Waxaa tafatiray Peter Sarnow, Dugsiga Caafimaadka ee Jaamacadda Stanford, Jaamacadda Stanford, California, oo la ansixiyay Diseembar 25, 2020 ( dib loo eegay Oktoobar 25, 2020)
Waxaan ka warbixineynaa isdhexgalka ka dhexeeya qayb-hoosaadyada soo-celinta coronaviruses-ka dhismooyinka qoraalka, kuwaas oo lagama maarmaan u ah soo-saarista iyo ilaalinta korriinka.Waxaan bixinay caddayn ah in domainka NiRAN ee la xidhiidha nsp12 uu leeyahay nucleoside monophosphate (NMP) dhaqdhaqaaqa wareejinta trans, oo loo aqoonsaday nsp9 (borotiinka xirida RNA) inuu yahay bartilmaameedkiisa.NiRAN waxa ay kicisaa isku xidhka unugyada NMP ee ku xidhan teerminus amino nsp9 taaso falcelin ku tiirsan Mn2+ ion iyo hadhaaga Asn ee la xafiday.Waxaa la ogaaday in dhaqdhaqaaqa NiRAN iyo nsp9 NMPylation ay lagama maarmaan u yihiin ku-noqoshada coronavirus.Xogtu waxay noo ogolaanaysaa in aan ku xidhno hawshan calaamadeeyaha fayraska buul-ku-jirka ah ee enzyme-ka iyo indho-indhayntii hore ee mala-awaalka ah in bilaabista isku-darka RNA ee fasalka fayraska RNA uu yahay mid shaqaynaya oo horumarsan.
RNA-ku-tiirsanaanta RNA polymerase (RdRps) ee Nidovirales (Coronaviridae, Arterioviridae, iyo 12 qoys oo kale) waxay ku xiran tahay amino-terminal (N-terminal) ee borotiinka aan qaabdhismeedka ahayn (nsp) ee laga sii daayo borotiinka, oo loo yaqaan NiRAN 1ab wuxuu ka kooban yahay fayraska ugu weyn ee protease (Mpro).Markii hore, fayraska halbowlaha ah ee NiRAN-RdRp nsp shaqadiisa GMPylation/UMPylation ayaa la soo sheegay, waxaana la soo jeediyay in la abuuro ku meel gaadh ah wareejinta nucleoside monophosphate (NMP) ee (hadda aan la garanayn) fayraska iyo/ama walxaha bioopolymerization unug.Halkan, waxaan ku tusineynaa in coronavirus (Human Coronavirus [HCoV]-229E iyo Syndrome Ba'an oo Ba'an oo Neefsi ah Coronavirus 2) nsp12 (NiRAN-RdRp) uu leeyahay Mn2+-ku-tiirsanaanta NMPylation, kaas oo laga soo qaatay nsp9 iyada oo loo marayo sameynta Mpro-dhexdhexaadin nsp9 Kadib N-terminal flanking nsps si proteolyt ahaan loo sii daayo, fosfooraska waxay ku xidhan tahay amine aasaasiga ah (N3825) ee N-terminal ee nsp9.Uridine triphosphate waa nucleotide la door bidaayo falcelintan, laakiin adenosine triphosphate, guanosine triphosphate, iyo cytidine triphosphate ayaa sidoo kale ku habboon wada-substrates.Daraasadaha is-beddelka iyadoo la adeegsanayo recombinant coronavirus nsp9 iyo nsp12 borotiinada iyo hidde-hagaajinta HCoV-229E mutants ayaa go'aamiyay hadhaaga lagama maarmaanka u ah dhexdhexaadinta NiRAN-dhexdhexaadinta nsp9 NMPylation iyo ku-noqoshada fayraska ee dhaqanka unugyada.Xogtu waxay xaqiijisay saadaasha hadhaaga goobta firfircoon ee NiRAN waxayna go'aamisay doorka muhiimka ah ee haraaga nsp9 N3826 ee nsp9 NMPylation iyo fayraska ku celcelinta vitro.Hadhaagani waa qayb ka mid ah taxanaha N-terminal NNE tripeptide ee la xafiday oo la caddeeyey inuu yahay hadhaaga kaliya ee is bedbeddela ee nsp9 iyo homologs ee qoyska coronavirus.Daraasadani waxay bixisaa aasaas adag oo loogu talagalay daraasadda shaqeynta ee waxqabadka NMPylation ee fayrasyada kale ee buulka leh waxayna soo jeedinaysaa bartilmaameedyada suurtagalka ah ee horumarinta daawooyinka fayraska.
Nidovirales togan-ku xayiran fayraska RNA waxa uu ku dhacaa laf dhabarta iyo laf dhabarta (1, 2).Amarka hadda waxaa ku jira 14 qoys (3), kuwaas oo qoyska Coronavirus si weyn loo baaray 20-kii sano ee la soo dhaafay.Waqtigaas, saddex coronaviruses zoonotic ayaa ka soo baxay xeryahooda xayawaanka waxayna sababeen dillaac ballaaran oo caabuqyada neef-mareenka ah ee bini'aadamka.Oo ay ku jiraan masiibooyinka joogtada ah ee ay sababaan cudurrada faafa ee daran.Cudurka neef-mareenka Coronavirus 2 (SARS-CoV-2) (4âââ7).Nidoviruses waxay wadaagaan urur genome ah oo caadi ah, iyo qaybta xuubka-ku-xidhan isku-duubnida-ku-soo-celinta (RTC) waxay ku qoran tahay 5-?²-terminal saddex-meelood laba iyo qaybta ugu muhiimsan ee qayb ka mid ah fayraska, iyo sidoo kale qalabyada qaarkood. .Borotiin, ku lifaaqan 3??² dhamaadka saddexaad ee genome (1).Marka laga reebo hal qoys oo ka mid ah fayrasyada planarian (Monoviridae) (8), dhammaan fayrasyada buulka leh waxay ku dhejiyaan qaybo RTC oo ah laba qaybood oo wax akhris ah oo furan (ORF) ORF1a iyo ORF1b, kuwaas oo laga tarjumay genomic RNA ee.ORF1a waxay dejisaa polyprotein (pp) 1a, iyo ORF1a iyo ORF1b si wada jir ah u koodka pp1ab.Iyada oo ka qaybqaadashada guud ee protease-ka ugu weyn (Mpro) ee ay dejisay ORF1a, pp1a iyo pp1ab labadaba si proteolytically ah ayaa looga baaraandegay noocyo kala duwan oo borotiinno aan qaabdhismeed ahayn (nsps), oo sidoo kale loo yaqaan 3CLpro, sababtoo ah waxay leedahay homology leh 3Cpro ee picornavirus ( 9).Nsps-yadaan waxaa loo maleynayaa in lagu soo ururiyay RTC firfircoon oo ballaaran, waxay kiciyaan isku-dhafka RNA (ku-noqoshada) iyo set of RNA subgenomic (qoraal), waxaana loo adeegsadaa isku-dubarid muujinta ORF ee ku taal hoose ee ORF1b (10? ? ?12).
Xudunta u ah RTC waxaa ka mid ah RNA-ku-tiirsanaanta RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) iyo dhowr enzymes farsamaynta RNA, kuwaas oo inta badan lagu calaamadeeyay ORF1b iyo qoyska coronavirus Waxay ka kooban tahay nsp12-nsp16 iyo nsp9-nsp12 ee qoyska Arterioviridae (fiiri tixraaca 10ââ 12).RdRp iyo HEL1 waxay ka dhigan yihiin laba (shan-meelood meel) xayndaabyada la ilaaliyo ee fayraska buulka shimbiraha waxayna leeyihiin isku-imaatinka fayraska kale ee RNA.Nuqulka xudunta ah ayaa la rumeysan yahay inay caawiyaan qaybo kale, oo ay ku jiraan dhowr nsps yar yar oo laga sii daayay gobolka karboxy-terminal (C-terminal) ee pp1a, hoosta Mpro (coronavirus nsp5 iyo fayraska halbowlaha nsp4, siday u kala horreeyaan).Waxay leeyihiin ilaalin xaddidan oo qoyska u gaar ah iyo hawlo kala duwan (oo lagu eegay tixraac 10ââ12).
Dhawaan, domain leh astaamo isku xigxig oo gaar ah ayaa laga helay terminus amino (N-terminus) oo ku dheggan RdRp dhammaan fayrasyada buulka leh, laakiin ma jiro fayrasyo kale oo RNA ah (16).Iyada oo ku saleysan meesha ay ku taal iyo nucleotide transferase (nucleoside monophosphate [NMP] transferase transferase), domainkan waxaa loo magacaabay NiRAN (Nestvirus RdRp-related nucleotide transferase).Isku darka laba-domain ee NiRAN-RdRp wuxuu ka kooban yahay nsp12 ee qoyska Coronaviridae iyo nsp9 ee qoyska Arterioviridae, iyo nestoviridae kale, NiRAN-RdRp ayaa la filayaa in loo sii daayo nsp ka madax banaan polyprotein-ka fayraska.Gudaha coronavirus-ka, domainka NiRAN waxa uu ka kooban yahay ??1/450 hadhaaga ah waxana uu ku xidhan yahay goobta C-terminal RdRp iyada oo loo sii marayo gobolka isku xidha (16?19).In Equine Arteritis Virus (EAV) (Arteriviridae), recombinant nsp9 waxay muujinaysaa Mn2+ ion-ku-tiirsanaanta (naftiisa) UMPylation iyo GMPylation, kuwaas oo ku xiran saddex saldhig oo isku xigta ee nestovirus, AN, BN iyo CN Hadhaaga ee isku xigxiga.Halka N ay u taagan tahay NiRAN) (16).Garabyada N-terminal ee motifyadan ayaa ah horudhac muxaafid ah oo ka yar.Qaar ka mid ah haraaga kuwan ayaa sidoo kale lagu keydiyaa kinases borotiinka fog, halkaas oo lagu muujiyey inay ku lug leeyihiin nucleoside triphosphate (NTP) xidhitaanka iyo dhaqdhaqaaqa kicinta (20, 21).Iyada oo la raacayo indha-indheyntan, dhowr hadhaaga goobta firfircoon ee muhiimka ah ee pseudokinase SelO ee sirinjigga Pseudomonas waxaa lagu soo ururin karaa SARS-CoV-2 nsp7/8/12/13 supercomplex.Hadhaaga Coronavirus ee la xafiday ee NiRAN oo lagu kor buufiyay qaab-dhismeedka elektarooniga ah.Barootiin dib-u-habayn (17).Waxaa la qiyaasayaa in dukumeentiga (naftiisa) U/GMPylation ay soo saari doonto xaalad ku-meel-gaar ah si loogu wareejiyo NMP (hadda aan la garanayn) substrate (16), iyo isku ekaanshaha qaabdhismeedka u dhexeeya NiRAN iyo protein kinase (17, 19) ) Miyuu mala-awaalku yahay in NiRAN waxay beddeshaa borotiinno kale.
Sifooyin badan, oo ay ku jiraan ururkeeda nidaamsan ee gaarka ah iyo kuwa gaarka ah oo ay la socdaan fayrasyo buul leh iyo kala soocida hidde-sidaha ee RdRp, waxay NiRAN ka dhigaan enzym sharciyeed macquul ah oo loogu talagalay fayrasyada buulkooda leh, kaas oo muhiim u ah soo bixitaankooda iyo aqoonsigooda.Markii hore, saddex hawlood oo suurtagal ah oo ku lug leh NiRAN si loo habeeyo tarjumaada genome/subgenomic ama nuqul/qorid ayaa loo yaqaan.Marka la tixgeliyo xogta yar iyo kuwa aan dhamaystirnayn ee la heli karo wakhtigaas, shaqo kastaa waxay leedahay faa'iidooyin iyo faa'iido darrooyinkeeda (16).Cilmi-baaristan, waxaan hiigsaneynaa inaan isku-darno biochemical-ka iyo dib-u-noqoshada daraasadaha hidde-sidaha ee coronaviruses-ka ee matalaya labada hidde, oo aan ku dhejinno natiijooyinkayaga asalka kobaca ee isbeddelka dabiiciga ah ee qoyska coronavirus, si aan u helno aragti ku saabsan boqortooyadan dahsoon.Waxaan ka warbixin horumarka waaweyn ee fahamka NiRAN iyada oo loo marayo aqoonsiga bartilmaameedyada dabiiciga ah ee RTC, taas oo (ka mid ah saddexda mala awaal ee la heli karo) ka qaybqaato doorka domain this bilaabay synthesis ee fayraska buul RNA.Cilmi-baaristan waxay sidoo kale fureysaa fursadaha doorarka kale ee NiRAN ee interface-ka martida loo yahay.
Si loo tilmaamo sifooyinka enzymatic ee fayraska corona nsp12 ee la xidhiidha NiRAN, waxaanu soo saarnay qaab dib-u-habayn ah oo ah coronavirus bini'aadamka 229E (HCoV-229E) nsp12 ee E. coli, oo leh sumadda His6 ee C-terminus, oo isku daray borotiinka leh [α32-P] Isku-dubarid NTP-ga joogitaanka MnCl2 sida lagu qeexay Qalabka iyo Hababka.Falanqaynta badeecada falcelinta waxay muujisay joogitaanka borotiin sumadeysan oo la guuraya nsp12 (106 kDa), taasoo muujineysa in coronavirus nsp12 uu kiciyo sameynta borotiinka kondhomka ah-NMP, oo doorbidaya la sameeyay uridine monophosphate (UMP) (Jaantus 1A) Iyo B)Falanqaynta tirada ayaa muujisay in marka la barbardhigo nucleotide kale, xoojinta calaamadda is-dhexgalka UMP ay kordheen 2 ilaa 3 jeer (Jaantus 1C).Xogtan ayaa la socota dhaqdhaqaaqa wareejinta NMP ee la saadaaliyay ee domainka NiRAN ee coronavirus (16), laakiin waxay muujineysaa in doorbidida nucleotide ee domainka NiRAN ee coronavirus iyo fayraska halbowlaha ay kala duwan yihiin.
Waxqabadka is-NMPylation ee HCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) waxaa lagu darey [α-32P] NTP iyadoo ay jirto 6 mM MnCl2 muddo 30 daqiiqo ah (eeg Qalabka iyo Hababka wixii faahfaahin ah).Alaabooyinka falcelinta waxaa kala saaray SDS-PAGE oo lagu midabeeyay Coomassie buluug dhalaalaya.(B) Barootiinka shucaaca leh waxaa lagu sawiraa sawir-qaadista fosfooraska.Jagooyinka nsp12-His6 iyo borotiinka molecular molecular markers (ee kilodaltons) ayaa lagu muujiyay A iyo B. (C) xoojinta shucaaca shucaaca (macnaha ± SEM) ayaa lagu go'aamiyay saddex tijaabo oo madax-bannaan.*P≤0.05.Xoogga isha (boqolkiiba) waxay la xiriirtaa UTP.
In kasta oo waxqabadyada ensaymka ee NiRAN ee la xidhiidha la muujiyey inay lagama maarmaan u yihiin ku-noqoshada EAV iyo SARS-CoV ee dhaqanka unugyada (16), shaqada gaarka ah ee NiRAN iyo bartilmaameedyada suurtagalka ah weli lama go'aamin.Isku ekaanshaha qaabdhismeedka dhowaan la sheegay ee u dhexeeya NiRAN iyo qoyska borotiinada leh laallaabyada borotiinka kinase-u eg (17, 22) ayaa nagu kalliftay inaan tijaabino mala-awaalka ah in NiRAN ay kiciso NMPylation borotiinnada kale.Waxaan soo saarnay bartilmaameedyo isku mid ah oo suurtagal ah, oo ay ku jiraan borotiinno aan qaab-dhismeed ahayn oo ay dejisay HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), mid kasta oo ka kooban sumadda C-terminal His6 (SI lifaaqa, Miiska S1) , Iyo ku shub borotiinadan [α32-P] uridine triphosphate ([α32-P] UTP) joogitaanka ama maqnaanshaha nsp12.Serum albumin iyo MBP-LacZα borotiinka isku dhafka ah ee laga soo saaray E. coli waxay u adeegeen sidii kontaroolada (Jaantuska 2A, jidadka 1 ilaa 7).Barootiinka shucaaca ah waxaa lagu falanqeeyay sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) iyo autoradiography, waxaana la ogaaday in ay jirto calaamad shucaac ah oo xooggan oo falcelinta ka kooban nsp12 iyo nsp9.Meesha calaamaduhu waxay u dhigantaa cufka molecular ee nsp9, taasoo muujinaysa nsp12-dhexdhexaadin UMPylation ee nsp9 (Jaantus 2B, track 7).Ma jiro borotiinno kale oo tijaabo ah oo la ogaaday inay yihiin UMPylated, taas oo keentay inaan ku soo gabagabeyno in nsp9 uu yahay substrate gaar ah oo nsp12 ah.Iyada oo la raacayo xogta is-NMPylation ee lagu muujiyey Jaantuska 1, nsp12 waxay awood u leedahay inay ku wareejiso dhammaan afarta NMP ee nsp9, inkasta oo waxtarkeedu ka duwan yahay, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Sawir).3 A iyo B).Marka la eego shuruudaha loo isticmaalo baaritaankan (gaabso falcelinta iyo wakhtiga soo-gaadhista, yaree xoogga nsp12; qalabka iyo hababka), is-NMPylation ee nsp12 lama ogaan karo (is barbar dhig Jaantuska 2B, Lane 7, iyo Jaantuska 1B), kaas oo Waxay caddeeyeen wax ku ool ah (iyo wareegyo badan) UMP waxay ka guurtay nsp12 una guurtay nsp9.Dhaqdhaqaaqa wareejinta UMP wuxuu u baahan yahay joogitaanka Mn2+ ions, sida ku cad Jaantuska 3C, halka kaliya dhaqdhaqaaqa wareejinta UMP ee ugu yar lagu arkay joogitaanka Mg2+, mana jirto wax dhaqdhaqaaq ah oo ka jira joogitaanka labada qaybood ee kale ee la tijaabiyey.Xog la mid ah ayaa lagu helay NMPylation assays ka kooban cytidine triphosphate (CTP), guanosine triphosphate (GTP), iyo adenosine triphosphate (ATP) (SI lifaaqa, Jaantus S1).
HCoV-229E nsp12-dhexdhexaadin UMPylation ee nsp9.Qaybaha borotiinka oo taxane ah (oo ay ku jiraan serum albumin, MBP-lacZα, iyo taxane HCoV-229E nsps ah oo lagu calaamadeeyay C-terminal His6 oo ay dejisay ORF1a) ayaa loo adeegsaday si loo qiimeeyo waxqabadka UMPylation ee HCoV-229E nsp12-His6⁺-dhexdhexaadin. borotiinka.Ku dheji borotiinka [α-32P] UTP 10 daqiiqo maqnaanshaha (A) ama joogitaanka (B) ee nsp12 sida lagu qeexay agabyada iyo hababka.Xagga sare ee A iyo B, SDS-polyacrylamide jel oo leh Coomassie Brilliant Blue ayaa lagu muujiyay, halka hoose ee A iyo B, sawir-qaadayaasha u dhigma ayaa la muujiyay.Booska calaamadeeyaha molecular mass protein (ee kilodaltons) ayaa laga bixiyaa dhanka bidix.Booska nsp12-His6 (B, sare) iyo calaamada shucaaca ee lagu arkay inta lagu gudajiro nsp12-His6 oo leh nsp9-His6 (B, lane 7) ayaa sidoo kale la tilmaamay, taas oo muujinaysa in [α-32P] UMP ilaa nsp9-His6 (12.9 kDa), kaas oo aan lagu arkin borotiinada kale ee la tijaabiyay.
HCoV-229E NiRAN-dhexdhexaadinta biochemical iyo astaamaha fayraska ee nsp9 NMPylation.(A iyo B) Doorka substrate-ka nucleotide ee loo isticmaalo falcelinta.Nsp12-His6 iyo nsp9-His6 waa isku dhafan yihiin oo la isku daray iyadoo ay jiraan [α-32P] NTP-yada kala duwan ee qiyaasta heerka NMPylation.(A, sare) Coomassie-stained nsp9-His6 waxaa kala soocay SDS-PAGE.(A, hoose) Autoradiograph ee isla aagga jel.(B) Dhaqdhaqaaqa qaraabada ah (macnaha ± SEM) ee joogitaanka cofactor nucleotide ee loo qoondeeyey waxaa lagu go'aamiyaa saddex tijaabo oo madax-bannaan.*P≤0.05.(C) Doorka ion birta.Muujiyay waa tijaabada caadiga ah ee NMPylation ee joogitaanka [α-32P] UTP iyo ayoonnada birta ee kala duwan, mid kasta oo leh fiirsashada 1 mM.Gudaha C, xagga sare, Coomassie midabaysan nsp9-His6 ayaa lagu muujiyay, iyo gudaha C, xagga hoose, autoradiography u dhigma ayaa lagu muujiyay.Baaxadda borotiinka calaamadeysan (oo ku jira kilodaltons) waxaa lagu muujiyey bidixda A iyo C. (D) Qaabka mutant ee HCoV-229E nsp12-His6 oo sidda beddelka amino acid ee la cayimay waxay ku jirtaa [α-32P] UTP, sida lagu sharraxay. Qalabka iyo Hababka.Nsp9-His6 ee shucaaca leh ee lagu soo saaray falcelinta NMPylation-ka waxaa lagu ogaadaa sawirka phosphorylation (D, sare).Dhaqdhaqaaqa qaraabada ah marka la barbar dhigo borotiinka nooca duurjoogta ah (wt) ayaa lagu muujiyay D, hoostana waxaa loo qaataa celcelis ahaan (± SEM) seddex tijaabo madax-bannaan.Xiddigohu waxay muujinayaan beddelka hadhaaga aan la ilaalin(E) Titer-ka fayraska ee dhaqanka sare ee unugyada p1 oo la helay 24 saacadood ka dib infekshanka waxaa lagu go'aamiyay baaritaanka huurada.Beddelka koodka ee qaybta NiRAN ee mutant HCoV-229E ee la farsameeyay ayaa la tilmaamay (lambarada soo hadhay waxay ku salaysan tahay booskooda pp1ab).Ku-noqoshada-dhimanaanta RdRp firfircoon ee goobta nsp12_DD4823/4AA ayaa loo isticmaalay xakameyn ahaan.
Si loo helo faham qoto dheer oo ku saabsan goobta firfircoon ee NiRAN oo loo go'aamiyo hadhaaga la xidhiidha waxqabadka nsp9-gaar ah wareejinta NMP, waxaanu samaynay falanqaynta isbeddelka, kaas oo aan ku beddelnay hadhaaga muxaafidka ah ee NiRAN AN, BN iyo CN motifs ( 16) Waa Ala ( lifaaqa SI, Sawirka S2).Intaa waxaa dheer, saameynta konserfatifka Arg-to-Lys ama Lys-to-Arg beddelka ayaa lagu qiimeeyay laba xaaladood.Xakamaynta (negative) ahaan, hadhaaga aan lagu kaydin karin qaybta NiRAN ee coronaviruses iyo fayrasyada kale ee buulka leh ayaa lagu beddelaa Ala. BN) iyo D4280A (CN) waxay si weyn u yareeyaan ama xitaa baabi'iyaan nsp9 NMPylation iyada oo loo marayo nsp12, halka borotiinnada leh beddelka muxaafidka (R4178K), K4116R) ay hayaan 60% iyo 80% dhaqdhaqaaqooda, taas oo muujinaysa in nasashada xayiraadaha dhinacooda silsiladuhu waa kuwo xasaasi u ah kiimikaad ahaan jidheed (Jaantuska 3D).Beddelida dhowr hadhaaga kale ee la keydiyay E4145A, D4273A, F4281A iyo D4283A aad ayey u dhib yartahay, iyo nsp9 UMPylation si dhexdhexaad ah ayaa loo dhimay.Natiijooyin la mid ah ayaa laga helay nsp9 NMPylation falcelinta ku lug leh NTP-yada kale (Jaantuska 3D iyo SI lifaaqa, Jaantuska S3), xaqiijinta in saamaynta la arkay ee beddelka amino acid gaar ah ay ka madax bannaan yihiin nooca nucleotide co-substrate la isticmaalo.Marka xigta, waxaan tijaabinay saameynta suurtagalka ah ee beddelladan nsp12 ee ku-noqoshada coronaviruses ee dhaqanka unugyada.Si taas loo gaaro, waxaan isticmaalnay habyaal DNA ah oo dhameystiran oo habboon (cDNA) oo lagu xiray fayraska tallaalka recombinant (23, 24) si aan u qorno 5-7 unug.Titration of fayraska faafa ee laga soo saaray unugyadan ayaa muujisay in badi HCoV-229E NiRAN mutants aysan ahayn wax macquul ah (Jaantuska 3E).Koox ka mid ah mutants fayraska aan shaqayn karin waxaa ka mid ah beddelka la muujiyay si loo baabi'iyo ama si weyn loo yareeyo dhaqdhaqaaqa wareejinta NMP ee vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), laakiin waxaa jira laba beddel oo kale (K4116R, E48045A) % la xafiday?Dhaqdhaqaaqooda in vitro NMPylation waxay soo jeedinayaan in xaddidaadyo dheeraad ah ay ku lug leeyihiin.Sidoo kale, laba isbeddel oo kale (R4178K, F4281A) oo sababay hoos u dhac dhexdhexaad ah ee NiRAN's in vitro NMPylation dhaqdhaqaaqa ayaa soo saaray fayras nool, si kastaba ha ahaatee, fayrasyadani waxay si weyn u yareeyeen titers iyada oo loo marayo ku celcelin.Iyadoo la raacayo xogta waxqabadka in vitro ee lagu muujiyey Jaantuska 3D, beddelaya afar hadhaaga kale oo aan lagu kaydin coronavirus iyo/ama fayrasyada kale ee buulka leh (K4113A, D4180A, D4197A, D4273A) (8, 16) waxay soo saareen fayras wax ku ool ah Farcanku, inkastoo ay leeyihiin titer si dhexdhexaad ah loo dhimay marka loo eego fayraska nooca duurjoogta ah (Jaantuska 3E).
Si loo barto in NiRAN-dhexdhexaadin NMP dhaqdhaqaaqa wareejinta ay ku xiran tahay domainka firfircoon ee RdRp, labada haraaga Asp ee la xafiday ee ku lug leh isuduwidda ion birta divalent (11) ee RdRp motif C waxaa lagu beddelay Ala. borotiinka ka soo baxay nsp12_DD4823/4AA ayaa sii hayn doona Dhaqdhaqaaqeeda nsp9 NMPylation, taas oo muujinaysa in nsp12-dhexdhexaadin gudaha vitro nsp9 NMPylation-ka aan u baahnayn dhaqdhaqaaqa polymerase (SI Lifaaqa, Jaantuska S4).
Ka dib markii aan aasaasnay dhaqdhaqaaqa NMP-ga gaarka ah ee NMP ee nsp12, waxaan isku daynay in aan ku sifeyno NMP-nsp9 soo jiidashada mass spectrometry (MS).Tirada guud ee borotiinka ee dib-u-habaynta HCoV-229E nsp9 waxay muujisay ugu sarreeya 12,045 Da (Jaantuska 4A).Ku darida nsp12 ma aysan bedelin tayada nsp9, taas oo muujinaysa in nsp12 iyo nsp9 aysan samayn doonin dhismo xasilloon oo hoos yimaada shuruudaha loo isticmaalo (denaturation) (Jaantus 4A).Marka ay joogaan UTP iyo GTP, cabbirka qiyaasta falcelinta ka kooban nsp9 iyo nsp12 siday u kala horreeyaan waxay muujisay in tirada borotiinka ee UTP ay dhaqaaqday 306 Da, iyo cufnaanta borotiinka ee GTP waxay dhaqaaqday 345 Da, taas oo muujinaysa in nsp9 molecule kasta uu xidho UMP ama GMP (Sawirka 4) C iyo D.Waxaa la qiyaasayaa in tamarta looga baahan yahay NiRAN-dhexdhexaadin nsp9 NMPylation ay ka timaaddo NTP hydrolysis iyo sii deynta pyrophosphate.In kasta oo falcelintan loo adeegsaday 10-jibbaaran molar-ka-dhaafka ah ee nsp9 (bartilmaameedka) marka loo eego nsp12 (enzyme) falcelintan, ku dhawaad dhammaystiran NMPylation ee nsp9 ayaa la arkay, taas oo muujinaysa in isdhexgalka u dhexeeya nsp12 iyo nsp9 uu yahay mid cimri gaaban, iyo nsp12 waxay NMPylateyn kartaa nsp9 badan. molecule in vitro.
NMPylation Keliya ee nsp9 iyadoo ay joogaan nsp12 iyo UTP ama GTP.Muujiyay waa kala-soocidda guud ee baaxadda borotiinka ee HCoV-229E nsp9 (SI lifaaqa, Shaxda S1) (AD).(A) nsp9 keligiis, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 iyadoo ay joogaan UTP, (D) nsp9 + nsp12-His6 iyadoo la joogo GTP.
Si loo go'aamiyo hadhaaga nsp9 UMPylated by nsp12, nsp9-UMP waxaa lagu kala jeexjeexay trypsin.peptides-ka soo baxay waxaa lagu kala saaray nano-high performance dareeraha chromatography (HPLC) waxaana lagu falanqeeyay tandem mass spectrometry (MS/MS) onlayn.Falanqaynta xogta iyadoo la adeegsanayo xirmada software-ka Byonic (Metrics Protein) waxay muujisay UMPylation ee N-terminal amino acid.Tan waxaa lagu xaqiijiyay gacantaQiyaasta tirada guud ee tandem ee peptide hore [UMP] NNEIMPGK (SI lifaaqa, Jaantuska S5A) ayaa daaha ka qaaday jajab dhan 421 m/z, taas oo muujinaysa in UMP ay ku xidhan tahay hadhaaga 1 ee nsp9.
Waqtiga N-terminus ee nsp9, Asn waxaa lagu keydiyaa xubnaha Orthocoronavirinae (SI lifaaqa, Jaantuska S6).In kasta oo aan aaminsanahay in N-terminal-ka aasaasiga ah amine nitrogen uu yahay kan ugu badan ee aqbala UMP, waxaan go'aansanay inaan ka helno caddayn dheeri ah oo ku saabsan xiritaanka NMP ee N-terminal.Sababtan awgeed, N-terminal peptide nsp9 aan NMPylated iyo NMPylated N-terminal peptide nsp9 oo ay nadiifisay HPLC waxaa laga soo qaatay joogitaanka acetone iyo sodium cyanoborohydride.Xaaladahan hoos yimaada, kaliya aminyada aasaasiga ah ee bilaashka ah ayaa lagu beddeli karaa propyl (25).N-terminal nsp9-ku-soo-saaray peptide oo leh taxanaha NNEIMPGK wuxuu ka kooban yahay laba amines aasaasiga ah, mid ka mid ah N-terminus ee Asn iyo kan kale ee silsiladda dhinaca ee Lys ee C-terminus.Sidaa darteed, kooxaha propyl waxaa lagu soo bandhigi karaa labada daraf.chromatogram-yada ion ee la soo saaray ee peptides-ka aan NMPylated ahayn ayaa lagu muujiyay lifaaqa SI, Jaantuska S5B.Sida la filayo, N-terminal iyo C-terminal (mono) propylated (SI lifaaqa, Jaantuska S5B, haadka sare) iyo peptides dipropylated (SI lifaaqa, Jaantus S5B, haadka hoose) waa la aqoonsan karaa.Habkani wuxuu is beddelaa isticmaalka NMPylated N-terminal peptide ee nsp9.Xaaladdan oo kale, kaliya C-terminal propylated peptides ayaa la aqoonsan karaa, laakiin N-terminal propylated peptides iyo peptides dipropylated aan la aqoonsanin (SI Lifaaqa, Jaantuska S5C), taas oo muujinaysa in UMP loo wareejiyay amine aasaasiga ah ee N-terminal Si looga hortago tan. kooxda inay isbedel sameeyaan.
Marka xigta, waxaan ku bedelnaa (Ala ama Ser) ama tirtirnaa hadhaaga la xafiday ee N-terminus ee nsp9 si loo qeexo caqabadaha gaarka ah ee bartilmaameedka ah.Iyada oo ku saleysan xogtayada MS ee muujineysa in NiRAN ay sameyso nsp9-NMP oo leh amine aasaasiga ah ee hadhaaga N-terminal ee nsp9, waxaan ku qiyaasnay in nsp9 NMPylation ay u baahan tahay protease sayidkiisa fayraska (Mpro, nsp5) si uu u sii daayo nsp9 N-terminal ka horudhaciisa polyprotein-ka.Si loo tijaabiyo mala-awaalkan, waxaanu soo saarnay borotiinka nsp7-11 oo ka kooban nsp9 ee E. coli waxaana aanu samaynay imtixaan NMPylation ah oo ay joogaan [α-32P] UTP (qalabka iyo hababka).Sida ku cad Jaantuska 5A (haadka 3), horreeyaha nsp7-11 ee aan la jarin laguma calaamadin nsp12.Taas bedelkeeda, haddii nsp7-11 lagu kala jeexo recombinant nsp5 si loo sii daayo nsp9 (iyo nsps kale) oo ka soo jeeda horudhac, borotiin sumadeysan oo la haajiray nsp9 ayaa la ogaadaa, taasoo xaqiijineysa gabagabadayada NiRAN iyo N-Xulashada qaabeynta nsp9-NMP .Amine aasaasiga ah ee terminal ee N-terminal Asn (booska 3825 ee pp1a/pp1ab).Gabagabadaan waxaa sidoo kale taageeray tijaabooyin la isticmaalayo nsp9 dhiska, kaas oo ka kooban hal ama laba hadhaaga dheeraad ah ee N-terminus.Labada xaaladoodba, NiRAN-dhexdhexaadin UMPylation ee nsp9 waa la tirtiray (SI Lifaaqa, Jaantuska S7).Marka xigta, waxaanu soo saarnay borotiin leh hal ama laba hadhaaga Asn oo laga tirtiray isku xigxiga 3825-NNEIMPK-3832 peptide ee N-terminal ee nsp9.Labada xaaladoodba, nsp9 UMPylation si buuxda ayaa loo xannibay (Jaantuska 5B), siinta caddayn dheeraad ah in nsp9 N-terminus dhabta ah uu u dhaqmo sidii NMP reseptor.
Habaynta boroteolytiga ee nsp9 iyo doorka hadhaaga N-terminal ee nsp12-dhexdhexaadinta UMPylation.(A) nsp9 UMPylation waxay u baahan tahay nsp9 N-terminal bilaash ah.Nsp7-11-His6 ayaa horay loogu sii daayay 30 °C ee kaydinta ogaanshaha NMPylation ee ka kooban UTP joogitaanka ama maqnaanshaha dib-u-habaynta Mpro (nsp5-His6).3 saacadood ka dib, billow NMPylation assay adiga oo ku daraya nsp12-His6 sida lagu qeexay Qalabka iyo Hababka.Falcelinta ka kooban nsp5-His6 (haadka 1) iyo nsp9-His6 (lane 2) ayaa loo adeegsaday xakameyn ahaan.10 daqiiqo ka dib, falcelinta waa la joojiyay waxaana isku darka falcelinta kala saaray SDS-PAGE.Borootiinka waxaa lagu sumeeyay Coomassie Brilliant Blue (A, sare).Horudhaca Nsp7-11-His6 iyo badeecada la warshadeeyay ee ka dhalatay kala goynta dhexdhexaadinta ee nsp5-His6 ayaa lagu muujiyay dhanka midig.Fadlan ogow (xajmiga yar ee ay leeyihiin) in nsp7 iyo nsp11-His6 aan lagu ogaan karin jeelkan, falcelintana waxaa lagu kabay nsp5-His6 (wadooyinka 1 iyo 4; booska nsp5-His6 waxaa lagu muujiyay goobada adag) ama nsp9-His6 (Lane 2) waxay ka kooban tahay qadar yar oo MBP ah (oo lagu tilmaamay wareegyada furan) sida wasakh haraaga ah sababtoo ah waxaa lagu muujiyay borotiinnada isku-dhafka MBP (SI lifaaqa, Shaxda S1).(B) Kala duwanaanshiyaha Nsp9-His6 waxa ka maqan hal ama laba N-terminal Asn hadhaaga ah (lambarada haraaga iyadoo loo eegayo booska pp1a/pp1ab) waxaana la safeeyey oo lagu dhex daray nsp12-His6 iyo [α-32P] UTP.B, SDS-PAGE oo leh Coomassie ayaa lagu muujiyay xagga sare, B, autoradiograph-ka u dhigma ayaa lagu muujiyay xagga hoose.Booska calaamadeeyaha miisaanka molecular (ee kilodaltons) ayaa lagu muujiyay dhanka bidix.(C) HCoV-229E nsp9-His6 N-terminal hadhaaga la kaydiyay ayaa lagu bedelay Ala ama Ser, isla qadarka borotiinka ayaa loo adeegsaday falcelinta UMPylation ee dhexdhexaadinta nsp12-His6.Alaabooyinka falcelinta waxaa kala saaray SDS-PAGE waxaana lagu midabeeyay Coomassie Brilliant Blue (C, top), iyo radio sumadeysan nsp9-His6 waxaa lagu ogaaday sawirka phosphorescence (C, dhexe).Isticmaalka borotiinka nooca duurjoogta ah (wt) ee tixraac ahaan (loo dhigay 100%), dhaqdhaqaaqa NMPylation qaraabo ah (macnaha ± SEM) ayaa laga soo xisaabiyay saddex tijaabo oo madax-bannaan.(D) Titers Virus-ka ku jira dhaqanka unugga p1 ee ka sarreeya unugyada Huh-7 ee uu ku dhacay unugyada HCoV-229E-nooca duurjoogta ah ee Huh-7, iyo mutants sidda beddelka amino acid ee loo qoondeeyey ee nsp9 ayaa lagu go'aamiyey huurada.RdRp motif-ka laba-jibbaaran ee ku-noqoshada-dhiman ee DD4823/4AA ayaa loo adeegsaday xakamayn taban.
N-terminus-ka nsp9 (gaar ahaan jagooyinka 1, 2, 3, iyo 6) ayaa aad loogu xafiday xubnaha qoyska hoose ee Orthocoronavirinae (SI lifaaqa, Jaantuska S6).Si loo barto doorka suurtogalka ah ee hadhaagan ee nsp12-dhexdhexaadin nsp9 NMPylation, laba haraaga Asn oo isku xigta ee N-terminus ee nsp9 ayaa lagu beddelay Ala ama Ser (kali ama isku dhafan).Marka la barbar dhigo nooca duurjoogta ah ee nsp9, ku beddelashada N3825 ee Ala ama Ser waxay keentay in ka badan laba laab hoos u dhac ku yimaada nsp12-dhexdhexaadinta UMPylation (Jaantus 5C).Iyada oo la raacayo gabagabadayada in NMPylation ay ka dhacdo amine aasaasiga ah ee N-terminal halkii ay ka ahaan lahayd silsiladda dhinaca ee hadhaaga N-terminal, waxaan aragnay hadhaaga NMPylation oo muhiim ah oo lagu beddelayo N3825A iyo N3825S.Waxa xiiso leh, haddii Asn-ka labaad lagu beddelo Ala ama Ser, nsp9 UMPylation si xoog leh ayaa loo dhimay (in ka badan 10 jeer), halka beddelka Ala ee boosaska 3, 4, iyo 6 uu leeyahay saameyn dhexdhexaad ah nsp9 UMPylation (Jaantus 2). ) .5C).Natiijooyin la mid ah ayaa la helay iyadoo la isticmaalayo ATP, CTP ama GTP (SI lifaaqa, Jaantuska S8).Si wada jir ah, xogtan ayaa tilmaamaysa doorka muhiimka ah ee N2826 (booska 2 ee nsp9) ee nsp9 NMPylation.
Si loo helo caddaymo dheeraad ah oo ku saabsan xidhiidhka shaqada ee u dhexeeya N-terminus ee nsp9 iyo NMPylation, waxaanu samaynay isku toosin taxane badan (MSA) ee taxanaha nsp9 ee qoyska Coronavirus (oo u dhexeeya 104 iyo 113 hadhaaga) (SI Lifaaqa, Jaantuska S6).Isku soo wada duuboo, 47 nooc oo ah (la yaqaan iyo putative) oo ah 5 genera oo ka mid ah qoyska Orthocoronavirinae ee saameeya naasleyda, shimbiraha, iyo xamaaratada kala duwan, kaliya 8 hadhaaga wadarta guud ayaa la ogaaday inay kala duwan yihiin.Isbeddellada ugu ballaaran, oo ay ku jiraan tirtiridda iyo gelinta, ayaa lagu arkay wareegyada u dhexeeya qaybaha qaab-dhismeedka sare ee nsp9, sida lagu go'aamiyay daraasadihii hore ee dhismaha (26 ??28).Shan hadhaaga ah oo aan is beddelin ayaa laga helay β strand iyo α helix ee qaybta C-terminal ee nsp9.Saddex hadhaaga ah oo aan is beddelin ayaa ka kooban NNE motif ee N terminus ee nsp9.Waxaa shaaca laga qaaday in Asn-ka labaad ee motif-kani uu yahay hadhaaga kaliya ee aan isbeddelayn, kaas oo sidoo kale la wadaago mala-awaalka nsp9 ee coronavirus-ka raha fog ee la xidhiidha, oo u taagan noocyada Microhyla letovirus 1 ee qoyska hoose ee Letovirinae ee Alphaletovirus.Ilaalinta haraaga ee nsp9 qaabdhismeedka sare waxaa lagu qiyaasi karaa tixgalin qaabdhismeed si loo ilaaliyo laalaabida ama sifooyinka RNA ee la yaqaan.Si kastaba ha ahaatee, sababtani uma muuqato inay khusayso ilaalinta NNE, ka horna daraasaddan, dabeecadda caqabadaha xaddidaya kala duwanaanshaha taxanaha tripeptide ayaa si buuxda loo daboolay.
Si loo go'aamiyo muhiimadda nsp9-NMPylation iyo ilaalinta NNE ee ku-noqoshada coronavirus, waxaan soo saarnay mutants HCoV-229E, kuwaas oo sita hal ama laba beddel oo ah haraaga nsp9 N-terminal, taas oo muujinaysa in nsp9 NMPylation ay waxyeello u leedahay gudaha vitro.Kahor intaanan bilaabin, waxaan isku dayeynaa inaan ka jawaabno su'aasha haddii beddeladan (oo u dhow nsp8|9 goobta la gooyo) ay saameeyaan habka borotiinka ee gobolka C-terminal pp1a.Qayb ka mid ah dhismooyinka polyprotein-ka ee nsp7-11 oo ay ku jiraan beddelaadyo u dhigma N-terminus ee nsp9 ayaa lagu soo saaray E. coli waxaana lagu gooyay dib-u-habaynta Mpro.Burburinta borotiinka ee afarta goobood (oo ay ku jirto goobta garabka nsp9) si weyn uma saameynayso beddelaad kasta oo la soo bandhigay (SI lifaaqa, Jaantuska S9), marka laga reebo isbeddellada qaabdhismeedka borotiinnadan ee farageliya Mpro-dhexdhexaadin nsp8|9 dillaac (ama mid kale) website.
Unugyada Huh-7 waxaa lagu wareejiyay dhererka genome-dhererka HCoV-229E RNA, iyagoo ku dhejinaya beddelka Ala ama Ser ee NNE tripeptides ee la ilaaliyo (N3825, N3826, iyo E3827) ee nsp9 N terminus, taasoo muujineysa in badi isbeddellada ay yihiin kuwo dilaa ah.Waxaan awoodnay inaan samatabbixinno fayraska annaga oo beddelnay Ser ama Ala ee N-terminal Asn (N2835A ama N2835S), laakiin waxaan ku guul darreysannay inaan ka soo kabsano fayraska iyada oo la adeegsanayo hal iyo laba-is-beddello kale oo isku xigxiga NNE (N3826A, N3826S, NN3825/6AA, NN3825/6SS) , E3827A) (Jaantuska 5D).
Natiijooyinkani waxay muujinayaan in ku-noqoshada coronaviruses ee dhaqanka unuggu ay xaddidan yihiin (isku mid ama la mid ah), xaddidaya isbeddelka dabiiciga ah ee goobaha nsp9 NMPylation ee jirka, iyo taageeridda doorka muhiimka ah ee jawaabtan ee wareegga nolosha ee coronaviruses.
Tijaabooyinkii ugu dambeeyay, waxaanu soo saarnay C-terminal His6 oo ku suntan SARS-CoV-2 nsp12 iyo nsp9, iyo laba nooc oo mutant ah oo nsp12 ah gudaha E. coli.Hadhaaga goobta firfircoon ee NiRAN iyo RdRp waa siday u kala horreeyaan Isticmaalka Ala beddelka (Jaantuska 6A iyo lifaaqa SI, Shaxda S2).K4465 gudaha SARS-CoV-2 nsp12 waxay u dhigantaa K4135 ee HCoV-229E (SI Lifaaqa, Jaantuska S2), kaas oo cadeeyay in looga baahan yahay waxqabadka NiRAN iyo ku celcelinta HCoV-229E (Jaantuska 3D iyo E).Hadhaagani waxa kale oo uu la mid yahay fayraska halbowlaha ah ee EAV nsp9 K94 hadhaaga, kaas oo hore loo muujiyay in uu lama huraan u yahay NiRAN is-UMPylation/self-GMPylation (16).Sida ku cad Jaantuska 6B, SARS-CoV-2 nsp12 waxay leedahay UMP waxqabad wareejin ah iyadoo la adeegsanayo nsp9 substrate ahaan, halka nsp12_K4465A goobta firfircooni aanu firfircoonayn.Beddelka labanlaabka ah ee isku xigxiga sifada SDD ee RdRp motif C ma saamayso dhaqdhaqaaqa wareejinta UMP (Jaantuska 6B), taasoo muujinaysa in hawsha RdRp aanay saamayn toos ah ku lahayn nsp9 UMPylation.Xog la mid ah ayaa la helay iyadoo la adeegsanayo CTP, GTP iyo ATP ( lifaaqa SI, Jaantuska S10).Isku soo wada duuboo, xogtan ayaa tilmaamaysa in NSP9 NMPylation dhexdhexaadisay NiRAN ay leedahay waxqabad muxaafid ah oo ku jira coronaviruses oo matalaya hiddaha kala duwan ee qoyska hoose ee orthocoronavirus.
SARS-CoV-2 nsp12-dhexdhexaadin NMPylation ee nsp9.(A) Coomassie midabaysan SDS-polyacrylamide jel oo muujinaya borotiinka dib-u-habaynta loo isticmaalo tijaabada NMPylation.Xakameyn ahaan, borotiin mutant ah oo leh bedel firfircoon oo goobta NiRAN (K4465A) iyo RdRp domain (DD5152/3AA) ee SARS-CoV-2 nsp12 ayaa la isticmaalay.Nambarada soo hara waxay ku salaysan tahay booska pp1ab.(B) Autoradiograph ee ogaanshaha UMPylation iyadoo la adeegsanayo nsp9-His6 iyo [α-32P] UTP sida substrate nsp12-His6 (nooca duurjoogta [wt] iyo mutant).Cufka molecular (ee kilodaltons) ee borotiinka calaamadeysan ayaa lagu muujiyay dhanka bidix.
Goobaha NiRAN waxaa guud ahaan lagu keydiyaa Nidovirales (16), taasoo muujineysa inay kiciyaan falcelinta enzymatic ee lagama maarmaanka u ah ku-noqoshada Nidovirus.Daraasaddan, waxaan awoodnay inaan cadeyno in domainka NiRAN ee coronavirus uu u wareejiyo NMP (oo laga soo saaray NTP) ilaa nsp9, borotiinka qarsoon ee RNA ku xira borotiinka ku lug leh taranka fayraska (26 ?? 29), si loo go'aamiyo inuu yahay bartilmaameed dabiici ah iyo lammaanaha coronavirus RTC.
Domain-ka NiRAN waxa uu wadaagaa saddex arrimood oo isku xiga (AN, BN, iyo CN), kuwaas oo ka kooban tiro aad u yar oo hadhaaga ah kuwaas oo lagu kaydiyay dhammaan qoysaska ku jira monophyletic-ka laakiin aad u kala duwan nidaamka Nidovirales (8, 16).Daraasadihii ugu dambeeyay waxay muujiyeen in ay qaab dhismeed ahaan la xiriiraan qoys aan si weyn loo aqoonsan oo borotiinno kinase-u eg, kuwaas oo markii hore loo yaqaan qoyska SelO (17, 19, 22, 30, 31).Borotiinnada laxiriira SelO waxay leeyihiin laalaabka kinase, laakiin waxaa ka maqan dhowr hadhaaga goobta firfircoon ee kinases-ka caadiga ah (22, 32).Iyada oo ku saleysan jihada gadaasha ee molecules ATP ee ku xidhan goobta firfircoon oo ay dejisay is dhexgalka gaarka ah, SelO waa la mala awaalay oo markii dambe la xaqiijiyay in lagu wareejiyo AMP (halkii fosfate) substrate-ka borotiinka (22), halka borotiin kale oo SelO u eg YdiU uu leeyahay dhawaanahan ayaa la tusay in uu kiciyo isku xidhka UMP ee Tyr iyo hadhaagiisa ee substrates borotiinka kala duwan (33).
Si loo xaqiijiyo loona balaadhiyo saadaasha hadhaaga goobta firfircoon ee fayraska NiRAN, waxaanu isticmaalnay biochemical iyo hababka hidde-sideyaasha si aan u samayno falanqaynta isbeddelka ee coronavirus nsp12 (Jaantuska 3D iyo E iyo SI lifaaqa, Jaantuska S3 iyo miiska) S1â S4).Xogtu waxay muujineysaa in beddelka HCoV-229E K4135, R4178 iyo D4280 ee Ala ay meesha ka saarayso waxqabadka wareejinta NMP ee vitro iyo fayraska ku celcelinta dhaqanka unugyada (Jaantuska 3D iyo E iyo lifaaqyada SI, Jaantus S3), iyagoo taageeraya joogitaankooda NTP γ-phosphate (K4135, R4178) iyo isuduwidda ion birta goobta firfircoon (D4280).Beddelka E4145A ee Glu-ga la ilaaliyo ee kala duwan ee fayraska buulka shimbiraha ee la saadaaliyay inuu dejiyo booska K4135 (17) ayaa la muujiyay si loo baabi'iyo taranka fayraska, laakiin waxaa la yaab leh, hawsha waxaa lagu hayaa in vitro NMPylation assay (Jaantuska 3D iyo E iyo Lifaaqa SI, Jaantuska S3 Iyo Shaxannada S1-S4).U fiirsi la mid ah ayaa la sameeyay markii beddelka u dhigma lagu soo bandhigay YdiU homolog of Salmonella typhimurium (E130A) (33).Isku soo wada duuboo, xogtani waxay taageertaa shaqada sharciyeynta ee hadhaaga la xafiday halkii ay ka ahaan lahayd shaqada catalytic.
Beddelka hadhaaga Phe ee la xafiday (F4281A) gudaha inta u dhexeysa nestovirus ee HCoV-229E NiRAN domain (8) waxay keentay hoos u dhac ku yimid dhaqdhaqaaqa NMPylation ee vitro iyo hoos u dhac weyn oo ku yimid ku-noqoshada fayraska ee dhaqanka unugyada (Jaantuska 3D, E iyo SI) lifaaqa, Sawirka S3).Xogtu waxay la socotaa shaqada muhiimka ah ee sharciyeynta hadhaagan, sida hadhaaga DFG ee isku midka ah ee Phe hadhaaga ee hore loo muujiyey.Kinases borotiinka qadiimiga ah, waa qayb ka mid ah wareegga isku xidhka Mg2+ waxayna caawisaa in la ururiyo oo habeeyo lafdhabarta???Looga baahan yahay waxqabad firfircooni leh (32, 34).Beddelka Ala iyo Arg ee hadhaaga K4116 (ee motif preAN), siday u kala horreeyaan, tirtiray ku-noqoshada fayraska iyo, sidii la filayay, waxay saameyn kala duwan ku yeelatay dhaqdhaqaaqa wareejinta NMP ee vitro, iyadoo ku xiran silsiladda dhinaca amino acid ee la soo bandhigay (Jaantuska 3D iyo E iyo lifaaqyada SI , Sawirka S3).Xogta shaqaynaysa waxay la socotaa macluumaadka qaabdhismeedka, taas oo muujinaysa in hadhaagani ay aasaaseen isdhexgalka ATP phosphate (17).Qaybta NiRAN ee qoysaska kale ee fayraska buul ku jira, booska HCoV-229E pp1a/pp1ab K4116 waxaa ku jira Lys, Arg ama His (8), taas oo muujinaysa in xaddidaadda shaqeynta ee hadhaaga gaarka ah la dejiyay.Beddelka D4188A iyo D4283A waxay meesha ka saaraysaa ama si adag u yaraynaysaa firfircoonida enzyme waxayna meesha ka saaraysaa ku celcelinta fayraska (Jaantuska 3).Labadan hadhaaga ah waxa lagu kaydiyaa badi (laakin ma wada aha) fayrasyada buulka leh (8), taas oo muujinaysa hawl qoys-gaar ah oo muhiim ah laakiin suurtogal ah in aan katalytik ahayn.Beddelka Ala ee haraaga kale ee Lys iyo Asp (K4113A, D4180A, D4197A iyo D4273A) kuwaas oo aan lagu kaydin Coronaviridae ama qoysaska kale ee Nestioviridae (8) ayaa loo adeegsaday kontarool ahaan.Sida la filayo, beddelladan ayaa inta badan loo dulqaadan karaa, iyada oo wax yar hoos u dhac ku yimi dhaqdhaqaaqa enzyme iyo taranka fayraska xaaladaha qaarkood (Jaantuska 3 iyo lifaaqa SI, Jaantuska S3).Isku soo wada duuboo, xogta mutagenesis-ka coronavirus-ku waxay aad ula socotaa is-GMP iyo xogta hidaha ee EAV NiRAN-RdRp (16), kaas oo EAV nsp9 (coronavirus nsp12 ortholog) haraaga K94 (oo u dhiganta HCoV-229E K4135) hawlaha muhiimka ah). R124 (ku habboon R4178), D132 (ku habboon D4188), D165 (ku habboon D4280), F166 (ku habboon F4281).Intaa waxaa dheer, xogta HCoV-229E mutagenesis waxay la socotaa oo laga ballaariyay xogtii hore ee la soo sheegay ee SARS-CoV ee xogta hidda-socodka (16), si la mid ah kuwa lagu arkay CN motif Phe-to-Ala mutant SARS-CoV_nsp12 The phenotype lagu sharaxay -F219A iyo HCoV-229E_F4281A (Jaantuska 3 D iyo E iyo SI lifaaqa, Jaantuska S3 iyo Shaxda S1-S4).
Marka la barbar dhigo EAV orthologs (16), kuwaas oo leh door cad UTP iyo GTP (ee falcelinta is-NMPylation), daraasaddeennu waxay muujinaysaa in fayraska NiRAN domain (oo ay matalaan HCoV-229E iyo SARS-CoV-2) ay si wax ku ool ah u noqon karaan la wareejiyay Dhammaan afarta NMPs, in kasta oo ay jirto door-bid yar oo loogu talagalay UMP (Jaantus 1 iyo 3).Qeexitaanka aadka u hooseeya ee isbahaysiga gaarka ah ee NTP wuxuu la jaan qaadayaa SARS-CoV-2 nsp7/8/12/13 qaab dhismeedka supercomposite ee dhowaan la soo sheegay, kaas oo ADP-Mg2+ ku xidho goobta firfircoon ee NiRAN, laakiin aan la socon qaybta adenine. ee samaynta is-dhexgalka gaarka ah (17).Daraasaddeena, nooca nucleotide ee loo isticmaalo falcelinta NMPylation ma laha saameyn kala duwan oo ku saabsan dhaqdhaqaaqa borotiinka mutant (SI Lifaaqa, Jaantuska S3), taas oo muujinaysa in mid ka mid ah haraagani aanu si dhow ula xiriirin xiritaanka nukleobase gaar ah.Saldhigga qaab-dhismeedka iyo muhiimadda bayooloji ee suurtagalka ah ee kala duwan ee doorbidida is-bahaysiga NTP ee lagu arkay qaybaha NiRAN ee coronaviruses iyo fayraska halbowlaha ah ayaa weli ah in la darso;Waxa laga yaabaa inay run yihiin ama ay sabab u tahay xaddidnaanta waxbarashadooda.Waqtigan xaadirka ah, lama saari karo in waxqabadka NMPylator ee suurtagalka ah ee fayraska halbowlaha ah ee NiRAN (marka la barbar dhigo waxqabadkii hore ee is-NMPylation) uu leeyahay door-hoosaadyo kala duwan, iyada oo la tixgelinayo in isku midka ah ee halbowlaha iyo coronavirus-ka. domainka NiRAN waa xadkiisaIsbarbardhigga taxanaha ku salaysan (16).Marka la barbar dhigo pseudokinase SelO, oo u adeegsata Mg2+ isku xidhe ahaan, hawsha coronavirus iyo fayraska halbowlaha NiRAN waxay ku xidhan tahay Mn2+ (16) (Jaantuska 3C iyo lifaaqa SI, Jaantuska S1).Ku-tiirsanaanta Mn2+ iyo doorbidida cad ee UTP waa muuqaal aan caadi ahayn oo ka mid ah borotiinka NMPylators, waxaana dhawaan lagu xaqiijiyay borotiinka YdiU ee Salmonella typhimurium, kaas oo kicinaya borotiinka adag ee Mn2+-ku-tiirsanaanta borotiinka Chaperone UMPylation si unugyada looga ilaaliyo walaaca kicinta barkada ATP. 33).
Isku ekaanshaha qaabdhismeed ee dhawaan la sharraxay ee u dhexeeya domainka coronavirus NiRAN iyo kinases borotiinka gacanta (17, 19) waxay siisaa taageero dheeraad ah awoodda NiRAN ee ay si wadajir ah ugu xidhidhiso NMP borotiinnada kale ee aan ku soo sheegnay daraasaddan.Waxaan diirada saarnay raadinta bartilmaameedyada suurtagalka ah ee NiRAN ee borotiinnada lagu calaamadeeyay HCoV-229E ORF1a, kuwaas oo la og yahay inay si toos ah ama si dadbanba u caawiyaan RTC's ORF1b-ku celcelinta (12, 35).Tijaabooyinkeenu waxay bixiyaan caddayn dhammaystiran oo ku saabsan waxtarka iyo gaarka ah ee NMPylation ee nsp9 (Jaantuska 2).Haddii borotiinka la beegsanayo loo isticmaalo mooska xad-dhaafka ah oo 8 ilaa 10 jeer ka sarreeya kan enzyme-ka (nsp12), waxa la xaqiijiyay in nsp9 uu yahay mid dhammaystiran (mono) NMPized (Jaantuska 4).Waxaan ku soo gabagabeynay in isdhexgalka u dhexeeya nsp12 iyo nsp9 uu yahay mid gaaban oo aan sameyn doonin dhismo xasilloon oo leh nsp9 (maqnaanshaha qaybo kale oo RTC ah).Gabagabadaan waxaa taageeray daraasadaha isdhexgalka borotiinka ee borotiinka SARS-CoV (35).Falanqaynta MS waxay aqoonsatay amine aasaasiga ah ee hadhaaga N-terminal ee nsp9 inay tahay goobta NMPylation (SI lifaaqa, Jaantuska S5).Samaynta dammaanadda fosfooraska iyo kooxda amino N-terminal waxay kala soocaysaa waxqabadka NMPylation ee dhexdhexaadinta NiRAN-ku-dhex-dhexaadka ah ee Pseudomonas syringae SelO-dhexdhexaadinta AMPylation falcelinta, kaas oo kicinaya samaynta O-linked AMP ee Ser, Thr, ama Tyr hadhaaga Peptide. 22), iyo S. typhimurium YdiU foomamka O-linked (la leh Tyr) iyo N-linked (oo leh) peptide-UMP ducts.Macluumaadka xaddidan ee laga heli karo qoyska SelO ee borotiinnada ayaa tilmaamaya in xubnaha qoyskan borotiinka weyn ay aad ugu kala duwan yihiin samaynta peptide-NMP adducts.Tani waa indho-indhayn xiiso leh oo mudan daraasad dheeraad ah.
Xogta laga helay daraasaddan ayaa noo horseeday in aan qiyaasno in NMPylation ee nsp9 ay u baahan tahay N-terminus bilaash ah.Marka la eego ku-noqoshada fayraska, tan waxaa bixin doona kala-goynta borotiinka ee goobta ka-habaynta nsp8|nsp9 ee ku-noqoshada polyprotein pp1a ee ay dhexdhexaadiyaan Mpro iyo pp1ab.Inta badan coronaviruses, farqiga u dhexeeya goobtan gaarka ah (VKLQ|NNEI ee HCoV-229E) iyo dhammaan goobaha kale ee coronavirus MPro waa Asn (halkii hadhaa kale oo yar, sida Ala, Ser Ama Gly) ay ku jiraan P1â???Goobta (36).Xogta jeexjeexa peptide ee lagu helay daraasadihii hore waxay muujisay in hufnaanta nadiifinta nsp8|nsp9 ay ka hooseyso kuwa goobaha kale, taas oo muujinaysa in 1) goobtan gaarka ah ay yeelan karto door nidaamsan habaynta wakhtiga isku dubaridka ah ee C-terminal pp1a, ama 2) a Doorka nsp9 N-terminus ee gaarka ah ee la xafiday ee fayraska (37).Xogtayada (Jaantuska 5A) waxay tustay in qaabka dib-u-habaynta ee nsp9 uu sido isku xigxiga dhabta ah ee N-terminal uu si wax ku ool ah NMPized by nsp12.Isku xigxiga N-terminal flanking waxaa meesha ka saaray factor Xa (nsp9-His6; lifaaqa SI, Shaxda S1) ama Mpro-dhexdhexaadin ah (nsp7-11-His6; Jaantuska 5A iyo SI lifaaqa, Shaxda S1).Muhiimad ahaan, horudhaca nsp9-ka kooban ee nsp7-11-His6 wuxuu muujiyay caabbinta NMPylation ee nsp12, taas oo la socota xogtayada, taas oo muujinaysa in nsp9-NMP duct lagu sameeyay iyada oo loo marayo N-terminal amine aasaasiga ah (SI lifaaqa, Jaantuska S5) .Si aan u helno faham qoto dheer oo ku saabsan gaar ahaan substrate NiRAN, waxaan markaas diirada saarnay hadhaaga N-terminal ee nsp9.Maqnaanshaha borotiinno kale, waxay yihiin kuwo qaab-dhismeed ahaan dabacsan, iyaga oo ka hortagaya in lagu ogaado qaabka aan calaamadeysan ee nsp9 (26 28, 38), oo muujinaya kala duwanaanshahooda dabiiciga ah ee xaddidan Tani waxay sabab u tahay taxanaha muhiimka ah ee gaarka ah (aan la xiriirin qaab-dhismeedka sare) shaqada nsp9 N-terminal jajab.Beddelka Ala ee hadhaaga kaydsan ee gobolkan (Jaantus 5C iyo D iyo lifaaqa SI, Jaantuska S8) ayaa muujinaya in N3826 ay lama huraan u tahay nsp9 NMPylation in vitro, halka beddelka N3825A iyo E3827A ay horseedayso hoos u dhaca NMPylation, halka beddelka M3829A iyo P3830A .Si cad u saameeya nsp9 NMPylation.In kasta oo beddelka N-terminal Asn (N3825A, N3825S) uu saameyn dhexdhexaad ah ku leeyahay nsp9 NMPylation iyo ku-noqoshada fayraska ee dhaqanka unugga (Jaantuska 5C iyo D), tirtirka taxanaha haraaga Asn ee N-terminal 3825-NN dipeptide Waxa la caddeeyey in ay halis u tahay fayrasyada, taas oo muujinaysa in hal haraaga Asn loo baahan yahay ka hor inta hadhaaga kale ee N-terminus, la doorbidayo Asn, inkastoo ay u muuqato in beddelka hadhaaga la midka ah qayb ahaan loo dulqaadan karo (Jaantuska 5B, C, iyo D).Waxaan ku soo gabagabeyneynaa in 3825-NN dipeptide, gaar ahaan haraaga N3826 ee la xafiday iyo lagama maarmaanka ah ee ku dhex jira tirada coronavirus ( lifaaqa SI, Jaantuska S6), ay hubiso xiritaanka saxda ah iyo jihaynta nsp9 N-terminus ee goobta firfircoon ee NiRAN.
Beddelka Ala (E3827A) ee Glu-ga la ilaaliyo ee dhammaan qoysaska hoose waxay sii haysaa nsp9 NMPylation in vitro laakiin waxay halis u tahay fayrasyada ku jira dhaqanka unugga (Jaantuska 5C iyo D), taasoo muujinaysa shaqada dheeraadka ah ee hadhaaga, tusaale ahaan, isdhexgalka muhiimka ah (NMPylated ama aan la beddelin ) nsp9 N-terminus iyo arrimo kale oo ku lug leh taranka fayraska.Isbeddellada Nsp9 ma aysan saameynin habka proteolytic ee nsp9 ama nsps kasta oo ku xiga (39) (SI Lifaaqa, Jaantuska S9), taas oo muujinaysa in dabeecadaha dilaaga ah ee dhowr isbeddellada nsp9 ee la arkay aysan keenin nidaam-darrada C-proteolytic process-terminal pp1a .
Xogta sare waxay bixinaysaa caddaynta in ka dib daawaynta Mpro-dhexdhexaadineed ee nsp8|9 ee goobta kala goynta ee pp1a/pp1ab, N-terminus ee nsp9 la UMPylated (ama qayb ahaan wax laga beddelay NMP kale).Intaa waxaa dheer, ilaalinta ugu wanaagsan ee N-terminus ee nsp9 (oo ay ku jiraan haraaga Asn ee kali ah iyo kala-duwanaanshaha qoyska coronavirus) iyo xogta hidda-socodka ee laga helay daraasaddan (Jaantus 3E iyo 5D) ayaa noo horseeday inaan soo gabagabeyno in nsp9 NMPylation ee lagu sharraxay. waxay la xidhiidha bayooloji waxayna lama huraan u tahay ku-noqoshada coronavirus.Cawaaqibta shaqeynta ee wax ka beddelkan ayaa weli ah in la darso, tusaale ahaan, ee ku saabsan horay loo sifeeyay (aan gaar ahayn) nsp9 (qaab aan la beddelin) RNA waxqabadka xidhitaanka (2628).N-terminal NMPylation waxa kale oo laga yaabaa inay saamayso isdhexgalka nsp9 ee borotiinka ama RNA substrates ama samaynta goleyaal afar heer oo kala duwan.Kuwaas waxaa lagu arkay daraasaadka qaab dhismeedka waxaana la xaqiijiyay inay si firfircoon ula xiriiraan soo-celinta coronavirus, in kasta oo gaar ahaan maqnaanshaha kiiska wax ka beddelka (26-ââ29, 40).
In kasta oo bartilmaameedka bartilmaameedka coronavirus-ka NiRAN uu wali u baahan yahay in lagu sifeeyo si faahfaahsan, xogtayadu waxay muujinaysaa in bartilmaameedka borotiinka gaarka ah ee goobta coronavirus NiRAN uu aad u cidhiidhi yahay.In kasta oo ilaalinta hadhaaga goobta muhiimka ah ee firfircoon (8, 16) ee qaybta NiRAN ee dhammaan qoysaska nidovirus ay si xooggan u taageerto waxqabadka NMPylator-ka la ilaaliyo borotiinadan, aqoonsiga substrate-ka ku xidhan hadhaaga jeebka ee domainkan Ilaalinta iyo ilaalinta ayaa ah mid lagu sifoobo , waxayna ku kala duwanaan kartaa qoysaska kala duwan ee ujeedooyinka Nidovirales.Sidoo kale, bartilmaameedyada khuseeya fayrasyada kale ee buulkooda leh weli lama go'aamin.Waxaa laga yaabaa inay noqdaan kuwo fogaan ah oo nsp9 ah ama borotiinno kale, sababtoo ah taxanaha ka baxsan shanta qaybood ee dib-u-celinta ee guud ahaan lagu xafido fayrasyada buulkoodu yar yahay (8), oo ay ku jiraan genome array u dhexeeya Mpro iyo NiRAN, iyaga ka mid ah, nsp9 waxay ku taallaa Fayruuska corona.
Intaa waxaa dheer, hadda kama saari karno suurtagalnimada in domainka NiRAN uu leeyahay bartilmaameedyo dheeraad ah (oo ay ku jiraan gacanta).Xaaladdan oo kale, waxaa mudan in la sheego in homologues-ka bakteeriyada ee borotiinkan soo baxaya ee NMPylators (NMPylators) (30, 31) ay u muuqdaan inay leeyihiin "maamulayaal sare"?NMP waxay habaysaa noocyo kala duwan oo borotiinno gacanta ah si ay u habeeyaan ama u baabi'iyaan hawlahooda hoose, si ay door uga ciyaaraan habab kala duwan oo bayooloji ah, sida jawaab celinta cadaadiska gacanta iyo redox homeostasis (22, 33).
Daraasaddan (Jaantus 2 iyo 4 iyo Lifaaqa SI, Jaantusyada S3 iyo S5), waxaan awoodnay inaan cadeyno in nsp12 ay u wareejisay qaybta UMP (NMP) hal boos (la xafiday) ee nsp9, halka borotiinnada kale aan wax laga beddelin loo isticmaalo shuruudaha, si wanaagsan loo qeexay (halkii dabacsanaan lahaa) gaar ahaan substrate-ka ayaa la taageerayaa.Iyadoo la raacayo tan, marka la barbar dhigo N-terminal nsp9 NMPylation, nsp12's dhaqdhaqaaqa NMPylation u gaar ah waa mid aad u hooseeya, ogaanshaha waxay u baahan tahay waqti dheer oo soo-gaadhista autoradiography, iyo koror 10-laab ah ee nsp12 ayaa la isticmaalaa.Intaa waxaa dheer, falanqayntayada MS waxay ku guuldareysatay inay bixiso caddaynta NMPylation ee nsp12, taas oo soo jeedinaysa in NiRAN domain is-NMPylation uu yahay (ugu fiican) hawl labaad.Si kastaba ha ahaatee, waa in la ogaadaa in daraasado kale ay bixiyeen caddayn horudhac ah oo ah in heerka is-AMPylation ee bakteeriyada NMPylator ay xakameyn karto hawlahooda NMPylation ee substrate-ka kale ee borotiinka (22, 33).Sidaa darteed, cilmi baaris dheeraad ah ayaa loo baahan yahay si loo baaro saameynta shaqeynta suurtagalka ah ee waxqabadyada is-NMPylation ee laga soo sheegay EAV nsp9 (16) iyo coronavirus nsp12 (daraasaddan), oo ay ku jirto saameynta chaperone-ka la mid ah ee la soo jeediyay ee isku laabma C-terminal RdRp domain ( 16)).
Markii hore, dhowr fikradood oo ku saabsan hawlaha hoose ee suurtogalka ah ee nidoviral NiRAN domain ayaa la tixgeliyey, oo ay ku jiraan RNA ligase, RNA -capped guanylate transferase iyo borotiinka borotiinka (16), laakiin midkoodna kuma habboona hawlaha hoose ee la heli karo.Xogta laga helay jagooyinka soo socda waa isku waqti iyada oo aan la samayn malo-awaal dheeraad ah.Xogta laga helay daraasaddan ayaa ah mid aad u waafaqaysa (laakin ma caddayn karto) in qaybta NiRAN ay ku lug leedahay bilawga borotiinka ee RNA.Waxaa hore loo rumaysnaa in shaqada domain-ka NiRAN ee 5 ??²-RNA daboolka ama falcelinta isku xidhka RNA ma saameeyaan kuwaas iyo Taageerada xogta kale.Sidaa darteed, tusaale ahaan, goobta firfircoon ee NiRAN waxaa loo arkaa inay ku lug leedahay Asp-ga la xafiday oo ah saldhig guud (D252 gudaha Pseudomonas syringae SelO; D4271 ee HCoV-229E pp1ab; D208 gudaha SARS-CoV-2 nsp12) (SI Lifaaqa, tirada 2 ).S2) (17, 22, 33), halka kicinta ku jirta ATP-ku-tiirsanaanta RNA ligase iyo RNA capping enzyme ay fuliso enzyme covalent (lysyl-N) - Dhexdhexaadiyaha NMP, kaas oo ku lug leh hadhaaga Lys ee aan Isbeddelin ( 41).Intaa waxaa dheer, qaas ahaan isku xigxiga ku saleysan ee cajiibka ah ee coronavirus NiRAN ee bartilmaameedyada borotiinka ee la ilaaliyo iyo gaarnimada dabacsan ee NTP co-substrates (doorbidayaa UTP) waxay ka soo horjeedaa NiRAN-dhexdhexaadinta enzyme capping ama RNA ligase-like.
Sida iska cad, shaqo badan oo dheeri ah ayaa loo baahan yahay si loo xaqiijiyo oo, haddii la caddeeyo, lagu faahfaahiyo doorka suurtogalka ah ee nsp9-UMP (nsp9-NMP) ee borotiinka ku jira RNA synthesis, kaas oo isku xiri doona dhowr xiiso leh laakiin (ilaa hadda) warbixinno hore ayaa la sheegay. .Indho-indhayn gooni ah.Tusaale ahaan, waxaa la go'aamiyay in dhamaadka xudunta xun-xun ee RNA ee coronavirus uu ku bilowdo xarig oligo (U) (42, 43).U fiirsashadani waxay la socotaa fikradda ah in isku-dhafka RNA-xun-xun uu bilaabay iyada oo lagu xirayo qaabka UMPylated ee nsp9 ee dabada poly(A) (kiciyeyaasha), taas oo laga yaabo inay kor u qaaddo xirmaheeda RNA Hawlaha iyo / ama isdhexgalka borotiin kale oo RTC ah.Qaybta UMP ee ay bixiso nsp9 ayaa markaa loo isticmaali karaa sidii "primer" ee nsp7/8/nsp12-dhexdhexaadin oligouridylation, iyadoo la adeegsanayo dabada 3??²-poly(A) ee genomic RNA ama oligo kale (A) ka kooban Waxay u adeegtaa qaab qaabaysan, oo la mid ah habka loo aasaasay borotiinka VPg ee picornavirus (44).Maxaa dhacaya haddii soo jeedintu tahay "mid aan caadi ahayn"????Bilawga (protein-ku-abuuray) isku-dhafka RNA-xun-xun wuxuu bixiyaa xiriirinta indho-indheynta, taasoo muujineysa in coronavirus-ka-xun-xun RNA uu leeyahay UMP (halkii UTP) dhamaadka (42), kaas oo loo arko inuu muujinayo in Nucleic acid Dicer ayaa kala jeexa dhamaadka fosforyaalka oo ay leedahay endonuclease gaar ah oo uridine aan la garanayn.Haddii la xaqiijiyo, hawshan nucleic acid hydrolytic waxay kaa caawin kartaa sii daynta oligomeric UMPylated form of nsp9 laga bilaabo 5 ² dhamaadka xadhig taban.Doorka suurtagalka ah ee nsp9 ee soo saarista borotiinka ayaa sidoo kale la jaan qaadaya daraasadihii hore ee hidde-sideyaasha, kuwaas oo muujiyay in nsp9 (iyo nsp8) ay si adag ula falgalaan oo si gaar ah ula falgalaan walxaha RNA ee la ilaaliyo ee u dhow dhamaadka 3 ee hiddo-wadaha coronavirus.45).Sida laga soo xigtay warbixintan, indha-indheyntan hore ayaa hadda dib loo baari karaa oo la ballaarin karaa iyada oo la adeegsanayo cilmi-baaris dheeraad ah.
Marka la soo koobo, xogtayadu waxay go'aamisay hawsha gaarka ah ee summada fayraska buul leh ee lahaanshaha ee ku xiran RdRp ee N-terminus.Gudaha coronavirus, hawshan cusub ee dhexdhexaadinta NiRAN ee UMPylator/NMPylator ee dhowaan la helay waxaa loo isticmaalaa in lagu tiirsado hadhaaga Mn2+ iyo kuwa ku xiga ee Asn oo sababa samaynta curaarta fosphoramidate (tamar yar) oo leh amine aasaasiga ah ee N-terminal.Iyada oo loo marayo kala-goynta Mpro-dhexdhexaadinta ee nsp8|9 goobta jeexjeexa, bartilmaameedka nsp9 waxaa loo isticmaali karaa NMPylation, taasoo muujinaysa isku xirka shaqeynta ee u dhexeeya protease iyo domainka NiRAN, kaas oo ku fidsan RdRp.Ilaalinta haraaga muhiimka ah ee nsp12 NiRAN ee firfircoon iyo bartilmaameedka nsp9, oo ay weheliso xogta laga helay laba coronaviruses oo ay ku jiraan SARS-CoV-2, ayaa bixisa caddayn xooggan oo ah in nsp9 NMPylation uu yahay astaamaha muxaafidka ee coronavirus sidoo kale waa tallaabo muhiim ah oo ku-noqoshada fayraska.Xogta la hayo ayaa noo horseedaysa inaan ku soo gabagabeyno in doorka gaarka ah ee qaabka NMPylated ee nsp9 ee borotiinka ay keentay RNA synthesis ay tahay xaalad macquul ah oo loogu talagalay coronavirus iyo fayrasyada kale ee buulka leh, iyo NiRAN waxay sidoo kale bartilmaameedsan kartaa borotiinno kale oo aan la aqoonsan.Habbee fayraska.Is dhexgalka martida.Haddii la xaqiijiyo, ku lug lahaanshaha asaasiga borotiinka ee fayraska RNA wuxuu kordhin doonaa isku xigxiga isku xigxiga ee MPro/3CLpro iyo RdRp domains ee u dhexeeya coronavirus-ka hore ee la ogaaday iyo picornavirus-sida supergroup (9), kuwaas oo hadda lagu mideeyay Pisonivirites ee dhawaan la aasaasay. 46) qaybta.
Xogtayadu waxay sidoo kale muujinaysaa in hawlaha asaasiga ah, xulashada iyo ilaalinta enzyme ee lagu aqoonsaday daraasaddan loo isticmaali karo bartilmaameedyada daawooyinka fayraska.Isku-dhafan oo farageliya xidhitaannada (iyo beddelka dambe) ee nsp9 N-terminus ee la ilaaliyo ee goobta firfircoon ee NiRAN waxaa loo horumarin karaa dawooyin fayruska ka-hortagga ah oo wax ku ool ah, oo ku habboon daaweynta xayawaanka iyo coronaviruses-ka bini'aadamka ee ka soo jeeda cudurro kala duwan (sub) , oo ay ku jiraan SARS-CoV-2 iyo Bariga Dhexe ee neef-mareenka Coronavirus.
Habka codeeynta ee borotiinka coronavirus ee lagu soo saaray daraasaddan waxaa xoojiyay RT-PCR iyadoo la adeegsanayo RNA ka go'an Huh-7 oo qaba HCoV-229E ama Vero E6 uu ku dhacay SARS-CoV-2, oo la geliyo iyadoo la adeegsanayo hababka cloning caadiga ah.pMAL-c2 (Shaybaadhka Biological New England) ama pASK3-Ub-CHis6 (47) muujinta muujinta (SI Lifaaqa, Shaxanka S1 iyo S2).Beddelka hal codonka ah waxaa soo bandhigay PCR-ku-saleysan mutagenesis-ku hagaya goobta (48).Si loo soo saaro borotiinka isku-dhafka MBP, unugyada E. coli TB1 ayaa lagu beddelay pMAL-c2 plasmid dhiska ku habboon (SI lifaaqa, Shaxda S1).Borotiinka isku dhafka ah waxaa lagu sifeeyay amylose affinity chromatography waxaana lagu kala gooyay factor Xa.Ka dib, borotiinka C-terminal His6-tagged waxa lagu sifeeyay Ni-immobilized affinity chromatography (Ni-IMAC) sida hore loogu sharraxay (49).Si loo soo saaro borotiinka isku-dhafka ah ee ubiquitin, unugyada E. coli TB1 waxay isticmaaleen pASK3-Ub-CHis6 plasmid dhiska ku haboon (SI Lifaaqa, Shaxanka S1 iyo S2) iyo pCGI plasmid DNA encoding ubiquitin-gaar ah C-terminal hydrolase 1 (Ubp1).Isbadal (47).C-terminal His6-tagged borotiinka coronavirus waa la nadiifiyey sidii hore loogu sharraxay (50).
Tijaabada is-NMPylation ee HCoV-229E nsp12-His6 ayaa la sameeyay sida lagu qeexay EAV nsp9 (16).Marka la soo koobo, nsp12-His6 (0.5 µM) waxay ka kooban tahay 50 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) -KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 in µM buffer, NTP-ga la cayimay iyo 0.17 µM waxay ku beegmeen [α32-P] NTP (3,000 Ci/mmol; Analytic Hartmann) at 30 °C ilaa 30 daqiiqo.Dhammaan tijaabooyinka kale ee NMPylation ee nsp12-dhexdhexaadinta nsp9 NMPylation, xaaladaha falcelinta waxaa lagu hagaajiyaa sida soo socota: nsp12-His6 (0.05 µM) iyo nsp9-His6 (4 µM) iyadoo ay joogaan 50 mM HEPES-KOH (pH 8.0) ), 5 mM DTT, 1 mM MnCl2, 25 µM waxay muujisay NTP, iyo 0.17 µM oo la mid ah [α32-P] NTP.Ka dib markii la isku duwo 10 daqiiqo at 30 ° C, muunadda falcelinta waxaa lagu qasi jiray muunad SDS-PAGE: 62.5 mM tris (hydroxymethyl) aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol iyo 0.005% bromophen buluug.Barootiinka waxaa la diiday kuleyliyaha 90 °C 5 daqiiqo waxaana loo kala saaray 12% SDS-BOG.Jeelku wuu go'an yahay oo lagu dheehday Coomassie Brilliant Blue xal (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), midab la sameeyay, oo soo bandhigay shaashadda sawir-qaadista fosfooraska 20 saacadood (si loo ogaado nsp12 ee NMPylation) ama (ugu badnaan) 2 saacadood (si loo qiimeeyo nsp9 NMPylation).Sawir qaade Typhoon 9200 ah (GE Healthcare) ayaa loo isticmaalay in lagu sawiro shaashadda waxaana ImageJ loo isticmaalay in lagu falanqeeyo xoogga calaamada.
Falanqaynta MS, 1 µM nsp12-His6 iyo 10 µM nsp9 (la'aanteed hexahistidine tag) ayaa loo adeegsaday falanqaynta NMPylation (SI lifaaqa, Shaxda S1) iyo xoojinta korodhka 500 µM UTP iyo GTP ayaa la isticmaalay.Iyada oo ku xidhan u-fiirsigooda iyo tayada borotiinka la filayo, nidaamka H-Class Waters ACQUITY H-Class HPLC oo ku qalabaysan tiirka MassPrep (Biyaha) ayaa loo isticmaalay in lagu nadiifiyo 1 ilaa 10 µL ee xalalka borotiinka kaydsan ee khadka.Borotiinka la macmalay waxaa lagu sii daayay isha isha elektiroonigga ah ee Synapt G2Si mass spectrometer (Biyaha) iyada oo loo marayo jaangooyooyinka soo socda ee buffer A (biyaha / 0.05% formic acid) iyo baffer B (acetonitrile / 0.045% formic acid), iyo heerkulka tiirka waa 60 ° C iyo heerka socodka 0.1 mL/daqiiqo: si gooni gooni ah leh oo leh 5% A 2 daqiiqo, ka dibna jaangooyo toosan ilaa 95% B 8 daqiiqo gudahood, oo ilaali 95% B 4 daqiiqo oo kale.
Ionooyin togan oo kala duwan oo u dhexeeya 500 ilaa 5000 m/z ayaa la ogaadaa.Glu-fibrinopeptide B waxaa la cabbiraa 45-kii sekan si toos ah loo saxo masraxa.Adeegso software-ka qalabka MassLynx oo leh MaxEnt1 kordhinta si aad u kala saartid celceliska spectrum ka dib markaad ka jarto saldhigga iyo fududaynta.
UMPylated HCoV-229E nsp9 waa la dheefshiiday iyada oo lagu daray heerka isku xigxiga ee trypsin la beddelay (Serva) waxaana lagu beeray habeenkii 37 ° C.Tiirka lafdhabarta ee Chromabond C18WP (lambarka qaybta 730522; Macherey-Nagel) ayaa loo isticmaalay in lagu miiro oo xooga saaro peptides-yada.Ugu dambeyntii, peptide-ku wuxuu ku milmay 25 µL oo biyo ah, kaas oo ka kooban 5% acetonitrile iyo 0.1% formic acid.
Tusaalooyinka waxaa lagu falanqeeyay MS iyadoo la isticmaalayo Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Nidaamka ugu dambeeya ee nanoâ HPLC (Dionex), oo ku qalabaysan dhamaadka caadada ah ee 50 cm ??Tiirka 75 μm C18 RP oo ay ku jiraan 2.4 μm kuul magnetic (Dr. Albin Maisch High Performance LC GmbH) Ku xidh spectrometer-ka tirada badan ee khadka tooska ah ee Proxeon nanospray;ku duri 6µL oo ah xalka dheefshiidka trypsin dhexroorka gudaha 300 µm ×??1 cm C18 PepMap tiirka-fiirsashada hore (Thermo Scientific).Isticmaalka biyaha/0.05% formic acid sida dareeraha, muunada si toos ah ayaa loo xayiray oo laga nadiifiyey heerka socodka 6 µL/daqiiqo.
Qaybaha soo socda ee biyaha / 0.05% formic acid (millaha A) iyo 80% acetonitrile / 0.045% formic acid (millaha B) ayaa loo isticmaalay si loo gaaro kala soocida peptides tryptic ee heerka socodka 300 nL / min: 4% B 5 daqiiqo, ka dibna 30 A gradient toosan ilaa 45% B daqiiqado gudahood, iyo koror toosan oo gaaraya 95% dareere B 5 daqiiqo gudahood.Ku xidh tiirka chromatographic bir nano-emitter ah (Proxeon), oo si toos ah ugu buufi eluent kaboolka kulaylka leh ee spectrometer mass iyadoo la adeegsanayo awood 2,300 V. Sawirka sahanka oo leh xal 60,000 ah ee Orbitrap mass analyzer ayaa la xidhiidha oo leh ugu yaraan saddex xogta MS/MS scans, si firfircoon looga saaray 30 ilbiriqsi, iyadoo la adeegsanayo isku dhac dabin toosan oo ion toos ah ama kala qaybsanaan shil tamar sare ah oo ay weheliso ogaanshaha orbitrap, xallintu waa 7,500.
Waqtiga boostada: Agoosto-03-2021