPylation ye-nsp9 yapheliswa (iSihlomelo se-SI, uMzobo S7).Emva koko, savelisa iprotheni enye okanye ezimbini i-Asn residues ecinywe kwi-3825-NNEIMPK-3832 yokulandelelana kwe-peptide kwi-N-terminal ye-nsp9.Kuzo zombini iimeko, i-nsp9 UMPylation ivalwe ngokupheleleyo (Umfanekiso 5B), inika ubungqina obongezelelweyo bokuba i-nsp9 N-terminus yangempela isebenza njenge-NMP receptor.
Ukusetyenzwa kweproteolytic ye-nsp9 kunye nendima yeentsalela ze-N-terminal kwi-nsp12-mediated UMPylation.(A) i-nsp9 UMPylation ifuna i-nsp9 N-terminal yasimahla.I-Nsp7-11-His6 ifakwe ngaphambili kwi-30 °C kwi-NMPylation ukufumanisa isithinteli esiqulethe i-UTP kubukho okanye ukungabikho kwe-recombinant Mpro (nsp5-His6).Emva kweeyure ezi-3, qalisa uvavanyo lwe-NMPylation ngokudibanisa i-nsp12-His6 njengoko kuchaziwe kwiZixhobo kunye neendlela.Ukusabela okuqulethe i-nsp5-His6 (i-layini 1) kunye ne-nsp9-His6 (i-layini 2) isetyenziswe njengolawulo.Emva kwemizuzu ye-10, impendulo yapheliswa kwaye umxube wokuphendula wahlulwa yi-SDS-PAGE.Iprotheyini yayingcoliswe yiCoomassie Brilliant Blue (A, phezulu).Isandulela se-Nsp7-11-His6 kunye nemveliso esetyenzisiweyo evela kwi-nsp5-His6 mediated cleavage iboniswe ekunene.Nceda uqaphele (ngenxa yobukhulu babo buncinci) ukuba i-nsp7 kunye ne-nsp11-His6 ayibonakali kule gel, kwaye impendulo yongezwa nge-nsp5-His6 (imizila 1 kunye ne-4; indawo ye-nsp5-His6 iboniswa ngesangqa esiqinileyo) okanye i-nsp9-His6 (i-Lane 2) iqulethe ixabiso elincinci le-MBP (ebonakaliswe ngeesangqa ezivulekileyo) njengokungcola okushiyekileyo ngenxa yokuba zivezwa njengeeprotheni ze-MBP ze-fusion (i-SI appendix, i-Table S1).(B) I-Nsp9-His6 eyahlukileyo ayinayo intsalela ye-N-terminal ye-Asn enye okanye ezimbini (intsalela yamanani ngokwendawo ekwi-pp1a/pp1ab) kwaye iyacocwa kwaye iqalwe nge-nsp12-His6 kunye ne- [α-32P] UTP.B, SDS-PAGE edyojwe ngeCoomassie iboniswe phezulu, B, i-autoradiograph ehambelana nayo iboniswe ezantsi.Indawo yokumakisha ubunzima bemolekyuli (kwi-kilodaltons) iboniswe ngasekhohlo.(C) I-HCoV-229E nsp9-His6 N-isiphelo se-N-terminal egciniweyo yatshintshwa nge-Ala okanye i-Ser, kwaye umlinganiselo ofanayo weprotheyini wasetyenziswa kwi-nsp12-His6 mediated UMPylation reaction.Iimveliso zokusabela zahlulwe yi-SDS-PAGE kwaye zifakwe i-Coomassie Brilliant Blue (C, phezulu), kunye ne-radiolabeled nsp9-His6 yafunyanwa yi-phosphorescence imaging (C, phakathi).Ukusebenzisa i-wild-type (wt) protein njengesalathiso (imiselwe kwi-100%), umsebenzi we-NMPylation onxulumeneyo (uthetha ± SEM) ubalwe kwimifuniselo emithathu ezimeleyo.(D) Ii-Virus titers kwi-p1 cell culture supernatant yeeseli ze-Huh-7 ezosulelwe yi-HCoV-229E wild-type Huh-7 cells, kunye nee-mutants eziphethe ii-amino acid substitutions ezichongiweyo kwi-nsp9 zamiselwa ngovavanyo lwe-plaque.I-replication-deficient RdRp motif C double mutant DD4823/4AA isetyenziswe njengolawulo olubi.
I-N-terminus ye-nsp9 (ingakumbi izikhundla 1, 2, 3, kunye ne-6) igcinwe kakhulu phakathi kwamalungu e-Orthocoronavirinae subfamily (i-SI appendix, Figure S6).Ukuze ufunde indima enokwenzeka yezi ntsalela kwi-nsp12-mediated nsp9 NMPylation, iintsalela ze-Asn ezimbini ezilandelelanayo kwi-N-terminus ye-nsp9 zatshintshwa nge-Ala okanye i-Ser (yodwa okanye ngokudibeneyo).Xa kuthelekiswa nohlobo lwasendle lwe-nsp9, ukubuyisela i-N3825 nge-Ala okanye i-Ser kubangele ukucutha okuphindwe kabini kwi-nsp12-mediated UMPylation (Figure 5C).Ngokungqinelana nesigqibo sethu sokuba i-NMPylation yenzeka kwi-N-terminal primary amine endaweni yetyathanga elisecaleni lentsalela ye-N-terminal, siqaphele intsalela ye-NMPylation ebalulekileyo ngokutshintshwa kwe-N3825A kunye ne-N3825S.Kuyathakazelisa ukuba, ukuba i-Asn yesibini ithathelwe indawo ngu-Ala okanye i-Ser, i-nsp9 UMPylation iyancitshiswa kakhulu (ngaphezu kwamaxesha e-10), ngelixa ukutshintshwa kwe-Ala kwizikhundla ze-3, 4, kunye ne-6 kunempembelelo ephakathi kwi-nsp9 UMPylation (Figure 2). ).5C).Iziphumo ezifanayo zifunyenwe kusetyenziswa i-ATP, i-CTP okanye i-GTP (i-appendix ye-SI, i-Figure S8).Ngokudibeneyo, ezi datha zibonisa indima ephambili ye-N2826 (isikhundla se-2 kwi-nsp9) kwi-nsp9 NMPylation.
Ukuze sifumane ubungqina obongezelelweyo bokulungelelaniswa okusebenzayo phakathi kwe-N-terminus ye-nsp9 kunye ne-NMPylation, senze ulungelelwaniso olulandelelanayo (MSA) lokulandelelana kwe-nsp9 yosapho lweCoronavirus (oluhluka phakathi kwe-104 kunye ne-113 yentsalela) (Isihlomelo seSI, Umfanekiso S6).Lilonke, kwiindidi ezingama-47 (ezaziwayo nezibekekayo) zenzala emi-5 ye-Orthocoronavirinae subfamily ezosulela izilwanyana ezanyisayo, iintaka, kunye nezilwanyana ezirhubuluzayo, zi-8 kuphela iintsalela ezifunyenwe zingaguquguquki.Olona tshintsho lubanzi kakhulu, kubandakanywa ukucinywa kunye nokufakwa, lubonwe kwimijikelezo phakathi kwezinto ezisisiseko zesakhiwo se-nsp9, njengoko kunqunywe kwizifundo zangaphambili zesakhiwo (26 ?? 28).Iintsalela ezihlanu ezingaguqukiyo zifunyenwe kwi-β strand kunye ne-α helix ye-C-terminal inxalenye ye-nsp9.Iintsalela ezintathu ezingaguquguqukiyo zenza i-NNE motif ye-N terminus ye-nsp9.Kutyhilwe ukuba i-Asn yesibini yale motif yintsalela engaguqukiyo kuphela, ekwabelwana ngayo yi-hypothetical nsp9 ye-coronavirus yesele ehlobene kude, kwaye imele i-Microhyla letovirus 1 iintlobo kwi-subfamily Letovirinae ye-Alphaletovirus.Ukugcinwa kweentsalela kwizinto ze-nsp9 zolwakhiwo lwesibini zinokulungelelaniswa ngokuqwalaselwa kwesakhiwo ukugcina ukusongwa okanye iipropati ezibophelelayo ze-RNA ezaziwayo.Nangona kunjalo, oku kuqikelelwa akubonakali kusebenza kulondolozo lwe-NNE, kwaye phambi kolu phononongo, uhlobo lwemithintelo ethintela ukwahluka kolandelelwano lwe-trieptide lwalusithekile ngokupheleleyo.
Ukuze kubonwe ukubaluleka kwe-nsp9-NMPylation kunye nolondolozo lwe-NNE kuphindaphindo lwe-coronavirus, siye savelisa i-HCoV-229E mutants, ephethe ukutshintshwa okukodwa okanye kabini kwe-nsp9 N-terminal iintsalela, ebonisa ukuba i-nsp9 NMPylation iyingozi kwi-vitro.Phambi kokuba siqale, sizama ukuphendula umbuzo wokuba ingaba olu tshintsho (kufutshane nesiza se-nsp8|9 sokucanda) luchaphazela ulungiso lweproteolytic yengingqi ye-C-terminal pp1a.Isethi ye-polyprotein ye-nsp7-11 yokwakha equlethe izinto ezihambelanayo ezihambelanayo kwi-N-terminus ye-nsp9 zaveliswa kwi-E. coli kwaye zisikwe kunye ne-Mpro ye-recombinant.Iproteolytic cleavage yeziza ezine (kubandakanywa nesiza seflanki ye-nsp9) ayichatshazelwanga kakhulu nangakuphi na ukufakwa endaweni (i-SI appendix, Figure S9), ngaphandle kotshintsho lwesakhiwo kwezi proteni eziphazamisana ne-Mpro-mediated nsp8|9 cleavage ( Okanye enye) iwebhusayithi.
Iiseli ze-Huh-7 zatshintshwa nge-genome-length HCoV-229E RNA, i-encoding Ala okanye i-Ser substitutions kwi-tripeptides ye-NNE egciniweyo (N3825, N3826, kunye ne-E3827) kwi-nsp9 N terminus, ebonisa ukuba uninzi lweenguqu zifa.Siye sakwazi ukuhlangula intsholongwane ngokutshintsha i-Ser okanye i-Ala ye-N-terminal Asn (N2835A okanye i-N2835S), kodwa asiphumelelanga ukubuyisela intsholongwane kunye nezinye iinguqulelo ezizimeleyo kunye neziphindwe kabini kulandelelwano lwe-NNE (N3826A, N3826S, NN3825 / 6AA, NN3825/6SS), E3827A) (Umfanekiso 5D).
Ezi ziphumo zibonisa ukuba ukuphindaphindwa kwee-coronavirus kwinkcubeko yethishu kuthintelwe (efanayo okanye efanayo), ukunciphisa uguqulo lwendalo lweendawo ze-nsp9 ze-NMPylation emzimbeni, kunye nokuxhasa indima ephambili yale mpendulo kumjikelo wobomi be-coronavirus.
Kwiseti yokugqibela yovavanyo, sivelise i-C-terminal His6 ebhalwe SARS-CoV-2 nsp12 kunye ne-nsp9, kunye neendlela ezimbini eziguqukayo ze-nsp12 kwi-E. coli.Iintsalela zesayithi ezisebenzayo kwi-domain ye-NiRAN kunye ne-RdRp ngokulandelanayo Sebenzisa i-Ala endaweni (Umfanekiso we-6A kunye ne-SI isihlomelo, iThebhile S2).I-K4465 kwi-SARS-CoV-2 nsp12 ihambelana ne-K4135 kwi-HCoV-229E (Isihlomelo se-SI, uMzobo S2), obonakalise ukuba uyafuneka kumsebenzi we-NiRAN kunye nokuphindaphinda kwe-HCoV-229E (Umfanekiso 3D kunye no-E).Le ntsalela ihambelana nentsholongwane ye-arterial EAV nsp9 K94 intsalela, eyayiboniswa ngaphambili ukuba iyimfuneko kwi-NiRAN self-UMPylation / self-GMPylation (16).Njengoko kubonisiwe kuMfanekiso 6B, i-SARS-CoV-2 nsp12 inomsebenzi wodluliselo we-UMP usebenzisa i-nsp9 njengenxalenye engaphantsi, ngelixa i-nsp12_K4465A isayithi eguqukayo esebenzayo ayisebenzi.Ukutshintshwa kabini kwi-SDD yokulandelelana kweempawu ze-RdRp motif C ayichaphazeli umsebenzi wokudluliselwa kwe-UMP (Umfanekiso 6B), ebonisa ukuba umsebenzi we-RdRp awunayo impembelelo ngqo kwi-nsp9 UMPylation.Idatha efanayo ifunyenwe ngokusebenzisa i-CTP, i-GTP kunye ne-ATP (i-appendix ye-SI, uMzobo S10).Ngamafutshane, ezi datha zibonisa ukuba i-NiRAN-mediated nsp9 NMPylation inomsebenzi wolondolozo kwi-coronavirus emele uluhlu olwahlukileyo lwe-orthocoronavirus subfamily.
I-SARS-CoV-2 nsp12-mediated NMPylation ye-nsp9.(A) Ijeli yeCoomassie ene-SDS-polyacrylamide ebonisa iprotein ephinda isetyenziswa kuvavanyo lwe-NMPylation.Njengolawulo, iprotheni eguquguqukayo enendawo esebenzayo endaweni ye-NiRAN domain (K4465A) kunye ne-RdRp domain (DD5152 / 3AA) ye-SARS-CoV-2 nsp12 yasetyenziswa.Intsalela yamanani isekelwe kwindawo ku-pp1ab.(B) I-Autoradiograph ye-UMPylation yokufumanisa usebenzisa i-nsp9-His6 kunye ne- [α-32P] UTP njenge-substrate ye-nsp12-His6 (uhlobo lwasendle [wt] kunye ne-mutant).Ubunzima bemolekyuli (kwi-kilodaltons) yeprotheyini ebhaliweyo iboniswe ngasekhohlo.
Imimandla ye-NiRAN igcinwa ngokubanzi kwi-Nidovirales (16), ebonisa ukuba i-catalyze reactions ze-enzymatic eziyimfuneko kuphindaphindo lwe-Nidovirus.Kolu phononongo, sikwazile ukubonisa ukuba i-domain ye-NiRAN ye-coronavirus idlulisela i-NMP (eyenziwe kwi-NTP) ukuya kwi-nsp9, iprotein ye-RNA engaqondakaliyo ebophelelayo ebandakanyeka ekuphindaphindweni kwentsholongwane (26 ?? 29), ukuyimisela njengento ekujoliswe kuyo yendalo kwaye iqabane le-coronavirus RTC.
I-domain ye-NiRAN yabelana ngeendlela ezintathu zokulandelelana (AN, BN, kunye ne-CN), eziqulethe inani elincinci kakhulu leentsalela ezigcinwe kuzo zonke iintsapho kwi-monophyletic kodwa i-Nidovirales order eyahluke kakhulu (8, 16).Uphononongo lwakutsha nje lubonise ukuba zinxulumene ngokwesakhiwo kusapho olukhulu olungachazwanga lweprotein kinase-like proteins, eyayibizwa ngokuba yintsapho yakwaSelO (17, 19, 22, 30, 31).Iiprotheyini ezinxulumene ne-SelO zine-kinase folds, kodwa zinqongophele iintsalela zesayithi ezininzi ezigciniweyo kwi-classical kinases (22, 32).Ngokusekwe kwi-reverse orientation ye-ATP molecules ebotshelelwe kwindawo esebenzayo kwaye izinziswe ngokusebenzisana okuthe ngqo, i-SelO yaqiniswa kwaye emva koko yaqinisekiswa ukuba idlulisele i-AMP (endaweni ye-phosphate) kwi-protein substrate (22), ngelixa enye i-bacterial SelO-like protein YdiU ine kutsha nje kubonisiwe ukwenza uncamathiselo olubambeneyo lwe-UMP ukuya eTyr kunye neentsalela Zakhe zeeprotein substrates ezahlukeneyo (33).
Ukuze kuqinisekiswe kwaye kwandiswe ingqikelelo yeentsalela zendawo esebenzayo ye-coronavirus ye-NiRAN, sisebenzise iindlela ze-biochemical kunye ne-reverse genetics ukwenza uhlalutyo lwenguqu kwi-coronavirus nsp12 (Umfanekiso 3D kunye ne-E kunye ne-SI isihlomelo, uMfanekiso S3 kunye netafile) S1â S4).Idatha ibonisa ukuba ukutshintshwa kwe-HCoV-229E K4135, i-R4178 kunye ne-D4280 kunye ne-Ala kuphelisa umsebenzi wokudluliselwa kwe-NMP kwi-vitro kunye nokuphindaphinda kwentsholongwane kwinkcubeko yeseli (Figure 3D kunye ne-E kunye ne-SI appendices, Figure S3), exhasa ubukho babo kwi-NTP γ-phosphate. ( K4135, R4178) kunye nokulungelelaniswa kwee-ion zetsimbi zesayithi ezisebenzayo (D4280).I-E4145A endaweni yeGlu egciniweyo kuluhlu lwentsholongwane yendlwane yentaka eqikelelwe ukuba izinzisa indawo ye-K4135 (17) yaboniswa ukuphelisa ukuphindaphindeka kwentsholongwane, kodwa ngokumangalisayo, umsebenzi wagcinwa kwi-in vitro NMPylation assay (Figure 3D kunye no-E kunye Isihlomelo se-SI, uMzobo S3 kunye neeThebhile S1-S4).Ukuqwalaselwa okufanayo kwenziwa xa ukutshintshwa okuhambelanayo kwaziswa kwi-YdiU homolog ye-Salmonella typhimurium (E130A) (33).Idityaniswe kunye, ezi datha zixhasa umsebenzi wolawulo wale ntsalela egciniweyo kunomsebenzi we-catalytic.
Ukutshintsha intsalela ye-Phe egciniweyo (F4281A) ngaphakathi koluhlu lwe-nestovirus kwisizinda se-HCoV-229E NiRAN (8) kubangele ukuncipha komsebenzi we-NMPylation kwi-vitro kunye nokuncipha okukhulu kokuphindaphinda kwentsholongwane kwinkcubeko yeseli (Umfanekiso 3D, E kunye ne-SI) isihlomelo, Umfanekiso S3).Idatha ihambelana nomsebenzi obalulekileyo wolawulo wale ntsalela, njenge-homologous DFG motif Phe residue eboniswe ngaphambili.Kwiprotheyini ye-classical kinases, iyingxenye ye-Mg2 + yokubopha i-loop kwaye inceda ukudibanisa nokulawula umqolo???Okufunekayo kumsebenzi osebenzayo we-catalytic (32, 34).Ukutshintsha i-Ala kunye ne-Arg yeentsalela ze-K4116 (kwi-motif ye-preAN), ngokulandelelanayo, yaphelisa ukuphindaphinda kwentsholongwane kwaye, njengoko bekulindelekile, yaba nemiphumo eyahlukileyo kumsebenzi wokudluliselwa kwe-NMP kwi-vitro, ngokuxhomekeke kwikhonkco yecala le-amino acid elaziswayo (Umfanekiso 3D kunye ne-E kunye ne-SI appendices. , Umfanekiso S3).Idatha esebenzayo ihambelana nolwazi lwesakhiwo, ebonisa ukuba le ntsalela iseke ukusebenzisana ne-ATP phosphate (17).Kwi-domain ye-NiRAN yezinye iintsapho ze-virus ezifakwe kwindlwane, indawo ye-HCoV-229E pp1a/pp1ab K4116 ihlalwe nguLys, Arg okanye i-His (8), ebonisa ukuba uthintelo olusebenzayo lwale ntsalela ethile ikhululekile.Ukutshintshwa kwe-D4188A kunye ne-D4283A kuphelisa okanye kunciphisa kakhulu umsebenzi we-enzyme kunye nokuphelisa ukuphindaphinda kwentsholongwane (Umfanekiso 3).Ezi ntsalela zimbini zilondolozwe kuninzi (kodwa ayizizo zonke) iintsholongwane ezihlala kwindlwane (8), nto leyo ebonisa umsebenzi obalulekileyo othe ngqo wosapho kodwa ongengowo-catalytic.Ukutshintshwa kwe-Ala kwezinye iintsalela ze-Lys kunye ne-Asp (K4113A, D4180A, D4197A kunye ne-D4273A) ezingalondolozwanga kwi-Coronaviridae okanye ezinye iintsapho ze-Nestioviridae (8) zisetyenziswe njengolawulo.Njengoko kulindelekile, ezi zitshintshi zibekezeleleka kakhulu, kunye nokunciphisa okuncinci kumsebenzi we-enzyme kunye nokuphindaphinda kwentsholongwane kwezinye iimeko (Umfanekiso we-3 kunye ne-SI isihlomelo, i-Figure S3).Lilonke, idatha ye-coronavirus mutagenesis ihambelana kakhulu ne-self-GMP kunye nedatha yemfuza ye-reverse ye-EAV NiRAN-RdRp (16), apho i-EAV nsp9 (i-coronavirus nsp12 ortholog) intsalela ye-K94 (ehambelana ne-HCoV- 229E K4135) imisebenzi ebalulekileyo), i-R124 (ihambelana ne-R4178), i-D132 (ihambelana ne-D4188), i-D165 (ihambelana ne-D4280), i-F166 (ihambelana ne-F4281).Ukongeza, idatha ye-HCoV-229E mutagenesis iyahambelana kwaye yandisiwe ukusuka kwidatha yemfuzo exelwe ngaphambili ye-SARS-CoV (16), ifana kakhulu naleyo iqwalaselwe kwi-CN motif ehambelanayo Phe-to-Ala mutant SARS-CoV_nsp12 i-phenotype echazwe -F219A kunye ne-HCoV-229E_F4281A (Umfanekiso 3 D kunye no-E kunye nesihlomelo se-SI, uMzobo S3 kunye neThebhile S1-S4).
Xa kuthelekiswa ne-EAV orthologs (16), enokhetho olucacileyo lwe-UTP kunye ne-GTP (kwi-self-NMPylation reaction), uphononongo lwethu lubonisa ukuba i-coronavirus NiRAN domain (emelwe yiHCoV-229E kunye ne-SARS-CoV-2) inokusebenza ngokufanelekileyo. zidluliselwe Zone ii-NMPs, nangona kukho ukhetho oluncinci lwe-UMP (iFigure 1 and 3).Ubume obusezantsi ngokwentelekiso obuthile be-NTP co-substrate iyahambelana neSars-CoV-2 nsp7/8/12/13 supercomposite isakhiwo esandula kuxelwa, apho i-ADP-Mg2+ ibophelela kwindawo esebenzayo ye-NiRAN, kodwa hayi ngeSahlulo se-adenine. yokwenziwa konxibelelwano oluthile (17).Kuphononongo lwethu, uhlobo lwe-nucleotide olusetyenziswa kwi-NMPylation reaction alunampembelelo eyahlukileyo kumsebenzi weprotheni eguqukayo (iSihlomelo se-SI, uMzobo we-S3), ebonisa ukuba akukho nanye yale ntsalela ehambelana ngokusondeleyo nokubophelela kwe-nucleobase ethile.Isiseko solwakhiwo kunye nentsingiselo yebhayoloji enokubakho yeenketho ezahlukeneyo ze-NTP ze-co-substrate eziqatshelwe kwimimandla ye-NiRAN ye-coronavirus kunye neentsholongwane ze-arterial zihlala zifundwa;zisenokuba yinyaniso okanye zisenokuba ngenxa yokusikelwa umda kwizifundo zabo.Okwangoku, ayinakunyanzelwa ukuba umsebenzi we-NMPylator onokuthi ubekho we-arterial virus domain NiRAN (xa kuthelekiswa nomsebenzi obonakaliswe ngaphambili we-NMPylation) unokhetho olwahlukileyo lwe-co-substrate, kuthathelwa ingqalelo ukuba ukufana phakathi kwe-arterial kunye ne-coronavirus. I-domain ye-NiRAN ikumda wayo.Thelekisa ulandelelwano (16).Xa kuthelekiswa ne-pseudokinase SelO, esebenzisa i-Mg2 + njenge-cofactor, umsebenzi we-coronavirus kunye ne-arterial virus ye-NiRAN ixhomekeke kwi-Mn2 + (16) (Umfanekiso we-3C kunye ne-SI isihlomelo, uMzobo S1).Ukuxhomekeka kwe-Mn2 + kunye nokukhethwa okucacileyo kwe-UTP yinto engaqhelekanga yeprotheyini ye-NMPylators, kwaye isanda kuqinisekiswa kwiprotheni ye-YdiU ye-Salmonella typhimurium, eyenza i-Mn2 + -ixhomekeke kwiprotheni exhomekeke kwi-UMPylation ukukhusela iiseli kwi-stress induction Cell ATP pool. 33).
Ukufana okusandul 'ukuchazwa kwesakhiwo phakathi kwesizinda se-coronavirus ye-NiRAN kunye ne-cellular protein kinases (17, 19) ibonelela ngenkxaso eyongezelelweyo kubuchule be-NiRAN bokudibanisa ngokubambisana i-NMP kwezinye iiproteni esizinike ingxelo kolu phononongo.Sigxininise kukhangelo lwethu lweethagethi ezinokwenzeka ze-NiRAN kwiiproteni ezifakwe kwi-HCoV-229E ORF1a, ezaziwa ngokunceda ngokuthe ngqo okanye ngokungathanga ngqo i-RTC's ORF1b-encoded replicase (12, 35).Imifuniselo yethu ibonelela ngobungqina obubambekayo be-NMPylation esebenzayo kunye nethe ngqo ye-nsp9 (Umfanekiso 2).Ukuba iprotheni ekujoliswe kuyo isetyenziselwa ukugqithisa kwe-molar eyi-8 kumaxesha e-10 aphezulu kune-enzyme (nsp12), iqinisekisiwe ukuba i-nsp9 iphelele (mono) i-NMPized (Umfanekiso 4).Sagqiba ekubeni ukusebenzisana phakathi kwe-nsp12 kunye ne-nsp9 kufutshane kwaye akuyi kwenza i-complex ezinzile kunye ne-nsp9 (ngokungabikho kwamanye ama-subunits e-RTC).Esi sigqibo sixhaswa zizifundo zonxibelelwano lweprotheyini kwi-SARS-CoV proteome (35).Uhlalutyo lwe-MS luchonge i-amine engundoqo yentsalela ye-N-terminal ye-nsp9 njengendawo ye-NMPylation (i-SI appendix, Figure S5).Ukuqulunqwa kwebhondi ye-phosphoramidate kunye neqela le-amino le-N-terminal lihlula umsebenzi we-NMPylation we-NiRAN-mediated ukusuka kwi-Pseudomonas syringae SelO-mediated AMPylation reaction, eyenza ukubunjwa kwe-O-linked AMP kwi-Ser, Thr, okanye iTyr intsalela ye-Peptide adduct ( 22), kunye ne-S. typhimurium YdiU iifom ze-O-linked (kunye neTyr) kunye ne-N-linked (kunye neyakhe) i-peptide-UMP adducts.Ulwazi oluncinci olukhoyo kwintsapho ye-SelO yeeprotheni ibonisa ukuba amalungu ale ntsapho enkulu yeprotheyini ahluke kakhulu ekubunjweni kwe-peptide-NMP adducts.Olu luqwalaselo olunika umdla olufuna ukufundwa ngakumbi.
Idatha efunyenwe kolu phononongo yasikhokelela ekubeni sicinge ukuba i-NMPylation ye-nsp9 ifuna i-N-terminus yamahhala.Kumxholo wophindaphindo lwentsholongwane, oku kuya kubonelelwa ngokuqhekeka kweproteolytic ye-nsp8|nsp9 yokusetyenzwa kwesiza kwi-replicase polyprotein pp1a ephakathi nguMpro kunye nepp1ab.Kwiindawo ezininzi zekoronavirus, umahluko phakathi kwesi siza sikhethekileyo (VKLQ|NNEI kwi-HCoV-229E) kunye nazo zonke ezinye iisayithi ze-coronavirus Mpro cleavage yi-Asn (kunokuba yenye intsalela encinci, efana ne-Ala, i-Ser okanye i-Gly) ithatha i-P1â???Indawo (36).Idatha ye-peptide cleavage efunyenwe kwizifundo zangaphambili ibonise ukuba ukusebenza kakuhle kwe-cleavage yendawo ye-nsp8|nsp9 yayingaphantsi kunezinye iisayithi, ebonisa ukuba i-1) le ndawo ikhethekileyo ingaba nendima yokulawula ekuqhutyweni kwexesha elilungelelanisiweyo le-C-terminal. pp1a ummandla, okanye 2) a Indima ye-nsp9 N-terminus ekhethekileyo egciniweyo kuphindaphindo lwentsholongwane (37).Idatha yethu (i-Figure 5A) ibonise ukuba ifom ye-recombinant ye-nsp9 ethwele ulandelelwano lwe-N-terminal yokwenyani yenziwe ngokufanelekileyo yi-NMPized ngu-nsp12.I-N-terminal flanking sequence isuswe yi-factor Xa (nsp9-His6; i-SI appendix, i-Table S1) okanye i-Mpro-mediated cleavage (nsp7-11-His6; Umfanekiso 5A kunye ne-SI appendix, iThebhile S1).Okubalulekileyo, i-precursor enganqunywanga ye-nsp9-equlethe i-nsp7-11-His6 ibonise ukuchasana ne-NMPylation ye-nsp12, ehambelana nedatha yethu, ebonisa ukuba i-nsp9-NMP adduct yenziwe nge-N-terminal primary amine (i-SI appendix, Figure S5) .Ukufumana ukuqonda okunzulu kwe-NiRAN substrate specificity, siye sagxila kwiintsalela ze-N-terminal ezikufutshane ze-nsp9.Xa kungabikho ezinye iiprotheyini, ziguquguquka ngokwesakhiwo, zithintele ukuba zingabonwa kwifomu engabhalwanga ye-nsp9 (26 28, 38), ebonisa ukuhluka kwazo okulinganiselwe kwendalo Oku kubangelwa ulandelelwano olubalulekileyo-oluthile (kungekhona isakhiwo sesibini esihambelanayo) umsebenzi we-nsp9 N-terminal fragment.Ukutshintshwa kwe-Ala kweentsalela ezigciniweyo kulo mmandla (Amanani 5C kunye no-D kunye ne-SI isihlomelo, uMfanekiso S8) ubonisa ukuba i-N3826 iyimfuneko kwi-nsp9 NMPylation in vitro, ngelixa i-N3825A kunye ne-E3827A endaweni ye-E3827A ikhokelela ekunciphiseni kwe-NMPylation, ngelixa i-M3829A ingekho kunye ne-P38 do38 .Ngokucacileyo ichaphazela i-nsp9 NMPylation.Nangona ukutshintshwa kwe-N-terminal Asn (N3825A, N3825S) kunempembelelo ephakathi kwi-nsp9 NMPylation kunye nokuphindaphinda kwentsholongwane kwinkcubeko yeseli (Umfanekiso 5C kunye no-D), ukususwa kokulandelelana kwe-Asn kwi-N-terminal 3825-NN dipeptide. ibonakalisiwe Iyingozi kwiintsholongwane, ebonisa ukuba intsalela ye-Asn iyafuneka ngaphambi kokuba enye intsalela kwi-N-terminus, ngokukhethekileyo i-Asn, nangona kubonakala ngathi ukutshintshwa kweentsalela ezifanayo kunokunyamezela ngokuyinxenye (Figure 5B, C, and D).Siphetha ukuba i-dipeptide ye-3825-NN, ngokukodwa i-N3826 egciniweyo kwaye iyimfuneko i-residu ngaphakathi kwendawo ye-coronavirus (i-appendix ye-SI, i-Figure S6), iqinisekisa ukubopha okuchanekileyo kunye nokuqhelaniswa kwe-nsp9 N-terminus kwindawo esebenzayo ye-NiRAN.
Ukutshintsha i-Ala (E3827A) ye-Glu egciniweyo yazo zonke ii-subfamilies igcina i-nsp9 NMPylation kwi-vitro kodwa ibulala iintsholongwane kwinkcubeko yeseli (Umfanekiso 5C kunye no-D), ebonisa umsebenzi owongezelelweyo wale ntsalela, umzekelo, kwintsebenziswano engundoqo (i-NMPylated okanye engalungiswanga ) nsp9 I-N-terminus kunye nezinye izinto ezibandakanyekayo ekuphindaphindeni intsholongwane.Utshintsho lwe-Nsp9 aluzange luchaphazele inkqubo ye-proteolytic ye-nsp9 okanye nayiphi na i-nsps ekufutshane (39) (iSihlomelo se-SI, uMzobo S9), ebonisa ukuba i-phenotypes ebulalayo yeenguqu ezininzi ze-nsp9 eziqatshelweyo azibangelwa kukungahambi kakuhle kwenkqubo ye-C proteolytic-terminal pp1a. .
Le datha ingentla ibonelela ngobungqina bokuba emva konyango lwe-Mpro-mediated ye-nsp8|9 indawo yokuqhawula kwi-pp1a/pp1ab, i-N-terminus ye-nsp9 inokuthi i-UMPylated (okanye ilungiswe ngokuyinxenye kunye nenye i-NMP).Ukongeza, ugcino olugqwesileyo lwe-N-terminus ye-nsp9 (kubandakanya isinye kunye neentsalela ze-Asn ezingaguqukiyo kusapho lwe-coronavirus) kunye nedatha eguqukayo yemfuza efunyenwe kolu phononongo (Amanani 3E kunye no-5D) asikhokelele ekubeni sigqibe kwelokuba i-nsp9 NMPylation echaziweyo. inxulumene nebhayoloji kwaye ibalulekile kuphindaphindo lwe-coronavirus.Iziphumo ezisebenzayo zolu hlengahlengiso zihlala zifundwa, umzekelo, malunga nokuchazwa kwangaphambili (okungacaciswanga) nsp9 (ifomu engalungiswanga) umsebenzi wokubopha i-RNA (2628).I-N-terminal NMPylation inokuchaphazela intsebenziswano ye-nsp9 kunye neprotheyini okanye i-RNA substrates okanye ukubunjwa kweendibano ezahlukeneyo zamanqanaba amane.Ezi ziqwalaselwe kwizifundo zolwakhiwo kwaye ziqinisekisiwe ukuba zisebenza ngokunxulumene nokuphindaphindwa kwe-coronavirus, nangona ngakumbi xa kungekho Kwimeko yolu hlengahlengiso (26- ââ29, 40).
Nangona okujoliswe kuko kwesizinda se-coronavirus ye-NiRAN kusafuneka kuchazwe ngokweenkcukacha ngakumbi, idatha yethu ibonisa ukuba iprotein ekujoliswe kuyo ye-coronavirus domain ye-NiRAN imxinwa kakhulu.Nangona ukugcinwa kweentsalela zesayithi ezisebenzayo (8, 16) kwi-domain ye-NiRAN yazo zonke iintsapho ze-nidovirus zixhasa ngamandla umsebenzi we-NMPylator egciniweyo ezi proteni, isazisi se-substrate ebophelelayo iintsalela zepokotho yesi sizinda Ukugcinwa kunye nokugcinwa kuhlala kuphawulwa. , kwaye inokwahluka phakathi kweentsapho ezahlukeneyo ngeenjongo zeNidovirales.Ngokufanayo, iithagethi ezifanelekileyo zezinye iintsholongwane ezihlala kwindlwane azikaqinisekiswa.Zisenokuba zii-orthologies ezikude ze-nsp9 okanye ezinye iiproteni, kuba ulandelelwano olungaphandle kwemimandla emihlanu ephindaphindayo egcinwa ngokubanzi kwiintsholongwane ezihlala kwindlwane ayigcinwanga kangako (8), kubandakanywa uluhlu lwegenome phakathi kweMpro neNiRAN, Phakathi kwazo, i-nsp9 ikwindawo intsholongwane ye-corona.
Ukongezelela, okwangoku asikwazi ukukhupha ukuba kunokwenzeka ukuba i-domain ye-NiRAN ineethagethi ezongezelelweyo (kubandakanywa neselula).Kule meko, kuyafaneleka ukukhankanya ukuba ii-homologues zebhaktheriya kule proteni ekhulayo i-NMPylators (NMPylators) (30, 31) ibonakala ngathi "inabalawuli abakhulu"?I-NMP imodareyitha iindidi zeeprotheni zeselula ukulawula okanye ukuphelisa imisebenzi yazo ephantsi, ngaloo ndlela idlala indima kwiinkqubo ezahlukeneyo zebhayoloji, njengempendulo yoxinzelelo lweselula kunye ne-homeostasis ye-redox (22, 33).
Kule sifundo (i-Figure 2 kunye ne-4 kunye neSihlomelo se-SI, iMifanekiso ye-S3 kunye ne-S5), sakwazi ukubonisa ukuba i-nsp12 idlulisele inxalenye ye-UMP (NMP) kwindawo enye (egciniweyo) kwi-nsp9, ngelixa ezinye iiprotheni azizange ziguqulwe kwi isetyenziswe Ngaphantsi kweemeko, i-substrate echazwe kakuhle (kunokuba ikhululekile) ixhaswa.Ngokuhambelana nale nto, xa kuthelekiswa ne-N-terminal nsp9 NMPylation, umsebenzi we-nsp12's we-NMPylation uphantsi kakhulu, ukufumanisa kwayo kufuna ixesha elide lokuvezwa kwe-autoradiography, kunye nokunyuka kwe-10 kwi-concentration ye-nsp12 isetyenziswa.Ukongezelela, uhlalutyo lwethu lwe-MS aluphumelelanga ukunika ubungqina be-NMPylation ye-nsp12, ebonisa ukuba i-NiRAN domain self-NMPylation yi (eyona nto ingcono) umsebenzi wesibini.Nangona kunjalo, kufuneka kuqatshelwe ukuba ezinye izifundo zinike ubungqina bokuqala bokuba isimo se-self-AMPylation ye-NMPylator yebhaktheriya inokulawula umsebenzi wabo we-NMPylation kwezinye iiprotheni substrates (22, 33).Ngoko ke, uphando olongezelelweyo luyafuneka ukuphanda iziphumo ezinokuthi zisebenze kwimisebenzi ye-self-NMPylation echazwe kwi-EAV nsp9 (16) kunye ne-coronavirus nsp12 (olu phononongo), kubandakanywa nempembelelo ecetywayo efana ne-chaperone ekusongweni kwe-C-terminal RdRp domain ( 16)).
Ngaphambili, iingqikelelo ezininzi malunga nemisebenzi enokwenzeka ezantsi ye-nidoviral domain ye-NiRAN iye yaqwalaselwa, kubandakanywa i-RNA ligase, i-RNA -capped guanylate transferase kunye neprotein priming activity (16), kodwa akukho nanye kuzo ehambelanayo nemisebenzi ekhoyo esezantsi.Ulwazi olufunyenwe kwezi zikhundla zilandelayo lufana kanye ngexesha elifanayo ngaphandle kokwenza iingcamango ezongezelelweyo.Idatha efunyenwe kulolu cwaningo ihambelana kakhulu (kodwa ayikwazi ukubonisa) ukuba i-domain ye-NiRAN ibandakanyeka ekuqalisweni kwe-protein-induced RNA synthesis.Ngaphambili kwakukholelwa ukuba umsebenzi we-domain ye-NiRAN kwi-5 ??²-i-RNA capping okanye i-RNA ligation reactions ayichatshazelwa zezi kunye neNkxaso yezinye iinkcukacha.Ke ngoko, umzekelo, indawo esebenzayo ye-NiRAN ithathwa njengebandakanya i-Asp egciniweyo njengesiseko jikelele (D252 kwiPseudomonas syringae SelO; D4271 kwi-HCoV-229E pp1ab; D208 kwi-SARS-CoV-2 nsp12) (Isihlomelo seSI, umzobo 2 ).I-S2) (17, 22, 33), ngelixa i-catalysis kwi-ATP-exhomekeke kwi-RNA ligase kunye ne-RNA capping enzyme iqhutyelwa yi-covalent enzyme-(lysyl-N) -NMP ephakathi, ebandakanya intsalela yeLys engatshintshiyo ( 41).Ukongeza, ulandelelwano olumangalisayo olusekwe ngokuthe ngqo kwe-coronavirus ye-NiRAN kwiithagethi zeprotheyini ezigciniweyo kunye nokucaciswa okuphumlileyo kwe-NTP co-substrates (ikhetha i-UTP) ichasa i-enzyme ye-capping ye-NiRAN-mediated okanye imisebenzi efana ne-RNA ligase.
Ngokucacileyo, mninzi umsebenzi owongezelelweyo ofunekayo ukuze kuqinisekiswe kwaye, ukuba kuqinisekisiwe, kucaciswe ngendima enokwenzeka ye-nsp9-UMP (nsp9-NMP) kwiprotein-induced RNA synthesis, eya kudibanisa uninzi olunomdla kodwa (ukuza kuthi ga ngoku) iingxelo ezixelwe ngaphambili. .Iingqwalasela ezizimeleyo.Umzekelo, kuye kwamiselwa ukuba isiphelo se-RNA engalunganga ye-coronavirus iqala nge-oligo (U) strand (42, 43).Olu qwalaselo luhambelana noluvo lokuba ukuhlanganiswa kwe-negative-strand RNA iqalwa ngokubophelela ifom ye-UMPylated ye-nsp9 kwi-poly(A) umsila (izixhokonxa), ezinokukhuthazwa yi-RNA yayo yokubopha Umsebenzi kunye / okanye ukusebenzisana kunye enye iprotheni ye-RTC.Isahlulo se-UMP esibonelelwe ngu-nsp9 sinokusetyenziswa njenge "primer" ye-nsp7/8/nsp12-mediated oligouridylation, kusetyenziswa i-3??²-poly(A) umsila kwi-genomic RNA okanye Enye i-oligo (A)-equlathe ulandelelwano. isebenza njenge template, efana nendlela esekwe kwiprotheni ye-picornavirus VPg (44).Kuthekani ukuba isindululo "asiqhelekanga"????Ukuqaliswa kwe-(iprotein-induced) negative-strand RNA synthesis ibonelela ngekhonkco kwimigqaliselo, ebonisa ukuba i-coronavirus negative-strand RNA ine-UMP (endaweni ye-UTP) ekupheleni kwayo (42), ethathwa njengebonisa ukuba i-nucleic acid Dicer iqhekeza isiphelo sephosphorylated yi-endonuclease engaziwayo ye-uridine.Ukuba iqinisekisiwe, lo msebenzi we-nucleic acid we-hydrolytic unokunceda ukukhulula i-oligomeric UMPylated form ye-nsp9 ukusuka kwi-5 ² ekupheleni komtya ongekazalwa.Indima enokwenzeka ye-nsp9 kwi-protein priming ikwahambelana nezifundo zofuzo zangaphambili, ezibonise ukuba i-nsp9 (kunye ne-nsp8) isebenzisana ngokunzulu kwaye ngokukodwa into egciniweyo ye-RNA ebambeleyo kufutshane nesiphelo se-3 se-coronavirus genome.45).Ngokwale ngxelo, olu qwalaselo lwangaphambili ngoku lungaphinda luphononongwe kwaye lwandiswe ngophando olongezelelweyo.
Isishwankathelo, idatha yethu igqibe umsebenzi othile wethegi ye-enzyme ene-nested virus edityaniswe ne-RdRp kwi-N-terminus.Kwi-coronavirus, lo msebenzi osandula ukufunyanwa we-NiRAN-mediated UMPylator/NMPylator usetyenziselwa ukuxhomekeka kwi-Mn2 + kunye neentsalela ze-Asn ezikufutshane kwaye kubangele ukubunjwa kwe-phosphoramidate (yamandla aphantsi) ibhondi kunye ne-N-terminal primary amine.Ngokucocwa kwe-Mpro-mediated kwindawo ye-nsp8|9 yokucoca, i-nsp9 ekujoliswe kuyo ingasetyenziselwa i-NMPylation, ebonisa ukudibanisa okusebenzayo phakathi kwe-protease kunye nesizinda se-NiRAN, esinabela kwi-RdRp.Ukugcinwa kweentsalela eziphambili kwindawo esebenzayo ye-nsp12 ye-NiRAN kunye nethagethi ye-nsp9, idityaniswe nedatha efunyenwe kwii-coronavirus ezimbini ezibandakanya i-SARS-CoV-2, ibonelela ngobungqina obunamandla bokuba i-nsp9 NMPylation yi-coronavirus Iimpawu zogcino zikwalinyathelo eliphambili lokuphindaphinda intsholongwane.Idatha ekhoyo isikhokelela ekubeni sigqibe kwelokuba indima ethile ye-NMPylated uhlobo lwe-nsp9 kwiprotein-induced RNA synthesis yimeko efanelekileyo ye-coronavirus kunye nezinye iintsholongwane ezihlala kwindlwane, kwaye i-NiRAN inokujolisa kwezinye iiproteni ezingaziwayo.Lawula intsholongwane.Intsebenziswano yomamkeli.Ukuba kuqinisekisiwe, ukubandakanyeka kweprotein primers kwi-viral RNA synthesis kuya kwandisa ulandelelwano oluhambelanayo lwe-Mpro/3CLpro kunye ne-RdRp domains phakathi kwe-coronavirus efunyenwe ngaphambili kunye ne-picornavirus-like supergroup (9), esele idityaniswe ngoku kwi-Pisonivirites esanda kusekwa. 46) kudidi.
Idatha yethu ikwabonisa ukuba imisebenzi esisiseko, ekhethiweyo kunye neyolondolozo ye-enzyme echongiweyo kolu phononongo ingasetyenziswa njengeethagethi zamachiza e-antiviral.Iikhompawundi eziphazamisana nokubophelela (kunye nokuguqulwa okulandelayo) kwe-nsp9 N-terminus egciniweyo kwindawo esebenzayo ye-NiRAN inokuphuhliswa ibe ngamachiza asebenzayo kunye namachiza e-antiviral, alungele unyango lwezilwanyana kunye nee-coronavirus zabantu ezivela kwiintlobo ezahlukeneyo zosulelo. , kuquka i-SARS-CoV-2 kunye ne-Middle East Respiratory Syndrome Coronavirus.
Ulandelelwano lwekhowudi yeprotheyini ye-coronavirus eveliswe kolu phononongo yandiswa yi-RT-PCR isebenzisa i-RNA ebekwe yodwa kwi-Huh-7 eyosulelwe yi-HCoV-229E okanye i-Vero E6 eyosulelwe yi-SARS-CoV-2, yaze yafakwa kusetyenziswa iinkqubo ze-cloning eziqhelekileyo.pMAL-c2 (New England Biological Laboratory) okanye pASK3-Ub-CHis6 (47) iVector yokubonakalisa (iSihlomelo seSI, iiThebhile S1 kunye ne-S2).I-codon substitutions enye yaziswa yi-PCR-based site-directed mutagenesis (48).Ukuvelisa i-protein ye-MBP ye-fusion, iiseli ze-E. coli ze-TB1 zatshintshwa kunye ne-pMAL-c2 efanelekileyo yokwakha i-plasmid (i-SI appendix, i-Table S1).Iprotheni yokudibanisa yahlanjululwa yi-amylose affinity chromatography kwaye yacandwa kunye ne-factor Xa.Emva koko, i-C-terminal His6-tagged protein yahlanjululwa yi-Ni-immobilized metal affinity chromatography (Ni-IMAC) njengoko kuchaziwe ngaphambili (49).Ukuvelisa iprotheni ye-ubiquitin fusion, iiseli ze-E. coli ze-TB1 zisebenzisa i-pASK3-Ub-CHIs6 i-plasmid efanelekileyo yokwakha (iSihlomelo se-SI, i-Tables S1 kunye ne-S2) kunye ne-pCGI ye-plasmid DNA encoding ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Inguqu (47).Iprotheyini ye-C-terminal His6-tagged coronavirus yacocwa njengoko bekuchaziwe ngaphambili (50).
Uvavanyo lwe-self-NMPylation lwe-HCoV-229E nsp12-His6 lwenziwa njengoko kuchaziwe kwi-EAV nsp9 (16).Ngamafutshane, i-nsp12-His6 (0.5 µM) iqulethe 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 µM buffer i-NTP ekhankanyiweyo kunye ne-0.17 µM zihambelana [α32-P]NTP (3,000 Ci/mmol; Hartmann Analytic) kwi-30 °C imizuzu engama-30.Kuzo zonke ezinye (eziqhelekileyo) iimvavanyo ze-NMPylation ze-nsp12-mediated nsp9 NMPylation, iimeko zokusabela zilungelelaniswa ngolu hlobo lulandelayo: nsp12-His6 (0.05 µM) kunye ne-nsp9-His6 (4 µM) kubukho be-50 mM HEPES-KOH (pH 8.0). ), 5 mM DTT, 1 mM MnCl2, 25 µM echaza i-NTP, kunye ne-0.17 µM ehambelana [α32-P]NTP.Emva kokufukanyelwa imizuzu eyi-10 nge-30 ° C, isampulu yokusabela ixutywe ne-SDS-PAGE i-buffer yesampula: 62.5 mM tris (hydroxymethyl) aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol kunye ne-0.005% bromophenol luhlaza.Iprotheyini yachithwa ngokufudumeza kwi-90 °C kwimizuzu ye-5 kwaye yahlulwe nge-12% ye-SDS-PAGE.Ijeli ilungisiwe kwaye ingcoliswe ngesisombululo se-Coomassie Brilliant Blue (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), idityanisiwe, kwaye yavezwa kwiscreen sokucinga sephosphorescent iiyure ezingama-20 (ukubona i-nsp12 evela kwi-NMPylation) okanye (ubuninzi) 2 Iiyure (ukuvavanya i-nsp9 NMPylation).Umfanekiso weTyphoon 9200 (GE Healthcare) yayisetyenziselwa ukuskena isikrini kwaye i-ImageJ yayisetyenziselwa ukuhlalutya ubungakanani bomqondiso.
Kuhlalutyo lwe-MS, i-1 µM nsp12-His6 kunye ne-10 µM nsp9 (ngaphandle kwethegi ye-hexahistidine) zisetyenzisiwe kuhlalutyo lwe-NMPylation (isihlomelo se-SI, iTheyibhile S1) kunye noxinzelelo olwandisiweyo lwe-500 µM UTP kunye ne-GTP zisetyenzisiwe.Ngokuxhomekeke ekugxininiseni kwabo kunye nomgangatho weprotheyini elindelekileyo, i-Waters ACQUITY H-Class ye-HPLC inkqubo exhotywe ngekholomu ye-MassPrep (Amanzi) yayisetyenziselwa i-desalt 1 kwi-10 µL ye-buffered protein solutions kwi-intanethi.Iprotheyini egalelwe ityuwa ikhutshiwe kumthombo we-ion ye-electrospray ye-Synapt G2Si mass spectrometer (Amanzi) ngokusebenzisa le mithamo ilandelayo ye-buffer A (amanzi/0.05% ye-asidi ye-formic) kunye ne-buffer B (i-acetonitrile/0.045% ye-asidi ye-formic), kunye nobushushu bekholomu bu I-60 ° C kunye nesantya sokuhamba kwe-0.1 mL / min: i-elution isocratically kunye ne-5% A ngemizuzu emi-2, emva koko i-gradient yomgca ukuya kwi-95% B ngaphakathi kwemizuzu eyi-8, kwaye igcine i-95% B eminye imizuzu emi-4.
Iiyoni ezintle ezinobunzima obuyi-500 ukuya kwi-5000 m/z zichongiwe.I-Glu-fibrinopeptide B ilinganiswa rhoqo kwimizuzwana engama-45 yokulungiswa kobunzima bokukhukuliseka okuzenzekelayo.Sebenzisa isoftware yesixhobo seMassLynx enolwandiso lweMaxEnt1 ukuze udibanise umndilili ophakathi emva kokutsala isiseko kunye nokugudiswa.
I-UMPylated HCoV-229E nsp9 yotyiswa ngokudibanisa i-trypsin elungisiweyo yolandelelwano (Serva) yaze yafukanyelwa ngobusuku kuma-37 °C.Ikholamu ye-Chromabond C18WP spin (inombolo yecandelo 730522; iMacherey-Nagel) yayisetyenziselwa ukulahla ityuwa kunye nokugxila kwiipeptides.Ekugqibeleni, i-peptide yachithwa kwi-25 µL yamanzi, equlethe i-5% ye-acetonitrile kunye ne-0.1% ye-asidi ye-formic.
Iisampulu zahlalutywa nguMS usebenzisa i-Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Inkqubo yokugqibela ye-nanoâ HPLC (i-Dionex), ixhotyiswe nge-custom end-50 cm ??I-75 μm ye-C18 RP ikholomu epakishwe nge-2.4 μm amaso amagnetic (uDkt. Albin Maisch High Performance LC GmbH) Qhagamshela kwi-spectrometer yobunzima kwi-intanethi ngomthombo we-Proxeon nanospray;tofa i-6 µL yesisombululo sokwetyisa i-trypsin kwi-300 µm yedayamitha yangaphakathi ×??I-1 cm ye-C18 ye-PepMap ikholamu yokugxilwa kwangaphambili (i-Thermo Scientific).Ukusebenzisa amanzi / i-0.05% ye-asidi ye-formic njenge-solvent, isampuli yabanjwa ngokuzenzekelayo kwaye yakhutshwa ityuwa kwinqanaba lokuhamba kwe-6 µL/min.
Oku kulandelayo kwe-gradients yamanzi / i-0.05% ye-asidi ye-formic (i-solvent A) kunye ne-80% ye-acetonitrile / 0.045% ye-asidi ye-formic (i-solvent B) isetyenziselwe ukufezekisa ukuhlukana kwe-tryptic peptides kwisantya sokuhamba kwe-300 nL / min: 4% B ye Imizuzu emi-5, emva koko i-30 i-gradient yomgca ukuya kwi-45% B ngaphakathi kwemizuzu, kunye nokunyuka komgca ukuya kwi-95% ye-solvent B ngaphakathi kwemizuzu emi-5.Qhagamshela ikholamu yechromatographic kwintsimbi ye-nano-emitter (Proxeon), kwaye utshize i-eluent ngokuthe ngqo kwi-capillary evuthayo ye-mass spectrometer usebenzisa amandla angama-2,300 V. Uvavanyo lwesaveyi ngesisombululo se-60,000 kwi-Orbitrap mass analyzer inxulumene ubuncinci bedatha emithathu ye-MS / MS scans, ekhutshwe ngokuguquguqukayo kwimizuzwana ye-30, usebenzisa i-ion linear trap collision induced dissociation okanye i-energy collision dissociation ephezulu idibaniswe nokufunyanwa kwe-orbitrap, Isisombululo yi-7,500.
Ixesha lokuposa: Aug-03-2021