Byahinduwe na Peter Sarnow, Ishuri Rikuru ry’Ubuvuzi rya Stanford, Kaminuza ya Stanford, muri Californiya, byemejwe ku ya 25 Ukuboza 2020 (bisubirwamo ku ya 25 Ukwakira 2020)
Turamenyesha imikoranire hagati ya subunits mugusubiramo coronavirus-transcription complex, zikenewe mukwigana no kubungabunga ubwihindurize.Twatanze ibimenyetso byerekana ko domaine ya NiRAN ifitanye isano na nsp12 ifite ibikorwa byo kwimura nucleoside monophosphate (NMP) muri trans, kandi twerekanye nsp9 (protein ihuza RNA) nkintego yayo.NiRAN ituma covalent attachment ya mois ya NMP kuri terminus yabitswe nsp9 amino muri reaction yishingikiriza kuri Mn2 + ion hamwe nibisigazwa bya Asn byegeranye.Byagaragaye ko ibikorwa bya NiRAN na nsp9 NMPylation ari ngombwa mu kwigana coronavirus.Amakuru aradufasha guhuza iki gikorwa cyibimenyetso byanduye virusi ya enzyme nicyerekezo cyabanjirije muri hypothesis ko gutangiza synthesis ya RNA mubyiciro bya virusi ya RNA bikora kandi bigahinduka.
RNA polymerase iterwa na RNA (RdRps) ya Nidovirales (Coronaviridae, Arterioviridae, n'indi miryango 12) ifitanye isano na amino-terminal (N-terminal) muri poroteyine idafite imiterere (nsp) yasohotse muri polyproteine, yitwa NiRAN 1ab igizwe na protease nyamukuru ya virusi (Mpro).Mbere, virusi ya arterial NiRAN-RdRp nsp nyirizina ya GMPylation / UMPylation yavuzwe, kandi hasabwa ko habaho inzibacyuho yo kwanduza nucleoside monophosphate (NMP) kuri virusi (kugeza ubu itazwi) na / cyangwa selile biopolymerisation.Hano, twerekana ko coronavirus (Umuntu Coronavirus [HCoV] -229E na Syndrome ikaze yubuhumekero bukabije Coronavirus 2) nsp12 (NiRAN-RdRp) ifite ibikorwa bya Mn2 + -bishingiye kuri NMPylation, bikomoka kuri nsp9 binyuze mugushinga Mpro-yunganirwa nsp9 Nyuma N-terminal ihanamye nsps irekurwa proteolytike, fosifora ihujwe na amine yibanze (N3825) kuri N-terminal ya nsp9.Uridine triphosphate ni nucleotide ikunzwe muri iki gisubizo, ariko adenosine triphosphate, guanosine triphosphate, na cytidine triphosphate nayo ikwiye hamwe na hamwe.Ubushakashatsi bwa mutation ukoresheje recombinant coronavirus nsp9 na proteine nsp12 hamwe na genoside yakozwe na geno ya HCoV-229E yagennye ibisigara bikenewe kugirango NiRAN yunganirwa nsp9 NMPylation no kwigana virusi mumico y'akagari.Amakuru yemeje ubuhanuzi bwibisigazwa bya NiRAN kandi byerekana uruhare rukomeye rwibisigazwa bya nsp9 N3826 muri nsp9 NMPylation no kwigana virusi muri vitro.Iki gisigara kiri mubice byabitswe N-terminal NNE tripeptide kandi byagaragaye ko aribisigisigi byonyine bidahinduka bya nsp9 hamwe naba homologologi mumuryango wa coronavirus.Ubu bushakashatsi butanga urufatiro rukomeye rwo kwiga imikorere ya NMPylation yizindi virusi zatewe kandi itanga intego zishoboka zo guteza imbere imiti ya virusi.
Indwara ya Nidovirales yanduye virusi ya RNA yanduza inyamaswa zidafite ubuzima n’inyamaswa (1, 2).Iri teka ririmo imiryango 14 (3), muri yo umuryango wa Coronavirus wizwe cyane mu myaka 20 ishize.Muri kiriya gihe, coronavirus eshatu zoonotic zasohotse mu nyamaswa kandi zitera indwara nini zandurira mu myanya y'ubuhumekero ku bantu.Harimo icyorezo gikomeje guterwa n'indwara zikomeye zandura.Indwara y'ubuhumekero Coronavirus 2 (SARS-CoV-2) (4âââ7).Nidovirus isangiye ishyirahamwe rusange rya genome, kandi subunit yikigo cyo kwigana-kwandikirana (RTC) igizwe na 5-? ²-terminal-bibiri bya gatatu hamwe na subunit nyamukuru yibice bigize virusi, hamwe nibindi bikoresho. .Poroteyine, igizwe muri 3 ?? ² iherezo rya gatatu rya genome (1).Usibye umuryango umwe wa virusi ya planari (Monoviridae) (8), virusi zose zashyizwe hamwe zifata subunits ya RTC mubice bibiri binini byo gusoma (ORF) ORF1a na ORF1b, bisobanurwa muri genomic RNA ya.ORF1a ikora polyproteine (pp) 1a, na ORF1a na ORF1b ifatanya pp1ab.Hamwe nubwitabire rusange muri protease nkuru (Mpro) yashizweho na ORF1a, pp1a na pp1ab byombi bitunganyirizwa muri proteolytique zitandukanye za poroteyine zitandukanye zidafite imiterere (nsps), izwi kandi nka 3CLpro, kuko ifite homologiya hamwe na 3Cpro ya picornavirus ( 9).Izi nsps zitekereza ko ziteranijwe muri RTC nini cyane, igahindura synthesis ya genomic RNA (kwigana) hamwe na RNA ya subgenomic RNA (transcription), kandi ikoreshwa muguhuza imvugo ya ORF iherereye hepfo ya ORF1b (10? ? 12).
Intangiriro ya RTC ikubiyemo RNA iterwa na RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) hamwe na enzymes nyinshi zitunganya RNA, zikaba ziba cyane muri ORF1b no mumuryango wa coronavirus Irimo nsp12-nsp16 na nsp9-nsp12 mumuryango wa Arterioviridae (reba umurongo wa 10ââ 12).RdRp na HEL1 byerekana ibyiciro bibiri (kimwe cya gatanu) cyabitswe muri virusi yinyoni yinyoni kandi bifite homologiya mubindi virusi ya RNA.Kwisubiramo kwingenzi byitwa ko byafashijwe nizindi subunits, harimo nsps ntoya zasohotse mu karere ka carboxy-terminal (C-terminal) mukarere ka pp1a, hepfo ya Mpro (coronavirus nsp5 na virusi ya arterial nsp4).Bafite imipaka yihariye yo kurinda umuryango nibikorwa bitandukanye (bisubirwamo muri 10â12).
Ugereranije vuba aha, domaine ifite imiterere yihariye ikurikirana yabonetse kuri amino terminus (N-terminus) yegeranye na RdRp muri virusi zose zatewe, ariko ntayindi virusi ya RNA (16).Ukurikije aho uherereye no kwimura nucleotide (nucleoside monophosphate [NMP] transfert), iyi domaine yitwa NiRAN (Nestvirus RdRp ijyanye na nucleotide transfert).Guhuza ibice bibiri bya NiRAN-RdRp bigize nsp12 mu muryango wa Coronaviridae na nsp9 mu muryango wa Arterioviridae, no mu zindi nestoviridae, NiRAN-RdRp biteganijwe ko izasohoka nka nsp yigenga ikomoka kuri polyproteine.Muri coronavirus, indangarugero ya NiRAN irimo ?? 1/450 gisigara kandi ihujwe na C-terminal RdRp ya domaine binyuze mukarere gahuza (16? 19).Muri virusi ya Arteritis Virusi (EAV) (Arteriviridae), recombinant nsp9 yerekana ibikorwa bya Mn2 + ion (biterwa) na UMPylation na GMPylation, biterwa nibice bitatu byabitswe bikurikirana muri nestovirus, AN, BN na CN Ibisigara bikurikiranye.Aho N bisobanura NiRAN) (16).N-terminal kuruhande rwiyi motifs ni motif idahwitse motif preAN.Bimwe muri ibyo bisigazwa kandi bibikwa muri poroteyine za kinase zifitanye isano rya kure, aho byagaragaye ko zigira uruhare mu bikorwa bya nucleoside triphosphate (NTP) guhuza no gukora catalitike (20, 21).Uhuje n'iki cyerekezo, ibisigisigi byinshi byingenzi bisigaye kurubuga muri pseudokinase SelO yo muri syringae ya Pseudomonas irashobora guteranyirizwa hamwe na SARS-CoV-2 nsp7 / 8/12/13 supercomplex iherutse gusohoka.Ibisigarira bya Coronavirus NiRAN bisigara hejuru ya microstructure ya electron.Poroteyine ya Recombinant (17).Biravugwa ko inyandiko (yonyine) U / GMPylation izatanga leta yinzibacyuho yo kwimura NMP muri substrate (kuri ubu itazwi) (16), kandi isano iri hagati ya NiRAN na protein kinase (17, 19)) Ese hypothesis ni NiRAN ihindura izindi poroteyine.
Ibintu byinshi, harimo ishyirahamwe ryihariye kandi ridasanzwe hamwe na virusi zatewe no gutandukanya genetike na RdRp, bituma NiRAN itanga umusemburo wingenzi ugenga virusi zanduye, zikaba ari ingenzi cyane kugaragara no kumenyekana.Mbere, ibikorwa bitatu bishoboka birimo NiRAN kugenzura genome / subgenomic ibisobanuro cyangwa kwigana / kwandukura byiswe.Iyo urebye amakuru make kandi atuzuye aboneka muricyo gihe, buri gikorwa gifite ibyiza byacyo (16).Muri ubu bushakashatsi, tugamije guhuza ubushakashatsi bwibinyabuzima na reaction ya genetique ya coronavirus ihagarariye genera zombi, kandi tugashyira ibyo twabonye mumiterere yubwihindurize bwimiterere ihindagurika ryimiterere yumuryango wa coronavirus, kugirango tumenye neza kariya gace kayobera.Turamenyesha iterambere ryinshi mu gusobanukirwa NiRAN binyuze mu kumenya intego kamere muri RTC, (muri hypotheses eshatu ziboneka) igira uruhare mu ruhare rwiyi domaine mugutangiza synthesis ya virusi RNA yatewe.Ubu bushakashatsi kandi butanga ibisobanuro ku zindi nshingano za NiRAN kuri interineti yakira virusi.
Kugirango turanga imiterere ya enzymatique ya virusi ya corona nsp12 ifitanye isano na NiRAN, twakoze uburyo bwa recombinant bwa coronavirus yumuntu 229E (HCoV-229E) nsp12 muri E. coli, hamwe na tagi ya His6 kuri C-terminus, hanyuma duhuza hamwe poroteyine hamwe na [α32-P] Incubate hamwe na NTP imbere ya MnCl2 nkuko byasobanuwe mu Bikoresho no mu buryo.Isesengura ryibicuruzwa byerekanaga ko hariho poroteyine ya radiyo ikora hamwe na nsp12 (106 kDa), byerekana ko coronavirus nsp12 itera ishingwa rya poroteyine ya covalent-NMP, cyane cyane ikorwa na monofosifate ya uridine (UMP) Kandi (Ishusho 1A) Kandi B).Isesengura ryinshi ryerekanye ko ugereranije nizindi nucleotide, ubukana bwibimenyetso bya UMP bwiyongereyeho inshuro 2 kugeza kuri 3 (Ishusho 1C).Aya makuru ajyanye nigikorwa cya NMP cyo guhanura ibikorwa bya NiRAN ya domaine ya coronavirus (16), ariko byerekana ko ibyifuzo bya nucleotide bya NiRAN ya domaine ya coronavirus na virusi ya arterial bitandukanye.
Kwikorera-NMPylation ya HCoV-229E nsp12..Ibicuruzwa bya reaction byatandukanijwe na SDS-PAGE hanyuma bisiga irangi rya Coomassie nziza.(B) Poroteyine ya radiyo yerekana amashusho ya fosifori.Imyanya ya nsp12-His6 hamwe na protein molekulari ya marike (muri kilodaltone) irerekanwa muri A na B. (C) Ubukomezi bwikimenyetso cya radio (bivuze ± SEM) cyagenwe uhereye kubushakashatsi butatu bwigenga.* P≤0.05.Imbaraga z'ikimenyetso (ijanisha) zifitanye isano na UTP.
Nubwo ibikorwa bya enzyme bifitanye isano na NiRAN byagaragaye ko ari ngombwa mu kwigana EAV na SARS-CoV mu muco w’akagari (16), imikorere yihariye ya NiRAN nintego zishobora kuba zitaramenyekana.Imiterere iherutse kuvugwa hagati ya NiRAN n'umuryango wa poroteyine zifite poroteyine kinase zimeze nk'imitsi (17, 22) byaduteye gusuzuma hypothesis ivuga ko NiRAN itera NMPylation yizindi poroteyine.Twashizeho urutonde rwintego zishobora guhuza ibitsina, harimo poroteyine zidafite imiterere zashyizweho na HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), buri kimwe kirimo C-terminal His6 (SI umugereka, Imbonerahamwe S1), Kandi shyiramo izo poroteyine hamwe na [α32-P] uridine triphosphate ([α32-P] UTP) nsp12 ihari cyangwa idahari.Bovine serum albumin na MBP-LacZα proteyine ya fusion ikorerwa muri E. coli yabaye nk'igenzura (Ishusho 2A, umurongo wa 1 kugeza 7).Poroteyine ya radiolabel yasesenguwe na sodium dodecyl sulfate-polyacrylamide gel electrophorei (SDS-PAGE) na autoradiography, kandi byagaragaye ko hari ikimenyetso gikomeye cya radiyoyakire mu reaction irimo nsp12 na nsp9.Umwanya wikimenyetso uhuye na molekuline ya nsp9, byerekana nsp12-ihuza UMPylation ya nsp9 (Ishusho 2B, inzira 7).Ntayindi poroteyine yipimishije wasangaga UMPylated, yatumye twemeza ko nsp9 ari substrate yihariye ya nsp12.Bihuye namakuru-yonyine-NMPylation yerekanwe ku gishushanyo 1, nsp12 irashobora kwimura NMP zose uko ari enye kuri nsp9, nubwo imikorere itandukanye, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP)) ( Ishusho).3 A na B).Ukurikije ibihe byakoreshejwe muri ubu bushakashatsi (gabanya igihe cyo kubyitwaramo no kwerekana igihe, gabanya ubukana bwa nsp12; ibikoresho nuburyo), kwikorera-NMPylation ya nsp12 ntishobora kuboneka (gereranya Ishusho 2B, umurongo wa 7, nishusho 1B), ibyo yerekanye ingirakamaro (Kandi izenguruka nyinshi) UMP yavuye kuri nsp12 yerekeza kuri nsp9.Igikorwa cyo kwimura UMP gisaba ko habaho Mn2 + ion, nkuko bigaragara ku gishushanyo cya 3C, mugihe ibikorwa bike byo kwimura UMP gusa byagaragaye imbere ya Mg2 +, kandi nta gikorwa na kimwe imbere y’ibindi bice bibiri byagereranijwe.Amakuru nkaya yabonetse mubisubizo bya NMPylation birimo cytidine triphosphate (CTP), guanosine triphosphate (GTP), na adenosine triphosphate (ATP) (umugereka wa SI, Ishusho S1).
HCoV-229E nsp12-ihuza UMPylation ya nsp9.Urukurikirane rwa poroteyine (harimo na bovine serum albumin, MBP-lacZα, hamwe na HCoV-229E nsps yanditseho C-terminal His6 yashizweho na ORF1a) yakoreshejwe mu gusuzuma ibikorwa UMPylation ya HCoV-229E nsp12-His6⁺-yunganirwa poroteyine.Shyiramo poroteyine hamwe na [α-32P] UTP mu minota 10 udahari (A) cyangwa uhari (B) wa nsp12 nkuko byasobanuwe mubikoresho nuburyo.Hejuru ya A na B, SDS-polyacrylamide gel yanditswemo na Coomassie Brilliant Ubururu irerekanwa, naho hepfo ya A na B, herekanwa autoradiogramu ihuye.Umwanya wa protein molekulari ya marike (muri kilodaltons) itangwa ibumoso.Umwanya wa nsp12-His6 (B, hejuru) hamwe na radiyo yerekana ibimenyetso byagaragaye mugihe cya incubation ya nsp12-His6 hamwe na nsp9-His6 (B, umurongo wa 7) nayo irerekanwa, ibyo bikaba byerekana ko [α-32P] UMP kugeza nsp9-His6 (12.9 kDa), itagaragaye ku zindi poroteyine zapimwe.
HCoV-229E NiRAN-yunganirwa na biohimiki na virusi biranga nsp9 NMPylation.(A na B) Uruhare rwa nucleotide co-substrate ikoreshwa mugukora.Nsp12-His6 na nsp9-His6 zivanze kandi zishizwemo imbere ya NTP zitandukanye [α-32P] NTPs muburyo busanzwe bwa NMPylation.(A, hejuru) Coomassie-irangi nsp9-His6 yatandukanijwe na SDS-PAGE.(A, hepfo) Autoradiograf yubuso bumwe bwa gel.(B) Igikorwa gifitanye isano (bivuze ± SEM) imbere ya cofactor yagenewe nucleotide yagenwe uhereye kubushakashatsi butatu bwigenga.* P≤0.05.(C) Uruhare rwa ioni.Herekanwa ikizamini gisanzwe cya NMPylation imbere ya [α-32P] UTP hamwe na ion zitandukanye zicyuma, buri kimwe gifite ubunini bwa mM 1.Muri C, hejuru, Coomassie yanditseho nsp9-His6 irerekanwa, naho muri C, hepfo, autoradiografi ihuye irerekanwa.Ingano ya poroteyine yanditseho (muri kilodaltons) yerekanwa ibumoso bwa A na C. (D) Imiterere ya mutant ya HCoV-229E nsp12-His6 itwara insimburangingo ya aside amine iri muri UTP, nkuko byasobanuwe mu Bikoresho no mu buryo.Radiolabel nsp9-His6 yakozwe muri reaction ya NMPylation igaragazwa no gufata amashusho ya fosifora (D, hejuru).Igikorwa kigereranije ugereranije na poroteyine yo mu bwoko bwa (wt) yerekanwa muri D, naho hepfo ifatwa nkikigereranyo (EM SEM) uhereye kubushakashatsi butatu bwigenga.Asterisks yerekana gusimbuza ibisigazwa bitabitswe.(E) Titer ya virusi mumico ndengakamere ya p1 selile yabonye amasaha 24 nyuma yo kwandura kugenwa na plaque.Gusimbuza codon muri domaine ya NiRAN ya injeniyeri ya injeniyeri ya HCoV-229E irerekanwa (nimero isigaye ishingiye kumwanya wabo muri pp1ab).Kwigana-kubura RdRp urubuga rukora mutant nsp12_DD4823 / 4AA yakoreshejwe nkigenzura.
Kugirango turusheho gusobanukirwa neza urubuga rukora rwa NiRAN no kumenya ibisigisigi bijyanye nigikorwa cyo kwimura NMP9 yihariye ya NMP, twakoze isesengura rya mutation, aho twasimbuye ibisigisigi bya conservateur muri motif ya NiRAN AN, BN na CN ( 16) Ni Ala (Umugereka wa SI, Igicapo S2).Byongeye kandi, ingaruka z’abasimbura Arg-to-Lys cyangwa Lys-to-Arg zasuzumwe mu bihe bibiri.Nkigenzura (ribi), ibisigara bitabitswe cyangwa bike bibitswe muri domaine ya NiRAN ya coronavirusi nizindi virusi zatewe zasimbuwe na Ala. Gusimbuza K4116A (muri motif preAN), K4135A (AN), R4178A (BN), D4188A (motif BN) na D4280A (CN) bigabanya cyane cyangwa bikuraho nsp9 NMPylation ikoresheje nsp12, mugihe poroteyine zifite insimburangingo (R4178K), K4116R) zigumana 60% na 80% byibikorwa byabo, ibyo bikaba byerekana ko kuruhuka kubuzwa kuruhande rwabo. iminyururu yunvikana muburyo bwa fiziki (Ishusho ya 3D).Gusimbuza ibindi bisigazwa byinshi byabitswe E4145A, D4273A, F4281A na D4283A ntabwo byangiza cyane, kandi nsp9 UMPylation iragabanuka gusa.Ibisubizo nkibi byabonetse muri nsp9 NMPylation reaction irimo izindi NTP (Igicapo cya 3D na SI umugereka, Igicapo S3), yemeza ko ingaruka zagaragaye kumasemburo yihariye ya aside amine zidashingiye kubwoko bwa nucleotide co-substrate ikoreshwa.Ibikurikira, twagerageje ingaruka zishoboka zinsimburangingo nsp12 mugusubiramo coronavirus mumico y'akagari.Kugira ngo ibyo bishoboke, twifashishije inyandikorugero ya ADN (cDNA) ikozwe neza ikozwe muri virusi ya recombinant vaccinia (23, 24) kugirango twandike selile 5 -7.Kwandika virusi yanduye ikorerwa muri utwo tugari byerekanaga ko ihinduka ryinshi rya HCoV-229E NiRAN ridashoboka (Ishusho 3E).Itsinda ryimiterere ya virusi idafite imbaraga zirimo ubundi buryo bwerekanwe gukuraho cyangwa kugabanya cyane ibikorwa byo kwimura NMP muri vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), ariko hariho ubundi buryo bubiri (K4116R, E4145A) 80 % yabitswe?Ibikorwa byabo muri vitro NMPylation byerekana ko harimo izindi mbogamizi zirimo.Mu buryo nk'ubwo, izindi mutation ebyiri (R4178K, F4281A) zatumye igabanuka rito mu bikorwa bya NiRAN mu bikorwa bya vitro NMPylation byabyaye virusi nzima, ariko, izo virusi zagabanije cyane titereri binyuze mu kwigana.Bihuye namakuru yibikorwa bya vitro yerekanwe kumashusho ya 3D, asimbuza ibindi bisigazwa bine bitabitswe muri coronavirus na / cyangwa izindi virusi zatewe (K4113A, D4180A, D4197A, D4273A) (8, 16) byabyaye virusi zifatika Urubyaro, nubwo rufite igabanutse ku buryo bugereranije ugereranije na virusi yo mu gasozi (Ishusho 3E).
Kugirango umenye niba ibikorwa byo kwimura NMP byahujwe na NMP biterwa na domaine ikora ya RdRp, ibisigisigi bibiri bya Asp byabitswe byagize uruhare mu guhuza ioni zingana nicyuma (11) muri RdRp motif C byasimbuwe na Ala.Ibikomoka kuri poroteyine nsp12_DD4823 / 4AA bigumana. ibikorwa byayo nsp9 NMPylation, byerekana ko nsp12-yunganirwa muri vitro nsp9 Igikorwa cya NMPylation ntigisaba ibikorwa bya polymerase (SI Umugereka, Igicapo S4).
Nyuma yo gushyiraho ibikorwa bya nsp9 byihariye byo kwimura NMP kuri nsp12, twagerageje kuranga inyongeramusaruro ya NMP-nsp9 na mass spectrometrie (MS).Poroteyine yuzuye yuzuye ya recombinant HCoV-229E nsp9 yerekanye impinga kuri 12.045 Da (Ishusho 4A).Kwiyongera kwa nsp12 ntabwo byahinduye ubuziranenge bwa nsp9, byerekana ko nsp12 na nsp9 bitazakora urwego ruhamye mubihe byakoreshejwe (denaturation) (Ishusho 4A).Imbere ya UTP na GTP, gupima imbaga ya reaction irimo nsp9 na nsp12 byerekanaga ko poroteyine ya UTP yimuye 306 Da, naho poroteyine ya GTP yimuye 345 Da, byerekana ko buri molekile ya nsp9 ihuza UMP cyangwa GMP (Ishusho 4) C na D).Bikekwa ko ingufu zisabwa kuri NiRAN-yunganirwa nsp9 NMPylation ituruka kuri hydrolysis ya NTP no kurekura pyrophosifate.Nubwo umubyimba wikubye inshuro 10 urenze nsp9 (intego) kurenza nsp12 (enzyme) wakoreshejwe muriki gisubizo, NMPylation yuzuye ya nsp9 yagaragaye, byerekana ko imikoranire hagati ya nsp12 na nsp9 ari iyigihe gito, kandi nsp12 irashobora NMPylate nyinshi nsp9 muri molekile ya vitro.
NMPylation imwe ya nsp9 imbere ya nsp12 na UTP cyangwa GTP.Yerekanwa ni poroteyine yuzuye ya poroteyine yuzuye ya HCoV-229E nsp9 (Umugereka wa SI, Imbonerahamwe S1) (AD)..
Kugirango umenye ibisigazwa bya nsp9 UMPylated by nsp12, nsp9-UMP yashizwemo na trypsin.Peptide yavuyemo yatandukanijwe na nano-hejuru yimikorere ya chromatografiya (HPLC) hanyuma isesengurwa na tandem mass spectrometry (MS / MS) kumurongo.Isesengura ryamakuru ukoresheje porogaramu ya Byonic (Protein Metrics) yerekanaga UMPylation ya N-terminal amino aside.Ibi byemejwe nintoki.Tandem mass mass ya preursor peptide [UMP] NNEIMPGK (umugereka wa SI, Igicapo S5A) yerekanye igice kuri 421 m / z, byerekana ko UMP ihuza ibisigisigi 1 bya nsp9.
Kuri N-terminus ya nsp9, Asn ibitswe mubanyamuryango ba Orthocoronavirinae (Umugereka wa SI, Igicapo S6).Nubwo twemera ko N-terminal primaire ya amine azote niyo ishobora kwemerwa na UMP, twahisemo kubona ibindi bimenyetso byerekana ko NMP ihuza N-terminal.Kubera iyo mpamvu, non-NMPylated na NMPylated N-terminal peptide nsp9 yatunganijwe na HPLC yakomotse imbere ya acetone na sodium cyanoborohydride.Muri ibi bihe, amine yambere yubusa arashobora guhindurwa hamwe na propyl (25).N-terminal nsp9 ikomoka kuri peptide hamwe nurukurikirane rwa NNEIMPGK irimo amine abiri yibanze, imwe kuri N-terminus ya Asn indi kumurongo wuruhande rwa Lys kuri C-terminus.Kubwibyo, amatsinda ya propyl arashobora gutangizwa kumpera zombi.Iion chromatogramu yakuweho ya peptide itari NMPylated irerekanwa kumugereka wa SI, Igicapo S5B.Nkuko byari byitezwe, N-terminal na C-terminal (mono) ikwirakwizwa (SI umugereka, Igicapo S5B, umurongo wo hejuru) hamwe na peptide ya dipropylated (SI umugereka, Igicapo S5B, umurongo wo hasi) urashobora kumenyekana.Ubu buryo burahinduka hamwe no gukoresha NMPylated N-terminal peptide ya nsp9.Muri iki gihe, hashobora kumenyekana gusa peptide ya C-terminal, ariko peptide ya N-terminal na peptide ya dipropylated ntabwo yamenyekanye (SI Umugereka, Igicapo S5C), byerekana ko UMP yimuriwe muri N-terminal amine primaire Kugira ngo birinde ibi itsinda kuva muguhindura.
Ibikurikira, dusimbuza (hamwe na Ala cyangwa Ser) cyangwa gusiba ibisigazwa byabitswe kuri N-terminus ya nsp9 kugirango dusobanure imbogamizi zihariye.Dushingiye ku makuru yacu ya MS yerekana ko NiRAN ikora inyongera ya nsp9-NMP hamwe na amine yibanze ya N-terminal isigara ya nsp9, twavuze ko nsp9 NMPylation isaba virusi ya protease (Mpro, nsp5) kurekura nsp9 N-terminal kuva polyprotein ibanziriza.Kugirango dusuzume iyi hypothesis, twakoze protein prursor nsp7-11 irimo nsp9 muri E. coli kandi dukora ikizamini gisanzwe cya NMPylation imbere ya [α-32P] UTP (ibikoresho nuburyo).Nkuko bigaragara ku gishushanyo cya 5A (umurongo wa 3), ibanziriza nsp7-11 ibanziriza ntabwo irangwa na nsp12.Ibinyuranye, niba nsp7-11 ikozwe na recombinant nsp5 kugirango irekure nsp9 (hamwe nizindi nsps) kuri preursor, hamenyekanye poroteyine ya radiolabel yimuka hamwe na nsp9, byemeza ko twanzuye ko NiRAN na N- Guhitamo gushiraho covalent nsp9-NMP. .Terminal primaire amine ya N-terminal Asn (umwanya 3825 muri pp1a / pp1ab).Uyu mwanzuro ushyigikiwe kandi nubushakashatsi ukoresheje nsp9 yubaka, ikubiyemo ibisigisigi kimwe cyangwa bibiri byiyongera kuri N-terminus.Muri ibyo bihe byombi, NiRAN yunganirwa na UMPylation ya nsp9 yavanyweho (Umugereka wa SI, Ishusho S7).Ibikurikira, twakoze proteine ifite ibisigisigi kimwe cyangwa bibiri bya Asn byasibwe kuri 3825-NNEIMPK-3832 peptide ikurikiranye kuri N-terminal ya nsp9.Muri ibyo bihe byombi, nsp9 UMPylation yarahagaritswe burundu (Igicapo 5B), itanga ikindi kimenyetso cyerekana ko nsp9 N-terminus nyayo ikora nka reseptor ya NMP.
Gutunganya proteolytike ya nsp9 nuruhare rwibisigisigi bya N-terminal muri nsp12-ihuza UMPylation.(A) nsp9 UMPylation isaba nsp9 N-terminal.Nsp7-11-His6 yabanjirijwe kuri 30 ° C muri buffer ya NMPylation irimo UTP ihari cyangwa idahari Mpro (nsp5-His6).Nyuma yamasaha 3, tangira NMPylation assay wongeyeho nsp12-His6 nkuko byasobanuwe mubikoresho hamwe nuburyo.Igisubizo kirimo nsp5-His6 (umurongo wa 1) na nsp9-His6 (umurongo wa 2) cyakoreshejwe nkigenzura.Nyuma yiminota 10, reaction yarangiye kandi ivangwa rya reaction ryatandukanijwe na SDS-PAGE.Poroteyine yandujwe na Coomassie Brilliant Ubururu (A, hejuru).Nsp7-11-His6 ibanziriza nibicuruzwa byatunganijwe biva kuri nsp5-His6 byahujwe na clavage byerekanwe iburyo.Nyamuneka menya (bitewe nubunini bwazo) ko nsp7 na nsp11-His6 bitagaragara muri iyi gel, kandi reaction ikongerwaho nsp5-His6 (umurongo wa 1 na 4; umwanya wa nsp5-His6 werekanwa numuzingi ukomeye) cyangwa nsp9-His6 (Umuhanda wa 2) ikubiyemo umubare muto wa MBP (werekanwa nuruziga rufunguye) nkumwanda usigaye kuko bigaragazwa nka poroteyine ya MBP fusion (SI umugereka, Imbonerahamwe S1)..B, SDS-PAGE yanditseho Coomassie irerekanwa hejuru, B, autoradiograf ijyanye irerekanwa hepfo.Umwanya wa marike ya marike (muri kilodaltons) irerekanwa ibumoso..Ibicuruzwa byitandukanije byatandukanijwe na SDS-PAGE hanyuma bisiga irangi rya Coomassie Brilliant Ubururu (C, hejuru), kandi radiolabel nsp9-His6 yatahuwe na amashusho ya fosifore (C, hagati).Ukoresheje poroteyine yo mu bwoko bwa (wt) nkibisobanuro (yashyizwe kuri 100%), ibikorwa bya NMPylation ugereranije (bivuze ± SEM) byabazwe uhereye kubushakashatsi butatu bwigenga..Kwigana-kubura RdRp motif C kabiri mutant DD4823 / 4AA yakoreshejwe nkigenzura ribi.
N-terminus ya nsp9 (cyane cyane imyanya 1, 2, 3, na 6) ibitswe cyane mubanyamuryango ba Orthocoronavirinae mu buryo butemewe (Umugereka wa SI, Igicapo S6).Kugirango twige uruhare rushoboka rwibisigisigi muri nsp12-yunganirwa nsp9 NMPylation, ibisigazwa bibiri bya Asn bikurikiranye kuri N-terminus ya nsp9 byasimbuwe na Ala cyangwa Ser (wenyine cyangwa hamwe).Ugereranije nubwoko bwishyamba nsp9, gusimbuza N3825 na Ala cyangwa Ser byatumye habaho kugabanuka inshuro zirenga ebyiri muri nsp12-yunganiwe na UMPylation (Ishusho 5C).Dukurikije umwanzuro twafashe ko NMPylation iboneka kuri N-terminal primaire amine aho kuba urunigi rwuruhande rwibisigisigi bya N-terminal, twabonye NMPylation isigaye isimburwa na N3825A na N3825S.Igishimishije, niba Asn ya kabiri isimbuwe na Ala cyangwa Ser, nsp9 UMPylation yagabanutse cyane (inshuro zirenga 10), mugihe gusimbuza Ala kumwanya wa 3, 4, na 6 bifite ingaruka zoroheje kuri nsp9 UMPylation (Ishusho 2 ).5C).Ibisubizo nkibi byabonetse ukoresheje ATP, CTP cyangwa GTP (umugereka wa SI, Ishusho S8).Hamwe na hamwe, aya makuru yerekana uruhare runini rwa N2826 (umwanya wa 2 muri nsp9) muri nsp9 NMPylation.
Kugirango tubone ibimenyetso byinyongera byerekana isano iri hagati ya N-terminus ya nsp9 na NMPylation, twakoze urutonde rwinshi (MSA) rwurutonde rwa nsp9 rwumuryango wa Coronavirus (rutandukanye hagati y ibisigisigi 104 na 113) (Umugereka wa SI, Ishusho S6).Muri rusange, mu bwoko 47 (buzwi kandi bushyira) amoko 5 ya genera 5 ya Orthocoronavirinae yanduza inyamaswa z’inyamabere zitandukanye, inyoni, hamwe n’ibikururanda, ibisigazwa 8 gusa muri rusange wasangaga bidahinduka.Impinduka nini cyane, harimo gusiba no gushiramo, byagaragaye mu ruziga hagati yimiterere yinyongera ya nsp9, nkuko byagenwe nubushakashatsi bwibanze (26 ?? 28).Ibisigisigi bitanu byahinduwe byabonetse muri β umurongo na α helix ya C-terminal igice cya nsp9.Ibisigisigi bitatu bidahinduka bigize NNE motif ya N terminus ya nsp9.Byagaragaye ko Asn ya kabiri yiyi motif aricyo gisigara cyonyine kidahinduka, nacyo gisangiwe na hypothetical nsp9 yubwoko bwibikeri coronavirus bifitanye isano rya bugufi, kandi bugereranya ubwoko bwa Microhyla letovirus 1 mubwoko bwa Letovirinae bwa Alphaletovirus.Kubungabunga ibisigazwa muri nsp9 ibice byubatswe byongeye birashobora gutondekwa kubitekerezo byubaka kugirango bigumane cyangwa bizwi na RNA ihuza ibintu.Nyamara, iyi mitekerereze isa nkaho idakoreshwa mukubungabunga NNE, kandi mbere yubu bushakashatsi, imiterere yimbogamizi zigabanya itandukaniro ryurutonde rwa tripeptide yari yarahishe rwose.
Kugirango tumenye akamaro ko kubungabunga nsp9-NMPylation no kubungabunga NNE mugusubiramo coronavirus, twakoze mutant ya HCoV-229E, itwara insimburangingo imwe cyangwa ebyiri zasimbuwe nsp9 N-terminal, byerekana ko nsp9 NMPylation yangiza muri vitro.Mbere yo gutangira, turagerageza gusubiza ikibazo niba ibyo bisimburwa (hafi ya nsp8 | 9 cleavage site) bigira ingaruka kuri proteolytike yo gutunganya C-terminal pp1a mukarere.Igice cya nsp7-11 polyprotein yubatswe irimo insimburangingo ijyanye na N-terminus ya nsp9 yakorewe muri E. coli hanyuma ikata hamwe na recombinant Mpro.Proteolytic clavage yibibanza bine (harimo nimbuga ya nsp9) ntabwo ihindurwa cyane nabasimbuye (SI umugereka, Igicapo S9), ukuyemo impinduka zubatswe muri izo poroteyine zibangamira Mpro-medrated nsp8 | 9 clavage (Cyangwa izindi) urubuga.
Ingirabuzimafatizo za Huh-7 zandujwe na genome ndende ya HCoV-229E RNA, ikubiyemo insimburangingo ya Ala cyangwa Ser muri tripeptide ya NNE yabitswe (N3825, N3826, na E3827) kuri nsp9 N, byerekana ko ihinduka ryinshi ryica.Twashoboye gutabara virusi dusimbuza Ser cyangwa Ala ya N-terminal Asn (N2835A cyangwa N2835S), ariko ntitwashoboye kugarura virusi hamwe n’indi ihinduka rimwe kandi kabiri mu buryo bwa NNE (N3826A, N3826S, NN3825 / 6AA, NN3825 / 6SS), E3827A) (Ishusho 5D).
Ibisubizo byerekana ko kwigana coronavirus mumico yumubiri bigabanijwe (kimwe cyangwa bisa), bigabanya ihinduka ryimiterere yimiterere ya nsp9 ya NMPylation mumubiri, kandi igashyigikira uruhare runini rwiki gisubizo mubuzima bwa coronavirus.
Mubushakashatsi bwanyuma, twakoze C-terminal His6 yanditseho SARS-CoV-2 nsp12 na nsp9, nuburyo bubiri bwa mutant bwa nsp12 muri E. coli.Urubuga rukora ibisigara muri domaine ya NiRAN na RdRp byakoreshwaga Koresha Ala aho (Ishusho 6A na SI umugereka, Imbonerahamwe S2).K4465 muri SARS-CoV-2 nsp12 ihuye na K4135 muri HCoV-229E (Umugereka wa SI, Igicapo S2), byagaragaye ko bisabwa mubikorwa bya NiRAN no kwigana HCoV-229E (Ishusho 3D na E).Iki gisigarira kandi gihuye na virusi ya arterial EAV nsp9 K94, mbere byagaragaye ko ari nkenerwa kuri NiRAN kwikorera-UMPylation / kwikorera-GMPylation (16).Nkuko bigaragara ku gishushanyo cya 6B, SARS-CoV-2 nsp12 ifite ibikorwa byo kwimura UMP ukoresheje nsp9 nka substrate, mugihe nsp12_K4465A ihinduka ryimikorere ya site idakora.Gusimbuza kabiri muri SDD biranga urutonde rwa RdRp motif C ntabwo bigira ingaruka kubikorwa byo kwimura UMP (Ishusho 6B), byerekana ko ibikorwa bya RdRp nta ngaruka zitaziguye muri nsp9 UMPylation.Amakuru nkaya yabonetse hakoreshejwe CTP, GTP na ATP (Umugereka wa SI, Igicapo S10).Muncamake, aya makuru yerekana ko NiRAN-yunganirwa nsp9 NMPylation ifite ibikorwa byo guharanira inyungu muri coronavirus zerekana genera zitandukanye za orthocoronavirus mu buryo butemewe.
SARS-CoV-2 nsp12-ihuza NMPylation ya nsp9..Nkigenzura, hakoreshejwe proteine ya mutant hamwe no gusimbuza urubuga rukora muri domaine ya NiRAN (K4465A) na RdRp (DD5152 / 3AA) ya SARS-CoV-2 nsp12.Umubare usigaye ushingiye kumwanya uri muri pp1ab..Ubwinshi bwa molekile (muri kilodaltons) ya proteine yanditseho irerekanwa ibumoso.
Ubusanzwe NiRAN ibitswe muri Nidovirales (16), byerekana ko itera reaction enzymatique zikenewe mugusubiramo Nidovirus.Muri ubu bushakashatsi, twashoboye kwerekana ko domaine ya NiRAN ya coronavirus yimura NMP (ikomoka kuri NTP) ikajya kuri nsp9, poroteyine ya RNA itangaje igira uruhare mu kwigana virusi (26 ?? 29), kugira ngo tumenye ko ari intego karemano kandi umufatanyabikorwa wa coronavirus RTC.
Indangantego ya NiRAN isangiye ibintu bitatu bikurikirana (AN, BN, na CN), bikubiyemo umubare muto cyane wibisigara bibikwa mumiryango yose murwego rwa monofyletike ariko itandukanye cyane na Nidovirales (8, 16).Ubushakashatsi buherutse gukorwa bwerekanye ko bufitanye isano n’umuryango ahanini udafite imiterere ya poroteyine kinase imeze nka poroteyine, zahoze zitwa umuryango wa SelO (17, 19, 22, 30, 31).Poroteyine zijyanye na SelO zifite ububiko bwa kinase, ariko zikabura ibisigisigi byinshi byabitswe bisigaye muri kinase ya kera (22, 32).Hashingiwe ku cyerekezo cyerekezo cya molekile ya ATP ihujwe kurubuga rukora kandi igahagarikwa n’imikoranire yihariye, SelO yashizwe mu majwi hanyuma yemeza ko yohereza AMP (aho kuba fosifate) muri poroteyine (22), mu gihe indi bagiteri SelO isa na poroteyine YdiU ifite. vuba aha yerekanwe guhagarika covalent attachment ya UMP kuri Tyr hamwe n ibisigisigi bya poroteyine zitandukanye (33).
Kugirango twemeze kandi twagure ubuhanuzi bwibisigazwa byibikorwa bya site ya coronavirus NiRAN, twakoresheje uburyo bwa biohimiki na revers genetique kugirango dukore isesengura rya mutation kuri coronavirus nsp12 (Ishusho ya 3D na E na SI umugereka, Ishusho S3 nimbonerahamwe) S1â S4).Amakuru yerekana ko gusimbuza HCoV-229E K4135, R4178 na D4280 na Ala bivanaho ibikorwa byo kohereza vitro NMP no kwigana virusi mu muco w’akagari (Ishusho ya 3D na E na SI, umugereka S3), bishyigikira ko bahari muri NTP γ-fosifate. (K4135, R4178) no guhuza ibikorwa byicyuma gikora (D4280).Isimburwa rya E4145A rya Glu yabitswe mu rwego rwa virusi y’inyoni y’inyoni yahanuwe ko izahagarika imyanya ya K4135 (17) yerekanwe gukuraho virusi, ariko igitangaje ni uko igikorwa cyagumishijwe muri vitro NMPylation (Ishusho ya 3D na E na SI umugereka, Igicapo S3 Nimbonerahamwe S1 - S4).Ubushakashatsi nk'ubwo bwakozwe igihe hasimburwaga kimwe muri YdiU homologi ya Salmonella typhimurium (E130A) (33).Ufatiye hamwe, aya makuru ashyigikira imikorere igenga iki gisigara cyabitswe aho kuba ibikorwa bya catalitiki.
Gusimbuza ibisigazwa bya Phe byabitswe (F4281A) murwego rwa nestovirus muri domaine ya HCoV-229E NiRAN (8) byatumye ibikorwa bya NMPylation bigabanuka muri vitro ndetse no kugabanuka kwa virusi mu muco w’akagari (Ishusho 3D, E na SI) umugereka, Igicapo S3).Ibyatanzwe bihuye nibikorwa byingenzi bigenga iki gisigisigi, nkibisanzwe bya DFG motif ya Phe ibisigara byerekanwe mbere.Muri poroteyine ya kera ya kinase, ni igice cya Mg2 + gihuza kandi gifasha guteranya no kugenzura urutirigongo???Birakenewe kubikorwa byiza bya catalitiki (32, 34).Gusimbuza Ala na Arg ibisigisigi bya K4116 (muri motif ya preAN), byavanyeho kwigana virusi kandi nkuko byari byitezwe, byagize ingaruka zitandukanye mubikorwa byo kwimura NMP muri vitro, bitewe nurunigi rwa aside amine yatangijwe (Ishusho ya 3D na E na SI umugereka) , Igicapo S3).Amakuru yimikorere arahuza namakuru yimiterere, byerekana ko iki gisigara cyashyizeho imikoranire na ATP fosifate (17).Muri domaine ya NiRAN yindi miryango yanduye virusi, umwanya wa HCoV-229E pp1a / pp1ab K4116 utwarwa na Lys, Arg cyangwa ibye (8), byerekana ko guhagarika imikorere yibi bisigazwa byoroheje.Gusimbuza D4188A na D4283A bikuraho cyangwa bigabanya cyane ibikorwa bya enzyme kandi bikuraho kwigana virusi (Ishusho 3).Ibisigisigi byombi bibitswe muri virusi nyinshi (ariko sibyose) zanduye (8), byerekana ibikorwa byingenzi byumuryango ariko birashoboka ko atari catalitike.Gusimbuza Ala ibindi bisigazwa byinshi bya Lys na Asp (K4113A, D4180A, D4197A na D4273A) bitabitswe muri Coronaviridae cyangwa indi miryango ya Nestioviridae (8) byakoreshejwe nkigenzura.Nkuko byari byitezwe, ibyo bisimburwa birashobora kwihanganira ahanini, hamwe no kugabanuka gake kubikorwa bya enzyme no kwigana virusi mubihe bimwe na bimwe (Ishusho ya 3 na SI umugereka, Ishusho S3).Muri rusange, amakuru ya coronavirus mutagenezesi arahuza cyane na self-GMP hamwe namakuru ya genetics ya EAV NiRAN-RdRp (16), aho EAV nsp9 (coronavirus nsp12 ortholog) ibisigisigi K94 (bihuye nibikorwa byingenzi bya HCoV- 229E K4135), R124 (bihuye na R4178), D132 (bihuye na D4188), D165 (bihuye na D4280), F166 (bihuye na F4281).Byongeye kandi, amakuru ya mutagenezi ya HCoV-229E arahuza kandi akanagurwa kuva amakuru yavuzwe mbere ya SARS-CoV yerekana amakuru ya genetiki (16), kimwe cyane cyane nayagaragaye kuri CN motif ihuye na Phe-to-Ala mutant SARS-CoV_nsp12 The phenotype yasobanuwe -F219A na HCoV-229E_F4281A (Ishusho ya 3 D na E na SI umugereka, Ishusho S3 nimbonerahamwe S1-S4).
Ugereranije na EAV orthologs (16), zikunda neza UTP na GTP (muburyo bwo kwiyita NMPylation), ubushakashatsi bwacu bwerekana ko domaine ya coronavirus NiRAN (ihagarariwe na HCoV-229E na SARS-CoV-2) ishobora gukora neza yimuye NMP zose uko ari enye, nubwo hari icyifuzo gito kuri UMP (Ishusho 1 na 3).Ugereranije ni bike cyane byihariye bya NTP bifatanyiriza hamwe bihuye na SARS-CoV-2 nsp7 / 8/12/13 imiterere ya supercomposite iherutse gutangazwa, aho ADP-Mg2 + ihuza urubuga rukora rwa NiRAN, ariko ntabwo rufite igice cya adenine yo gushiraho imikoranire yihariye (17).Mu bushakashatsi bwacu, ubwoko bwa nucleotide ikoreshwa muri reaction ya NMPylation nta ngaruka zinyuranye zigira ku bikorwa bya poroteyine ya mutant (SI Umugereka, Igicapo S3), byerekana ko nta na kimwe muri ibyo bisigazwa bifitanye isano rya bugufi no guhuza nucleobase runaka.Ishingiro ryimiterere nibisobanuro biologiya yibintu bitandukanye bya NTP bifatanyiriza hamwe kugaragara muri domaine ya NiRAN ya coronavirus na virusi ya arterial iracyakomeza kwigwa;birashobora kuba ukuri cyangwa birashobora guterwa n'imbogamizi zinyigisho zabo.Kugeza ubu, ntidushobora kwirengagiza ko ibikorwa bishobora kuba NMPylator ya virusi ya arterial virusi NiRAN (ugereranije nigikorwa cyambere cyaranzwe no kwiyitaho NMPylation) ifite ubundi buryo bwo guhuza ibice, ukurikije ko isano iri hagati ya arterial na coronavirus Indangantego ya NiRAN iri ku mbibi zayo.Gereranya ukurikije Urutonde (16).Ugereranije na pseudokinase SelO, ikoresha Mg2 + nka cofactor, ibikorwa bya coronavirus na virusi ya arterial NiRAN biterwa na Mn2 + (16) (Ishusho ya 3C na SI umugereka, Ishusho S1).Kwishingikiriza kwa Mn2 + no kugaragara cyane kuri UTP ni ikintu kidasanzwe kiranga poroteyine NMPylators, kandi giherutse kwemezwa muri poroteyine YdiU ya Salmonella typhimurium, itanga intungamubiri za Mn2 + zishingiye kuri poroteyine chaperone UMPylation kugira ngo irinde ingirabuzimafatizo za selile ATP ( 33).
Imiterere iherutse gusobanurwa hagati ya coronavirus NiRAN hamwe na protein kinase ya selile (17, 19) itanga ubufasha bwinyongera kubushobozi bwa NiRAN bwo guhuza NMP nizindi poroteyine twatangaje muri ubu bushakashatsi.Twibanze ku gushakisha intego za NiRAN kuri poroteyine zashyizweho na HCoV-229E ORF1a, zizwiho gufasha mu buryo butaziguye cyangwa butaziguye RTC ya ORF1b ikoreshwa na kodegisi (12, 35).Ubushakashatsi bwacu butanga ibimenyetso bifatika kuri NMPylation nziza kandi yihariye ya nsp9 (Ishusho 2).Niba poroteyine yintego ikoreshwa mukurenza umubyimba wikubye inshuro 8 kugeza 10 kurenza iyo enzyme (nsp12), hemezwa ko nsp9 yuzuye (mono) NMPized (Ishusho 4).Twanzuye ko imikoranire hagati ya nsp12 na nsp9 ari iyigihe gito kandi ntabwo izakora urwego ruhamye hamwe na nsp9 (mugihe hatariho izindi subunits za RTC).Uyu mwanzuro ushyigikiwe nubushakashatsi bwa poroteyine kuri proteome ya SARS-CoV (35).Isesengura rya MS ryagaragaje amine yibanze ya N-terminal isigara ya nsp9 nkurubuga rwa NMPylation (SI umugereka, Igicapo S5).Ishirwaho ryumubano wa fosifora hamwe nitsinda rya N-terminal amino ritandukanya ibikorwa bya NMPylation byahujwe na NiRAN hamwe na Pseudomonas syringae SelO-yunganirwa na AMPylation reaction, itera ishyirwaho rya O-ihuza AMP kuri Ser, Thr, cyangwa Tyr ibisigazwa bya Peptide ( 22), na S. typhimurium YdiU ikora O-ihuza (hamwe na Tyr) na N-ihuza (hamwe na) peptide-UMP.Amakuru make aboneka kumuryango wa SelO ya proteine yerekana ko abagize uyu muryango munini wa poroteyine batandukanye cyane mugushinga peptide-NMP.Iki ni indorerezi ishimishije ikwiye kwigwa.
Amakuru yabonetse muri ubu bushakashatsi yaduteye gushidikanya ko NMPylation ya nsp9 isaba N-terminus yubuntu.Mu rwego rwo kwigana virusi, ibi bizatangwa na proteolytic cleavage ya nsp8 | nsp9 itunganyirizwa muri kopi ya polyproteine pp1a yunganirwa na Mpro na pp1ab.Muri coronavirus nyinshi, itandukaniro riri hagati yuru rubuga rwihariye (VKLQ | NNEI muri HCoV-229E) hamwe nizindi mbuga zose za coronavirus Mpro cleavage ni Asn (aho kuba ikindi gisigara gito, nka Ala, Ser cyangwa Gly) ifata P1â???Aho uherereye (36).Peptide clavage data yabonetse mubushakashatsi bwambere yerekanaga ko imikorere ya clavage ya nsp8 | nsp9 yari munsi yizindi mbuga, byerekana ko 1) uru rubuga rwihariye rushobora kugira uruhare runini mugutunganya neza C-terminal pp1a karere, cyangwa 2) a Uruhare rwihariye rwabitswe nsp9 N-terminus mu kwigana virusi (37).Amakuru yacu (Igicapo 5A) yerekanaga ko uburyo bwa recombinant bwa nsp9 butwara N-terminal nyayo yakurikiranye neza NMPized na nsp12.Urutonde rwa N-terminal rwakuweho kubintu Xa (nsp9-His6; SI umugereka, Imbonerahamwe S1) cyangwa Mpro-medrated clavage (nsp7-11-His6; Igicapo 5A na SI umugereka, Imbonerahamwe S1).Icy'ingenzi, nsp9 idakubiyemo irimo prursor nsp7-11-His6 yerekanye ko irwanya NMPylation ya nsp12, ibyo bikaba bihuye namakuru yacu, byerekana ko inyongeramusaruro ya nsp9-NMP ikorwa binyuze muri N-terminal yibanze (SI umugereka, Ishusho S5) .Kugirango tubashe gusobanukirwa byimbitse ya NiRAN substrate yihariye, noneho twibanze kubisigisigi bya N-terminal bisigaye bya nsp9.Mugihe habuze izindi poroteyine, zirahinduka muburyo bwubaka, zikabuza kumenyekana muburyo butemewe bwa nsp9 (26 28, 38), byerekana itandukaniro ryabo ridasanzwe Ibi biterwa nurwego rukomeye rwihariye (ntabwo rufite urwego rwa kabiri rujyanye) imikorere ya nsp9 N-terminal igice.Ala gusimbuza ibisigazwa byabitswe muri kano karere (Igicapo 5C na D na SI umugereka, Igicapo S8) kigaragaza ko N3826 ari ngombwa kuri nsp9 NMPylation muri vitro, naho N3825A na E3827A bituma igabanuka rya NMPylation, mugihe M3829A na P3830A zidashobora kugabanuka. .Biragaragara ko bigira ingaruka kuri nsp9 NMPylation.Nubwo gusimbuza N-terminal Asn (N3825A, N3825S) bifite ingaruka zoroheje kuri nsp9 NMPylation no kwigana virusi mumico y'utugari (Igicapo 5C na D), gusiba urutonde rwibisigisigi bya Asn muri N-terminal 3825-NN dipeptide byagaragaye ko byica virusi, byerekana ko igisigara kimwe cya Asn gisabwa mbere yikindi gisigara kuri N-terminus, byaba byiza Asn, nubwo bigaragara ko gusimbuza ibisigazwa bisa bishobora kwihanganira igice (Ishusho 5B, C, na D).Twanzuye ko dipeptide ya 3825-NN, cyane cyane ibisigisigi byabitswe kandi byingenzi bya N3826 murwego rwa coronavirus (umugereka wa SI, Igicapo S6), byemeza guhuza neza no kwerekana icyerekezo cya nsp9 N-terminus kurubuga rukora rwa NiRAN.
Gusimbuza Ala (E3827A) kuri Glu yabitswe mumiryango yose igumana nsp9 NMPylation muri vitro ariko byica virusi mumico y'utugari (Ishusho 5C na D), byerekana imikorere yinyongera yibi bisigara, kurugero, mubikorwa byingenzi (NMPylated cyangwa idahinduwe) ) nsp9 N-terminus nibindi bintu bigira uruhare mu kwigana virusi.Ihinduka rya Nsp9 ntabwo ryagize ingaruka kuri proteolytic ya nsp9 cyangwa nsps iyo ari yo yose yegeranye (39) (SI Umugereka, Igicapo S9), byerekana ko fenotipike yica ihinduka ryimiterere ya nsp9 yagaragaye itatewe no kudakwirakwizwa kwa C proteolytic process-terminal pp1a .
Amakuru yavuzwe haruguru aratanga ibimenyetso byerekana ko nyuma yubuvuzi bwa Mpro bwunganirwa nsp8 | 9 clavage site muri pp1a / pp1ab, N-terminus ya nsp9 irashobora UMPylated (cyangwa yahinduwe igice hamwe nindi NMP).Byongeye kandi, kubungabunga neza N-terminus ya nsp9 (harimo ibisigisigi bya Asn bidasanzwe kandi bidahinduka mu muryango wa coronavirus) hamwe namakuru ya genetique ya reaction yabonetse muri ubu bushakashatsi (Ishusho 3E na 5D) yatumye twemeza ko ibisobanuro nsp9 NMPylation byasobanuwe ni ibinyabuzima bifitanye isano kandi ni ngombwa mu kwigana coronavirus.Ingaruka zimikorere yiri hinduka ziracyakomeza kwigwa, kurugero, kubyerekeye ibyasobanuwe mbere (bidasanzwe) nsp9 (form idahinduwe) ibikorwa bya RNA bihuza (2628).N-terminal NMPylation irashobora kandi kugira ingaruka kumikoranire ya nsp9 hamwe na poroteyine cyangwa RNA insimburangingo cyangwa ishingwa ryinteko zitandukanye.Ibi byagaragaye mubushakashatsi bwubatswe kandi byemejwe ko bifitanye isano no kwigana coronavirus, nubwo cyane cyane iyo hatabayeho Mugihe cyo guhindura (26- â € 29, 40).
Nubwo intego yihariye ya coronavirus NiRAN iracyakeneye kurangwa muburyo burambuye, amakuru yacu yerekana ko intego ya poroteyine yihariye ya coronavirus NiRAN ari nto cyane.Nubwo kubungabunga ibisigazwa byingenzi byibikorwa byingenzi (8, 16) murwego rwa NiRAN mumiryango yose ya nidovirus ishyigikiye byimazeyo ibikorwa bya NMPylator yabungabunzwe izo poroteyine, umwirondoro wa substrate uhuza ibisigazwa byumufuka wiyi domeni Kubungabunga no kubungabunga bikomeje kurangwa , kandi irashobora gutandukana mumiryango itandukanye ya Nidovirales intego.Muri ubwo buryo, intego zijyanye nizindi virusi zatewe ntikiramenyekana.Bashobora kuba ari orthologs ya kure ya nsp9 cyangwa izindi poroteyine, kubera ko urukurikirane ruri hanze ya domaine eshanu zisubirwamo zikunze kubikwa muri virusi zatewe ntizibitswe neza (8), harimo na genome array hagati ya Mpro na NiRAN, Muri bo, nsp9 iherereye muri coronavirus.
Mubyongeyeho, ntidushobora guhakana ko bishoboka ko domeni ya NiRAN ifite intego zinyongera (harimo na selile).Kuri iki kibazo, birakwiye ko tuvuga ko bagiteri homologues muri iyi proteine igaragara NMPylators (NMPylators) (30, 31) isa nkaho ifite "umuyobozi mukuru"?NMP ihindura poroteyine zitandukanye za selile kugirango igenzure cyangwa ikureho ibikorwa byayo byo hasi, bityo igire uruhare mubikorwa bitandukanye byibinyabuzima, nko gukemura ibibazo bya selile na redox homeostasis (22, 33).
Muri ubu bushakashatsi (Isanamu 2 na 4 na SI Umugereka, Ishusho S3 na S5), twashoboye kwerekana ko nsp12 yimuye igice cya UMP (NMP) kumwanya umwe (wabitswe) muri nsp9, mugihe izindi poroteyine zidahinduwe muri Byakoreshejwe Mubihe, bisobanuwe neza (kuruta kurekura) substrate yihariye irashyigikirwa.Bihuye nibi, ugereranije na N-terminal nsp9 NMPylation, nsp12′s ibikorwa bya NMPylation ni bike cyane, kubimenya bisaba igihe kirekire cyo kwerekana autoradiografiya, kandi byikubye inshuro 10 kwibanda kuri nsp12.Byongeye kandi, isesengura ryacu rya MS ryananiwe gutanga ibimenyetso kuri NMPylation ya nsp12, ibyo bikaba byerekana ko NiRAN domaine yonyine-NMPylation ari (nibyiza) igikorwa cya kabiri.Ariko, twakagombye kumenya ko ubundi bushakashatsi bwatanze ibimenyetso byibanze byerekana ko imiterere-ya-AMPylation ya bagiteri NMPylator ishobora kugenzura ibikorwa byabo bya NMPylation kubindi bikoresho bya poroteyine (22, 33).Kubwibyo, harakenewe ubushakashatsi bwinshi kugirango hakorwe iperereza ku ngaruka zishobora guterwa n’ibikorwa byo kwikorera-NMPylation byatangajwe kuri EAV nsp9 (16) na coronavirus nsp12 (ubu bushakashatsi), harimo n'ingaruka ziteganijwe nka chaperone ku kuzenguruka kwa C-terminal RdRp ( 16)).
Mbere, ibitekerezo byinshi byerekeranye nimirimo ishoboka yo kumanuka ya domeni ya nidoviral NiRAN yatekerejweho, harimo RNA ligase, RNA ifata guanylate transfert hamwe nigikorwa cya priming priming (16), ariko ntanumwe murimwe ushobora guhuza nibikorwa bihari byo hasi.Amakuru yabonetse mumwanya ukurikira nigihe kimwe rwose udatanze ibindi bitekerezo.Amakuru yabonetse muri ubu bushakashatsi arahuza cyane (ariko ntashobora kwerekana) ko domaine ya NiRAN igira uruhare mugutangiza intungamubiri za poroteyine ziterwa na RNA.Mbere byizerwaga ko imikorere ya domaine ya NiRAN muri 5 ??²-RNA gufata cyangwa RNA yogusubiramo ntabwo bigira ingaruka kuri hamwe no Gushyigikira andi makuru.Kubwibyo, kurugero, urubuga rukora rwa NiRAN rufatwa nkuruhare rwa Asp rwabitswe nkibanze rusange (D252 muri syringae Peludomonas SelO; D4271 muri HCoV-229E pp1ab; D208 muri SARS-CoV-2 nsp12) (Umugereka wa SI, ishusho 2 ).S2). 41).Byongeye kandi, uburyo budasanzwe bushingiye ku rutonde rwa coronavirus NiRAN ku ntego za poroteyine zabitswe hamwe no kworoherwa kwa NTP gufatanya (guhitamo UTP) irwanya NiRAN-yifashishijwe na capping enzyme cyangwa imirimo ya RNA ligase.
Ikigaragara ni uko imirimo myinshi yinyongera irakenewe kugirango igenzurwe kandi, niba bigaragaye, irambuye ku ruhare rushoboka rwa nsp9-UMP (nsp9-NMP) muri sintezike ya RNA iterwa na poroteyine, izahuza raporo zishimishije ariko (kugeza ubu) zavuzwe mbere .Kwitegereza wenyine.Kurugero, hemejwe ko iherezo rya RNA itari nziza ya coronavirus itangirana numugozi wa oligo (U) (42, 43).Iri genzura rihuye nigitekerezo kivuga ko synthesis ya RNA itemewe-itangizwa no guhuza uburyo bwa UMPylated bwa nsp9 umurizo wa poly (A) umurizo (imbarutso), ushobora kuzamurwa na RNA ihuza Igikorwa na / cyangwa imikoranire hamwe indi poroteyine ya RTC.Igice cya UMP gitangwa na nsp9 noneho gishobora gukoreshwa nka "primer" kuri nsp7 / 8 / nsp12-yunganiwe na oligouridylation, ukoresheje umurizo wa 3 ?? ²-poly (A) muri genomic RNA cyangwa Indi oligo (A) -kurikirana ikora nk'icyitegererezo, gisa nuburyo bwashyizweho kuri poroteyine ya picornavirus VPg (44).Byagenda bite niba icyifuzo ari "kidasanzwe"????Itangizwa rya (protein-iterwa) na synthesis ya RNA itanga umurongo uhuza kwitegereza, byerekana ko coronavirus negative-strand RNA ifite UMP (aho kuba UTP) kumpera yayo (42), ifatwa nkikerekana ko the aside nucleic Dicer ikuraho amaherezo ya fosifora na endonuclease ya uridine itazwi.Niba byemejwe, iki gikorwa cya hydrolytike acide nucleic kirashobora gufasha kurekura uburyo bwa oligomeric UMPylated ya nsp9 kuva 5 ² iherezo ryumutwe mubi.Uruhare rushoboka rwa nsp9 mukubyara poroteyine narwo ruhuye nubushakashatsi bwibanze bwerekeranye na genetics, bwerekanye ko nsp9 (na nsp8) ikorana cyane cyane na cis ikora RNA ikora hafi yimpera ya 3 ya genona ya coronavirus.45).Nk’uko iyi raporo ibigaragaza, ibyo twabonye mbere birashobora kongera gusuzumwa no kwagurwa binyuze mu bundi bushakashatsi.
Muncamake, amakuru yacu yagennye ibikorwa byihariye bya virusi yanduye virusi ya enzyme ihujwe na RdRp kuri N-terminus.Muri coronavirus, iki gikorwa cyavumbuwe na NiRAN cyahujwe na UMPylator / NMPylator gikoreshwa mu gushingira kuri Mn2 + hamwe n’ibisigisigi bya Asn byegeranye kandi bigatera ishingwa rya fosifora yo mu bwoko bwa fosifora hamwe na amine y'ibanze ya N-terminal.Binyuze muri Mpro-yunganirwa na clavage kurubuga rwa nsp8 | 9, intego ya nsp9 irashobora gukoreshwa kuri NMPylation, byerekana guhuza imikorere hagati ya protease na domaine ya NiRAN, igera kuri RdRp.Kubungabunga ibisigazwa byingenzi muri site ikora ya nsp12 NiRAN hamwe nintego ya nsp9, ihujwe namakuru yakuwe muri coronavirus ebyiri harimo na SARS-CoV-2, itanga ibimenyetso bifatika byerekana ko nsp9 NMPylation ari coronavirus Ibiranga konserwatori nabyo ni intambwe yingenzi mu kwigana virusi.Amakuru aboneka atuma twemeza ko uruhare rwihariye rwa NMPylated ya nsp9 muri synthesis ya RNA iterwa na poroteyine ni ibintu byumvikana kuri coronavirus nizindi virusi zanduye, kandi NiRAN irashobora no kwibasira izindi poroteyine zitamenyekana.Tunganya virusi.Imikoranire.Niba byemejwe, uruhare rwa primers za poroteyine muri virusi ya RNA bizongera ubwinshi bwurwego rwa Mpro / 3CLpro na RdRp hagati ya coronavirus na picornavirus imeze nka supergroup (9), ubu ikaba yarahujwe muri Pisonivirites iherutse gushingwa ( 46) mu cyiciro.
Amakuru yacu arerekana kandi ko ibikorwa byibanze, gutoranya no guharanira inyungu za enzyme byagaragaye muri ubu bushakashatsi bishobora gukoreshwa nkibitego byibiyobyabwenge bya virusi.Imvange zibangamira guhuza (no guhindura nyuma) ya nsp9 N-terminus yabitswe mu kibanza gikora cya NiRAN irashobora gutezwa imbere mu miti igabanya ubukana bwa virusi itera virusi, ikwiranye no kuvura inyamaswa na coronavirus zandurira mu bwoko butandukanye (sub). , harimo SARS-CoV-2 na Syndrome de Coronvirus yo mu burasirazuba bwo hagati.
Urutonde rwa code ya poroteyine ya coronavirus yakozwe muri ubu bushakashatsi yongerewe ingufu na RT-PCR ikoresheje RNA yitandukanije na Huh-7 yanduye HCoV-229E cyangwa Vero E6 yanduye SARS-CoV-2, hanyuma yinjizwamo hakoreshejwe uburyo busanzwe bwo gukoroniza.pMAL-c2 (Laboratoire y’ibinyabuzima yo mu Bwongereza Nshya) cyangwa pASK3-Ub-CHis6 (47) imvugo yerekana (SI Umugereka, Imbonerahamwe S1 na S2).Gusimbuza codon imwe yatangijwe na PCR ishingiye kurubuga rwa mutagenezi (48).Kugirango habeho poroteyine ya MBP fusion, E. coli TB1 selile zahinduwe hamwe na pMAL-c2 plasmid yubaka (SI umugereka, Imbonerahamwe S1).Poroteyine ya fusion yasukuwe na amylose affinity chromatografiya kandi ifatanyijemo ibintu Xa.Ibikurikira, C-terminal His6-yashizwemo poroteyine yahanaguwe na Ni-immobilized metal affinity chromatography (Ni-IMAC) nkuko byasobanuwe mbere (49).Kugirango habeho poroteyine ya ubiquitin, selile E. E. coli TB1 yakoresheje pASK3-Ub-CHis6 plasmid yubaka (SI Umugereka, Imbonerahamwe S1 na S2) hamwe na ADN ya pCGI plasmid ADN ikubiyemo ubiquitin yihariye C-terminal hydrolase 1 (Ubp1).Guhinduka (47).C-terminal His6-yashizwemo poroteyine ya coronavirus yasukuwe nkuko byasobanuwe mbere (50).
Ikizamini cyo kwikorera-NMPylation ya HCoV-229E nsp12-His6 cyakozwe nkuko byasobanuwe muri EAV nsp9 (16).Muri make, nsp12-His6 (0.5 µM) irimo 50 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic aside (HEPES) -KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 µM buffer, incubate byerekanwe NTP na 0.17 µM bihuye [α32-P] NTP (3000 Ci / mmol; Hartmann Analytic) kuri 30 ° C muminota 30.Mubindi byose (bisanzwe) NMPylation yerekana nsp12-yunganirwa nsp9 NMPylation, imiterere yimyitwarire yahinduwe kuburyo bukurikira: nsp12-His6 (0.05 µM) na nsp9-His6 (4 µM) imbere ya mM 50 HEPES-KOH (pH 8.0 ), 5 mM DTT, 1 mM MnCl2, 25 µM yerekanye NTP, na 0.17 µM bihuye [α32-P] NTP.Nyuma yo gushiramo iminota 10 kuri 30 ° C, icyitegererezo cya reaction cyavanze na buffer ya sample ya SDS-PAGE: tris 62.5 mM tris (hydroxymethyl) aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol na 0.005% bromophenol ubururu.Poroteyine yatandukanijwe no gushyushya 90 ° C mu minota 5 hanyuma itandukanywa na 12% SDS-PAGE.Gele irakosowe kandi irangi hamwe na Coomassie Brilliant Igisubizo cyubururu (40% methanol, 10% acide acetike, 0,05% Coomassie Brilliant Blue R-250), irangi, kandi ihura na ecran ya fosifore yamasaha 20 (kugirango umenye nsp12 kuva NMPylation) cyangwa (ntarengwa) Amasaha 2 (gusuzuma nsp9 NMPylation).Inkubi y'umuyaga 9200 (GE Healthcare) yakoreshejwe mu gusikana ecran naho ImageJ yakoreshejwe mu gusesengura ubukana bw'ikimenyetso.
Kubisesengura rya MS, hakoreshejwe 1 µM nsp12-His6 na 10 µM nsp9 (idafite tagi ya hexahistidine) yakoreshejwe mu isesengura rya NMPylation (umugereka wa SI, Imbonerahamwe S1) kandi hiyongereyeho 500 µM UTP na GTP.Bitewe nubunini bwabo hamwe nubwiza bwa poroteyine ziteganijwe, sisitemu y'amazi H-yo mu rwego rwa HPLC ifite inkingi ya MassPrep (Amazi) yakoreshejwe mu kumanura 1 kugeza 10 µL by'ibisubizo bya poroteyine kuri interineti.Poroteyine yanduye ivanwa muri electrospray ion isoko ya Synapt G2Si mass spectrometer (Amazi) ikoresheje icyiciro gikurikira cya buffer A (amazi / 0,05% acide formique) na buffer B (acetonitrile / 0.045% acide formique), kandi ubushyuhe bwinkingi ni 60 ° C hamwe nigipimo cya 0.1 mL / min: gusiba muburyo bwa demokarasi hamwe na 5% A muminota 2, hanyuma umurongo ugana kuri 95% B muminota 8, hanyuma ugakomeza 95% B muminota 4.
Iion nziza ifite intera ya 500 kugeza 5000 m / z iragaragara.Glu-fibrinopeptide B ipimwa buri masegonda 45 kugirango ikosore byikora.Koresha porogaramu ya MassLynx hamwe niyagurwa rya MaxEnt1 kugirango ugabanye impuzandengo yikigereranyo nyuma yo gukuramo ibyingenzi no koroshya.
UMPylated HCoV-229E nsp9 yarimishijwe no kongeramo trypsin (Serva) ikurikirana-ikurikirana hanyuma ikarara ijoro kuri 37 ° C.Inkingi ya Chromabond C18WP (igice nimero 730522; Macherey-Nagel) yakoreshejwe mu kumanura no kwibanda kuri peptide.Hanyuma, peptide yashongeshejwe muri 25 µL y'amazi, yarimo acetonitrile 5% na aside aside ya 0.1%.
Ingero zasesenguwe na MS hakoreshejwe Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Sisitemu yanyuma ya nanoâ HPLC (Dionex), ifite ibikoresho byabugenewe byashizwe kuri cm 50 ??75 μm C18 RP inkingi yuzuye amasaro ya magnetiki 2,4 μm (Dr. Albin Maisch High Performance LC GmbH) Ihuze na mass spectrometer kumurongo ukoresheje isoko ya Proxeon nanospray;shyiramo 6 µL yumuti wa trypsin igogora muri 300 µm diametre y'imbere × ??1 cm C18 PepMap ibanziriza kwibanda (Thermo Scientific).Ukoresheje amazi / 0,05% acide ya formeque nkumuti, icyitegererezo cyahise gifatwa kandi kivanwaho umuvuduko wa 6 µL / min.
Imiyoboro ikurikira y'amazi / 0,05% ya aside irike (solvent A) na 80% acetonitrile / 0,045% acide formique (solvent B) yakoreshejwe kugirango igere ku gutandukanya peptide ya tryptic ku gipimo cya 300 nL / min: 4% B kuri Iminota 5, hanyuma 30 Umurongo ugereranya kugeza 45% B muminota mike, hanyuma umurongo wiyongera kuri 95% solvent B muminota 5.Huza inkingi ya chromatografiya nicyuma kitagira umwanda nano-emitter (Proxeon), hanyuma utere eluent mu buryo butaziguye kuri capillary yashyutswe ya mass spectrometer ukoresheje ubushobozi bwa 2.300 V. Gusikana ubushakashatsi hamwe nibisubizo 60.000 mubisesengura rusange bya Orbitrap bifitanye isano. hamwe nibura amakuru atatu ya MS / MS scan, ukuyemo imbaraga mumasegonda 30, ukoresheje umurongo wa ion trap kugongana byatewe no gutandukana cyangwa gutandukana kwingufu nyinshi hamwe no gutahura orbitrap, Icyemezo ni 7.500.
Igihe cyo kohereza: Kanama-03-2021