E hlophisitsoe ke Peter Sarnow, Sekolo sa Bongaka sa Univesithi ea Stanford, Univesithi ea Stanford, California, e amohetsoe ka la 25 Tšitoe 2020 (e hlahlobiloe ka la 25 Mphalane 2020)
Re tlaleha tšebelisano lipakeng tsa li-subunits mabapi le phetisetso ea li-coronaviruses-transcription complexes, tse bohlokoa bakeng sa phetisetso le paballo ea ho iphetola ha lintho.Re fane ka bopaki ba hore sebaka sa NiRAN se amanang le nsp12 se na le ts'ebetso ea transase ea nucleoside monophosphate (NMP) ho trans, mme e khethile nsp9 (protheine e tlamang ea RNA) e le sepheo sa eona.NiRAN e matlafatsa sehokelo se kopaneng sa sehlopha sa NMP ho nsp9 amino terminus e bolokiloeng ka karabelo e itšetlehileng ka li-ion tsa Mn2+ le masala a bolokiloeng a haufi a Asn.Ho fumanoe hore ts'ebetso ea NiRAN le nsp9 NMPylation li bohlokoa bakeng sa phetisetso ea coronavirus.Lintlha li re lumella ho hokahanya ketsahalo ena ea letšoao la enzyme ea sehlaha le litebello tse fetileng khopolong ea hore ho qalisoa ha motsoako oa RNA sehlopheng sa livaerase tsa RNA ho sebetsa le ka mokhoa oa ho iphetola ha lintho.
RNA polymerase (RdRps) e itšetlehileng ka RNA ea Nidovirales (Coronaviridae, Arterioviridae, le malapa a mang a 12) e hokahane le sebaka sa amino-terminal (N-terminal) sebakeng sa protheine e seng sebopeho (nsp) e tsoang ho polyprotein, e bitsoang NiRAN. 1ab e entsoe ka viral main protease (Mpro).Nakong e fetileng, ts'ebetso ea arterial virus NiRAN-RdRp nsp's GMPylation/UMPylation e ile ea tlalehoa, 'me ho ile ha etsoa tlhahiso ea ho hlahisa nakoana bakeng sa ho fetisetsa nucleoside monophosphate (NMP) ho kokoana (eo hajoale e sa tsejoeng) le / kapa cell biopolymerization Things.Mona, re bontša hore coronavirus (Human Coronavirus [HCoV]-229E and Severe Acute Respiratory Syndrome Coronavirus 2) nsp12 (NiRAN-RdRp) e na le ts'ebetso ea NMPylation e itšetlehileng ka Mn2+, e nkiloeng ho nsp9 ka ho thehoa ha Mpro-mediated nsp9 After N-terminal flanking nsps e lokolloa ka proteolytically, phosphoramidate e kopantsoe le amine ea mantlha (N3825) ho N-terminal ea nsp9.Uridine triphosphate ke nucleotide e ratoang karabelong ena, empa adenosine triphosphate, guanosine triphosphate, le cytidine triphosphate le tsona ke li-co-substrates tse loketseng.Lithuto tsa phetoho e sebelisang li-recombinant coronavirus nsp9 le liprotheine tsa nsp12 le liphetoho tse entsoeng ka liphatsa tsa lefutso tsa HCoV-229E li fumane hore na masala a hlokahalang bakeng sa NMPylation ea NiRAN-mediated nsp9 NMPylation le ho ikatisa ha vaerase moetlong oa lisele.Lintlha li netefalitse ho bolela esale pele ka masala a sebaka se sebetsang sa NiRAN le ho tseba karolo ea bohlokoa ea masala a nsp9 N3826 ho nsp9 NMPylation le replication ea kokoana-hloko ka vitro.Masala ana ke karolo ea tatellano e bolokiloeng ea N-terminal NNE tripeptide mme e ipakile e le eona feela masala a sa fetoheng a nsp9 le li-homologs tsa eona lelapeng la coronavirus.Boithuto bona bo fana ka motheo o tiileng bakeng sa boithuto bo sebetsang ba tšebetso ea NMPylation ea livaerase tse ling tse ahileng sehlaheng mme e fana ka maikutlo a sepheo se ka bang teng bakeng sa nts'etsopele ea litlhare tse thibelang kokoana-hloko.
Kokoana ea RNA ea Nidovirales positive-stranded e tšoaetsa mefuta e sa tšoaneng ea li-vertebrates le tse se nang lesapo la mokokotlo (1, 2).Taelo eo hajoale e kenyelletsa malapa a 14 (3), ao lelapa la Coronavirus le ithutoang haholo lilemong tse 20 tse fetileng.Ka nako eo, li-zoonotic coronaviruses tse tharo li ile tsa hlaha ho liphoofolo tse ngata 'me tsa baka ho phatloha ho hoholo ha tšoaetso e matla ea phefumoloho ho batho.Ho kenyeletsoa mafu a seoa a sa khaotseng a bakoang ke mafu a tšoaetsanoang a matla a matla.Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (4âââ7).Li-Nidoviruses li arolelana mokhatlo o tloaelehileng oa liphatsa tsa lefutso, 'me karoloana ea membrane-bound replication-transcription complex (RTC) e kentsoe ka har'a 5-?²-terminal two-thirds le karolo e ka sehloohong ea sebopeho sa karoloana ea vaerase, hammoho le lisebelisoa tse ling. .Protheine, e kentsoeng karolong ea 3??² qetellong ea genome (1).Ntle le lelapa le le leng la livaerase tsa planarian (Monoviridae) (8), livaerase tsohle tse sehiloeng li kenyelletsa li-subunits tsa RTC ka liforeimi tse peli tse kholo tse bulehileng tsa ho bala (ORF) ORF1a le ORF1b, tse fetoletsoeng ho tsoa ho genomic RNA ea.ORF1a e kopanya polyprotein (pp) 1a, le ORF1a le ORF1b e kopanya pp1ab.Ka karolo e akaretsang ea protease e kholo (Mpro) e kenyellelitsoeng ke ORF1a, pp1a le pp1ab ka bobeli li sebetsoa ka mokhoa oa proteolytically ho ba liprotheine tse seng tsa sebopeho (nsps), tse tsejoang hape e le 3CLpro, hobane e na le homology le 3Cpro ea picornavirus ( 9).Ho nahanoa hore li-nsps tsena li bokelloa ho RTC e kholo e matla, e etsa hore ho be le motsoako oa genomic RNA (replication) le sehlopha sa subgenomic RNA (transcription), 'me e sebelisetsoa ho hokahanya polelo ea ORF e ka tlaase ho noka ea ORF1b (10? ? ?12).
RTC ea mantlha e kenyelletsa RNA e itšetlehileng ka RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) le li-enzyme tse 'maloa tse sebetsanang le RNA, tse kentsoeng haholo ho ORF1b le lelapeng la coronavirus E na le nsp12-nsp16 le nsp9-nsp12 lelapeng la Arterioviridae (sheba tšupiso 10ââ 12).RdRp le HEL1 li emela libaka tse peli (karolo ea bohlano) tse bolokiloeng tsa kokoana-hloko ea sehlaha 'me li na le homology har'a likokoana-hloko tse ling tsa RNA.Replicase ea mantlha ho lumeloa hore e thusoa ke likaroloana tse ling, ho kenyelletsa le li-nsps tse nyane tse lokollotsoeng sebakeng sa carboxy-terminal (C-terminal) sa pp1a, tlase ho Mpro (coronavirus nsp5 le arterial virus nsp4, ka ho latellana).Ba na le ts'ireletso e khethehileng ea lelapa le mesebetsi e fapaneng (e hlahlobiloe ho tšupiso ea 10ââ12).
Haufinyane tjena, sebaka se nang le litšobotsi tse ikhethang tsa tatellano ea tatellano se ile sa fumanoa sebakeng sa amino terminus (N-terminus) e bapileng le RdRp ho livaerase tsohle tse hahelletsoeng, empa ha ho livaerase tse ling tsa RNA (16).E itšetlehile ka sebaka sa eona le nucleotide transferase (nucleoside monophosphate [NMP] transferase) mosebetsi, sebaka sena se bitsoa NiRAN (Nestvirus RdRp-related nucleotide transferase).Motsoako oa li-domeine tse peli tsa NiRAN-RdRp o etsa nsp12 lelapeng la Coronaviridae le nsp9 lelapeng la Arterioviridae, 'me ho tse ling tsa nestoviridae, NiRAN-RdRp e lebelletsoe ho lokolloa e le nsp e ikemetseng ho tsoa ho viral polyprotein.Ho coronavirus, sebaka sa NiRAN se na le masala a ??1/450 mme se hokahane le sebaka sa C-terminal RdRp ka sebaka sa linker (16?19).Ho Equine Arteritis Virus (EAV) (Arteriviridae), recombinant nsp9 e bonts'a mesebetsi ea Mn2+ ion-dependent (self) UMPylation le GMPylation, e itšetlehileng ka metheo e meraro e bolokiloeng ea tatelano ho nestovirus, AN, BN le CN Masala ka tatellano.Moo N e emelang NiRAN) (16).Karolo ea N-terminal flanking ea motifs ena ke preAN e sa tsitsang haholo.A mang a masala ana a boetse a bolokiloe ka har'a protheine kinases e amanang le hole, moo ho bonts'itsoeng hore e ameha ho tlamang ha nucleoside triphosphate (NTP) le ts'ebetso ea catalytic (20, 21).Tumellanong le tlhokomeliso ena, masala a 'maloa a mantlha a sebetsang ho pseudokinase SelO ho tsoa ho Pseudomonas syringae a ka bokelloa le SARS-CoV-2 nsp7/8/12/13 supercomplex e sa tsoa hatisoa.Conserved Coronavirus NiRAN masalla a holimo ka har'a sebopeho sa elektronike.Recombinant protheine (17).Ho nahanoa hore litokomane (self) U / GMPylation e tla hlahisa boemo ba nakoana ho fetisetsa NMP ho substrate (eo hona joale e sa tsejoeng) (16), le ho tšoana ha sebopeho pakeng tsa NiRAN le protein kinase (17, 19) ) Na khopolo eo NiRAN e fetola liprotheine tse ling.
Likarolo tse ngata, ho kenyeletsoa kamano ea eona e ikhethileng le e ikhethileng ea livaerase tse lutseng sehlaheng le karohano ea liphatsa tsa lefutso ho tsoa ho RdRp, li etsa hore NiRAN e be enzyme ea bohlokoa ea taolo bakeng sa livaerase tse hahelletsoeng, tse bohlokoa ho hlahella le boitsebahatso ba tsona.Pejana, ho ne ho bitsoa mesebetsi e meraro e kenyelletsang NiRAN ho laola phetolelo ea genome/subgenomic kapa replication/transcription.Ha ho nahanoa ka lintlha tse haellang le tse sa phethahalang tse neng li fumaneha ka nako eo, ts'ebetso e 'ngoe le e' ngoe e na le melemo le mathata a eona (16).Patlisisong ena, re ikemiselitse ho kopanya lithuto tsa biochemical le ho khutlisa liphatsa tsa lefutso tsa li-coronavirus tse emelang meloko e 'meli, le ho beha seo re se fumaneng molemong oa phetoho ea tlhaho ea lelapa la coronavirus, e le hore re tsebe ho utloisisa sebaka sena se makatsang.Re tlaleha tsoelo-pele e kholo kutloisisong ea NiRAN ka ho tsebahatsa liphofu tsa tlhaho ho RTC, eo (har'a likhopolo-taba tse tharo tse teng) e tlatsetsang karolo ea sebaka sena ho qala ho kopanya likokoana-hloko tsa RNA.Patlisiso ena e boetse e bula menyetla ea likarolo tse ling tsa NiRAN ho sebopeho sa moamoheli oa vaerase.
E le ho tsebahatsa thepa ea enzymatic ea kokoana-hloko ea corona e amanang le nsp12 e amanang le sebaka sa NiRAN, re hlahisitse mofuta oa recombinant oa human coronavirus 229E (HCoV-229E) nsp12 ho E. coli, e nang le tag ea His6 ho C-terminus, 'me re kopanya protheine e nang le [α32-P] Kopanya hammoho le NTP boteng ba MnCl2 joalokaha ho hlalositsoe ho Lisebelisoa le Mekhoa.Tlhahlobo ea sehlahisoa sa karabelo e bonts'a boteng ba protheine e nang le radiolabeled e tsamaeang le nsp12 (106 kDa), e bonts'a hore coronavirus nsp12 e susumetsa sebopeho sa li-adducts tsa covalent protein-NMP, tse entsoeng ka ho khetheha ka uridine monophosphate (UMP) (Setšoantšo sa 1A) B).Tlhahlobo ea palo e bontšitse hore ha e bapisoa le li-nucleotide tse ling, matla a pontšo ea ho kenngoa ha UMP a eketsehile ka 2 ho ea ho linako tsa 3 (Setšoantšo sa 1C).Lintlha tsena li lumellana le ts'ebetso ea phetisetso ea NMP e boletsoeng esale pele ea sebaka sa NiRAN sa coronavirus (16), empa e bonts'a hore likhetho tsa nucleotide tsa sebaka sa NiRAN sa coronavirus le vaerase ea arterial li fapane.
Ts'ebetso ea ho iketsetsa NMPylation ea HCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) e ile ea kenngoa ka [α-32P] NTP e khethiloeng ka pel'a 6 mM MnCl2 bakeng sa metsotso ea 30 (sheba Lisebelisoa le Mekhoa bakeng sa lintlha).Lihlahisoa tsa karabelo li ile tsa aroloa ke SDS-PAGE 'me tsa silafatsoa ka boputsoa bo botala ba Coomassie.(B) Protheine ea radiolabeled e bonoa ka litšoantšo tsa phosphorus.Maemo a nsp12-His6 le li-protein molecular mass markers (ka kilodaltons) li bontšoa ho A le B. (C) Matla a lets'oao la radioactive (ho bolela ± SEM) le ile la khethoa ho tsoa litekong tse tharo tse ikemetseng.*P≤0.05.Matla a pontšo (peresente) a amana le UTP.
Leha mesebetsi ea enzyme e amanang le NiRAN e bonts'itsoe e le ea bohlokoa bakeng sa phetisetso ea EAV le SARS-CoV moetlong oa lisele (16), ts'ebetso e ikhethileng ea NiRAN le lipheo tse ka bang teng ha li so tsejoe.Ho tšoana ho sa tsoa tlalehoa pakeng tsa NiRAN le lelapa la liprotheine tse nang le li-protein kinase-like folds (17, 22) li re susumelletse hore re leke khopolo-taba ea hore NiRAN e susumetsa NMPylation ea liprotheine tse ling.Re hlahisitse litebello tse ka bang teng homologous, ho kenyeletsoa liprotheine tseo e seng tsa sebopeho tse kentsoeng ke HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), e 'ngoe le e 'ngoe e na le tag ea C-terminal His6 (SI sehlomathiso, Tafole S1) , Le ho kenya liprotheine tsena ka [α32-P] uridine triphosphate ([α32-P]UTP) boteng kapa bo le sieo ba nsp12.Bovine serum albumin le MBP-LacZα fusion protheine e hlahisoang ka E. coli e sebelitse e le taolo (Setšoantšo sa 2A, litsela tsa 1 ho isa ho 7).Protheine e nang le radiolabeled e ile ea hlahlojoa ke sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) le autoradiography, 'me ho ile ha fumanoa hore ho na le pontšo e matla ea radioactive karabelo e nang le nsp12 le nsp9.Boemo ba pontšo bo lumellana le boima ba molek'hule ea nsp9, e bontšang nsp12-mediated UMPylation ea nsp9 (Setšoantšo sa 2B, pina ea 7).Ha ho liprotheine tse ling tsa tlhahlobo tse fumanoeng li le UMPylated, tse entseng hore re fihlele qeto ea hore nsp9 ke karolo e itseng ea nsp12.Tumellanong le boitsebiso ba ho iketsetsa NMPylation bo bontšitsoeng ho Setšoantšo sa 1, nsp12 e khona ho fetisetsa li-NMP tse 'nè ho nsp9, le hoja katleho e fapane, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Setshwantsho).3 A le B).Tlas'a maemo a sebelisitsoeng tekong ena ( khutsufatsa karabelo le nako ea ho pepeseha, fokotsa khatello ea nsp12; lisebelisoa le mekhoa), "self-NMPylation" ea nsp12 e ne e ke ke ea fumanoa (bapisa Figure 2B, lane 7, le Figure 1B), e ipakile e sebetsa hantle (Le mekhahlelo e mengata) UMP e tlohile ho nsp12 ho ea ho nsp9.Ts'ebetso ea phetisetso ea UMP e hloka boteng ba li-ion tsa Mn2 +, joalo ka ha ho bonts'itsoe ho Setšoantšo sa 3C, athe ke ts'ebetso e fokolang feela ea phetisetso ea UMP e ileng ea bonoa ka boteng ba Mg2 +, mme ha ho ts'ebetso boteng ba li-divalent tse ling tse peli tse lekiloeng.Lintlha tse tšoanang li ile tsa fumanoa litlhahlobong tsa NMPylation tse nang le cytidine triphosphate (CTP), guanosine triphosphate (GTP), le adenosine triphosphate (ATP) (SI appendix, Figure S1).
HCoV-229E nsp12-mediated UMPylation ea nsp9.Letoto la li-protein substrates (ho kenyeletsoa bovine serum albumin, MBP-lacZα, le letoto la HCoV-229E nsps le ngotsoeng ka C-terminal His6 e kenyellelitsoeng ke ORF1a) li ile tsa sebelisoa ho lekola tšebetso ea UMPylation ea HCoV-229E nsp12-His6⁺-mediated. protheine.Kenyelletsa protheine ka [α-32P] UTP bakeng sa metsotso ea 10 ho ba sieo (A) kapa ho ba teng (B) ea nsp12 joalokaha ho hlalositsoe ka thepa le mekhoa.Ka holimo ho A le B, ho bontšoa gel ea SDS-polyacrylamide e nang le Coomassie Brilliant Blue, 'me ka tlase ho A le B, ho bontšoa li-autoradiograms tse tsamaellanang.Boemo ba protheine molecular mass marker (ka kilodaltons) bo fanoe ka ho le letšehali.Boemo ba nsp12-His6 (B, top) le pontšo ea radioactive e hlokometsoeng nakong ea ho kenngoa ha nsp12-His6 le nsp9-His6 (B, lane 7) e boetse e bontšoa, e bontšang hore [α-32P]UMP ho nsp9-His6 (12.9 kDa), e neng e sa hlokomeloe bakeng sa liprotheine tse ling tse lekiloeng.
HCoV-229E NiRAN-mediated biochemical and virological characterization of nsp9 NMPylation.(A le B) Karolo ea "nucleotide co-substrate" e sebelisitsoeng karabelong.Nsp12-His6 le nsp9-His6 li tsoakoa 'me li kenngoa ka pel'a li-NTP tse fapaneng tsa [α-32P] tekong e tloaelehileng ea NMPylation.(A, top) Coomassie-stained nsp9-His6 e arotsoe ke SDS-PAGE.(A, tlase) Autoradiograph ea sebaka se le seng sa gel.(B) Mosebetsi o lekanyelitsoeng (o bolela ± SEM) ka pel'a "cofactor" e khethiloeng ea nucleotide e khethoa ho tsoa litekong tse tharo tse ikemetseng.*P≤0.05.(C) Karolo ea li-ion tsa tšepe.Ho bontšitsoe tlhahlobo e tloaelehileng ea NMPylation ka pel'a [α-32P] UTP le li-ion tse fapaneng tsa tšepe, e 'ngoe le e' ngoe e na le 1 mM.Ho C, holimo, Coomassie e silafalitsoeng ke nsp9-His6 e bonts'oa, 'me ho C, tlase, ho bontšoa autoradiography e lumellanang.Boholo ba protheine e ngotsoeng (ka kilodaltons) bo bontšoa ka ho le letšehali la A le C. (D) Mofuta o fetotsoeng oa HCoV-229E nsp12-His6 o nang le sebaka sa amino acid e boletsoeng e ho [α-32P]UTP, joalo ka ha ho hlalositsoe. ho Thepa le Mekhoa.Nsp9-His6 e entsoeng ka radiolabeled ka NMPylation reaction e fumanoa ka phosphorylation imaging (D, top).Ts'ebetso e lekanyelitsoeng ha e bapisoa le protheine ea mofuta oa hlaha (wt) e bonts'oa ho D, 'me tlase e nkuoa e le karolelano (±SEM) ho tsoa litekong tse tharo tse Ikemetseng.Linaleli li bonts'a libaka tsa masalla a sa bolokehang.(E) The virus titer in the culture supernatant of p1 cell fumanweng lihora tse 24 ka mor'a hore tšoaetso e khethoe ke plaque assay.Liphetoho tsa codon sebakeng sa NiRAN sa phetoho e entsoeng ka boenjiniere ea HCoV-229E (nomoro e setseng e ipapisitse le boemo ba bona ho pp1ab).Sebaka se sebetsang sa RdRp se sa sebetseng se sa sebetseng nsp12_DD4823/4AA se sebelisitsoe joalo ka taolo.
E le hore re fumane kutloisiso e tebileng ea sebaka se sebetsang sa NiRAN le ho fumana masala a amanang le mosebetsi oa nsp9-specific NMP transferase, re ile ra etsa tlhahlobo ea phetoho ea phetoho, moo re ileng ra nkela masala a ts'oarellang ho NiRAN AN, BN le CN motifs. 16) Ke Ala (SI sehlomathiso, Setšoantšo sa S2).Ho feta moo, tšusumetso ea li-conservative Arg-to-Ls kapa Lys-to-Arg e ile ea hlahlojoa maemong a mabeli.E le taolo (e mpe), masala a sa bolokoang kapa a sa bolokoa ka tlase ho sebaka sa NiRAN sa coronaviruses le livaerase tse ling tsa sehlaheng li nkeloa sebaka ke Ala. BN) le D4280A (CN) e fokotsa haholo kapa e felisa nsp9 NMPylation ka nsp12, ha liprotheine tse nang le li-conservative substitutes (R4178K), K4116R) li boloka 60% le 80% ea mosebetsi oa tsona, e leng se bontšang hore ho phomola ha lithibelo ka lehlakoreng la bona. liketane ke physicochemically sensitive (Figure 3D).Ho fetola masala a mang a 'maloa a bolokiloeng E4145A, D4273A, F4281A le D4283A ha ho kotsi haholo, 'me nsp9 UMPylation e fokotsehile ka mokhoa o itekanetseng.Liphetho tse tšoanang li ile tsa fumanoa ho nsp9 NMPylation reactions e amang NTPs tse ling (Figure 3D le SI appendix, Figure S3), e tiisang hore liphello tse hlokometsoeng ho li-substitutes tse itseng tsa amino acid li itšetlehile ka mofuta oa nucleotide co-substrate e sebelisitsoeng.Ka mor'a moo, re ile ra leka tšusumetso e ka bang teng ea li-substitutes tsena tsa nsp12 mabapi le phetisetso ea li-coronavirus ho setso sa lisele.Ho fihlela sena, re sebelisitse litempele tse tlatselletsang tsa DNA (cDNA) tse entsoeng ka liphatsa tsa lefutso tse kopantsoeng ka har'a kokoana-hloko ea recombinant vaccinia (23, 24) ho ngola lisele tse 5 -7.Tlhaloso ea kokoana-hloko e tšoaetsanoang e hlahisoang liseleng tsena e bontšitse hore liphetoho tse ngata tsa HCoV-229E NiRAN li ne li sa khonehe (Setšoantšo sa 3E).Sehlopha sa likokoana-hloko tse sa sebetseng li kenyelletsa mekhoa e meng e bontšitsoeng ho felisa kapa ho fokotsa haholo ts'ebetso ea NMP transferase in vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), empa ho na le mekhoa e meng e 'meli (K4116R, E4105A) % e bolokiloe?Ts'ebetso ea bona ea in vitro NMPylation e fana ka maikutlo a hore lithibelo tse ling lia ameha.Ka mokhoa o ts'oanang, liphetoho tse ling tse peli (R4178K, F4281A) tse bakileng phokotseho e itekanetseng ts'ebetsong ea NiRAN's in vitro NMPylation e hlahisitse likokoana-hloko tse phelang, leha ho le joalo, likokoana-hloko tsena li fokolitse li-titers haholo ka ho ikatisa.Ho tsamaellana le data ea in vitro e bonts'itsoeng ho Setšoantšo sa 3D, ho nkela masalla a mang a mane a sa bolokehang ho coronavirus le/kapa livaerase tse ling tsa sehlaha (K4113A, D4180A, D4197A, D4273A) (8, 16) e hlahisitse livaerase tse sebetsang Bana, leha ba na le titer e fokotsehileng ka mokhoa o itekanetseng ha e bapisoa le kokoana-hloko ea mofuta o hlaha (Setšoantšo sa 3E).
Bakeng sa ho ithuta hore na ts'ebetso ea NiRAN-mediated NMP transferase e ipapisitse le sebaka se sebetsang sa RdRp, masala a mabeli a bolokiloeng a Asp a amehang khokahanyong ea li-ion tsa tšepe tsa divalent (11) ho RdRp motif C li ile tsa nkeloa sebaka ke Ala. ts'ebetso ea eona ea NMPylation ea nsp9, e bontšang hore ts'ebetso ea nsp12-mediated in vitro nsp9 NMPylation ha e hloke mosebetsi oa polymerase (SI Sehlomathiso, Figure S4).
Kamora ho theha ts'ebetso e ikhethileng ea NMP ea nsp9-specific NMP bakeng sa nsp12, re lekile ho tšoaea tlhahiso ea NMP-nsp9 ka mass spectrometry (MS).Palo e felletseng ea boima ba protheine ea recombinant HCoV-229E nsp9 e bonts'itse tlhoro ho 12,045 Da (Setšoantšo sa 4A).Ho eketsoa ha nsp12 ha hoa ka ha fetola boleng ba nsp9, e bontšang hore nsp12 le nsp9 li ke ke tsa theha mohaho o tsitsitseng tlas'a maemo a sebelisoang (denaturation) (Setšoantšo sa 4A).Boteng ba UTP le GTP, tekanyo ea boima ba karabelo e nang le nsp9 le nsp12 ka ho latellana e bontšitse hore protheine ea UTP e tsamaile 306 Da, 'me boima ba protheine ea GTP bo tsamaile 345 Da, e bontšang hore molek'hule e' ngoe le e 'ngoe ea nsp9 e tlama UMP kapa GMP. (Setšoantšo sa 4) C le D).Ho nahanoa hore matla a hlokahalang bakeng sa NiRAN-mediated nsp9 NMPylation a tsoa ho NTP hydrolysis le pyrophosphate release.Le hoja molar e fetang 10 ea nsp9 (sepheo) ho feta nsp12 (enzyme) e sebelisitsoe karabelong ena, hoo e ka bang NMPylation e feletseng ea nsp9 e ile ea hlokomeloa, e bontšang hore tšebelisano pakeng tsa nsp12 le nsp9 ke ea nakoana, 'me nsp12 e ka NMPylate ho feta nsp9 molek'hule ea in vitro.
NMPylation e le 'ngoe ea nsp9 ka pel'a nsp12 le UTP kapa GTP.Ho bonts'itsoe ke protheine ea boima ba 'mele e felletseng ea HCoV-229E nsp9 (SI sehlomathiso, Tafole S1) (AD).(A) nsp9 feela, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 ka pel’a UTP, (D) nsp9 + nsp12-His6 ka pel’a GTP.
Ho fumana masala a nsp9 UMPylated ke nsp12, nsp9-UMP e ne e petsotsoe ka trypsin.Li-peptide tse hlahisitsoeng li ile tsa aroloa ke nano-high performance liquid chromatography (HPLC) 'me tsa hlahlojoa ke tandem mass spectrometry (MS/MS) inthaneteng.Tlhahlobo ea data e sebelisang sephutheloana sa software sa Byonic (Protein Metrics) e bontšitse UMPylation ea N-terminal amino acid.Sena se netefatsoa ka letsoho.Tandem mass spectrum ea precursor peptide [UMP]NNEIMPGK (SI appendix, Figure S5A) e senotse sekhechana sa 421 m/z, se bontšang hore UMP e tlama masalla 1 a nsp9.
N-terminus ea nsp9, Asn e bolokiloe har'a litho tsa Orthocoronavirinae (SI appendix, Figure S6).Leha re lumela hore N-terminal primary amine nitrogen ke eona e ka amohelang UMP, re nkile qeto ea ho fumana bopaki bo eketsehileng ba ho tlama ha NMP ho N-terminal.Ka lebaka lena, peptide e seng NMPylated le NMPylated N-terminal nsp9 e hloekisitsoeng ke HPLC e nkiloe ka boteng ba acetone le sodium cyanoborohydride.Tlas'a maemo ana, ke li-amine tsa mantlha tsa mahala feela tse ka fetoloang ka propyl (25).Peptide e nkiloeng ke N-terminal nsp9 e nang le tatellano ea NNEIMPGK e na le li-amine tse peli tsa mantlha, e 'ngoe e le N-terminus ea Asn le e' ngoe e lehlakoreng la ketane ea Lys ho C-terminus.Ka hona, lihlopha tsa propyl li ka hlahisoa lipheletsong ka bobeli.Li-chromatogram tsa ion tse nkiloeng tsa li-peptide tse seng tsa NMPylated li bonts'itsoe sehlomathisong sa SI, Setšoantšo sa S5B.Joalokaha ho lebeletsoe, N-terminal le C-terminal (mono) propylated (SI appendix, Figure S5B, top lane) le dipropylated peptide (SI appendix, Figure S5B, lane e ka tlaase) e ka tsejoa.Paterone ena e fetoha ha ho sebelisoa peptide ea NMPylated N-terminal ea nsp9.Tabeng ena, ke feela C-terminal propylated peptide e ka khetholloang, empa N-terminal propylated peptide le dipropylated peptide ha e tsejoe (SI Appendix, Figure S5C), e bontšang hore UMP e fetiselitsoe ho N-terminal primary amine Ho thibela sena. sehlopha ho tloha ho etsa liphetoho.
Ka mor'a moo, re nkela sebaka (ka Ala kapa Ser) kapa re hlakole masala a bolokiloeng ho N-terminus ea nsp9 ho hlalosa lithibelo tse ikhethileng.Ho ipapisitsoe le lintlha tsa rona tsa MS tse bonts'ang hore NiRAN e theha adduct ea nsp9-NMP e nang le amine ea mantlha ea masala a N-terminal ea nsp9, re ile ra nahana hore nsp9 NMPylation e hloka viral master protease (Mpro, nsp5) ho lokolla nsp9 N-terminal ho tloha. selelekela sa eona sa polyprotein.E le ho hlahloba khopolo-taba ena, re hlahisitse protheine ea precursor nsp7-11 e nang le nsp9 ho E. coli 'me ra etsa tlhahlobo e tloaelehileng ea NMPylation ka pel'a [α-32P] UTP (lisebelisoa le mekhoa).Joalo ka ha ho bonts'itsoe ho Setšoantšo sa 5A (lane ea 3), selelekela sa nsp7-11 se sa khaotsoang ha se na radiolabele ka nsp12.Ka lehlakoreng le leng, haeba nsp7-11 e ts'oaroa ke recombinant nsp5 ho lokolla nsp9 (le tse ling tse nsps) ho selelekela, protheine e nang le radiolabele e tsamaeang le nsp9 e fumanoe, e netefatsa qeto ea rona ea hore NiRAN le N- Selection ea sebopeho sa covalent nsp9-NMP adducts. .Terminal primary amine ea N-terminal Asn (boemo ba 3825 ho pp1a/pp1ab).Qeto ena e boetse e tšehetsoa ke liteko tse sebelisang moaho oa nsp9, o nang le masala a le mong kapa a mabeli ho N-terminus.Maemong ana ka bobeli, UMPylation ea NiRAN-mediated ea nsp9 e ile ea felisoa (SI Sehlomathiso, Figure S7).Ka mor'a moo, re ile ra hlahisa protheine e nang le masala a le 'ngoe kapa a mabeli a Asn a hlakotsoeng ho 3825-NNEIMPK-3832 peptide sequence ho N-terminal ea nsp9.Maemong ana ka bobeli, nsp9 UMPylation e ne e koetsoe ka ho feletseng (Setšoantšo sa 5B), ho fana ka bopaki bo eketsehileng ba hore N-terminus ea sebele ea Nsp9 e sebetsa e le NMP receptor.
Ts'ebetso ea proteolytic ea nsp9 le karolo ea masala a N-terminal ho NSP12-mediated UMPylation.(A) nsp9 UMPylation e hloka nsp9 N-terminal ea mahala.Nsp7-11-His6 e kentsoe pele ho 30 °C ho NMPylation detector buffer e nang le UTP boteng kapa bosieo ba recombinant Mpro (nsp5-His6).Kamora lihora tse 3, qala tlhahlobo ea NMPylation ka ho kenyelletsa nsp12-His6 joalo ka ha ho hlalositsoe ho Lisebelisoa le Mekhoa.Karabelo e nang le nsp5-His6 (lane 1) le nsp9-His6 (lane 2) e ile ea sebelisoa e le taolo.Kamora metsotso ea 10, karabelo e ile ea felisoa mme motsoako oa karabelo o arotsoe ke SDS-PAGE.Protheine e ne e silafalitsoe ke Coomassie Brilliant Blue (A, holimo).Selelekela sa Nsp7-11-His6 le sehlahisoa se sebelitsoeng se hlahisoang ke nsp5-His6 mediated cleavage li bonts'itsoe ka ho le letona.Ka kopo elelloa (ka lebaka la boholo ba tsona bo nyane) hore nsp7 le nsp11-His6 ha li bonoe ka har'a gel ena, 'me karabelo e tlatselletsoa ka nsp5-His6 (tsela ea 1 le ea 4; boemo ba nsp5-His6 bo bontšoa ke selikalikoe se tiileng) kapa nsp9-His6 (Lane 2) e na le palo e nyenyane ea MBP (e bontšitsoeng ke li-circles tse bulehileng) e le litšila tse setseng hobane li hlahisoa e le liprotheine tsa MBP fusion (SI sehlomathiso, Letlapa la S1).(B) Phapang ea Nsp9-His6 ha e na masala a le mong kapa a mabeli a N-terminal Asn (mesaletsa ea linomoro ho latela boemo ba pp1a/pp1ab) 'me e hloekisoa le ho kenngoa ka nsp12-His6 le [α-32P] UTP.B, SDS-PAGE e nang le Coomassie e bonts'itsoe ka holimo, B, autoradiograph e tsamaellanang e bonts'oa ka tlase.Boemo ba letšoao la boima ba molek'hule (ka kilodaltons) bo bontšoa ka ho le letšehali.(C) HCoV-229E nsp9-His6 N-terminal conserved residues e ile ea nkeloa sebaka ke Ala kapa Ser, 'me palo e tšoanang ea protheine e ile ea sebelisoa ho nsp12-His6 mediated UMPylation reaction.Lihlahisoa tsa karabelo li ile tsa aroloa ke SDS-PAGE 'me tsa silafatsoa ka Coomassie Brilliant Blue (C, top),' me radiolabeled nsp9-His6 e ile ea bonoa ke phosphorescence imaging (C, middle).Ho sebelisa protheine ea mofuta o hlaha (wt) e le tšupiso (e behiloeng ho 100%), ts'ebetso e amanang le NMPylation (e bolelang ± SEM) e ne e baloa ho tsoa litekong tse tharo tse ikemetseng.(D) Li-titer tsa vaerase ho p1 cell culture supernatant ea lisele tsa Huh-7 tse tšoaelitsoeng ke lisele tsa HCoV-229E tse hlaha tsa Huh-7, le liphetoho tse nkileng li-substitutes tse khethiloeng tsa amino acid ho nsp9 li ile tsa khethoa ka tlhahlobo ea plaque.RdRp motif C e feto-fetohang habeli DD4823/4AA e sa sebetseng hantle e ile ea sebelisoa e le taolo e mpe.
N-terminus ea nsp9 (haholo-holo maemo a 1, 2, 3, le 6) e bolokiloe haholo har'a litho tsa lelapa le lenyenyane la Orthocoronavirinae (SI sehlomathiso, Figure S6).E le ho ithuta karolo e ka bang teng ea masala ana ho nsp12-mediated nsp9 NMPylation, masala a mabeli a latellanang a Asn ho N-terminus ea nsp9 a ile a nkeloa sebaka ke Ala kapa Ser (a le mong kapa a kopane).Ha ho bapisoa le mofuta o hlaha oa nsp9, ho nkela N3825 sebaka ka Ala kapa Ser ho felletse ka phokotso e fetang habeli ea Nsp12-mediated UMPylation (Figure 5C).Tumellanong le qeto ea rona ea hore NMPylation e etsahala ho N-terminal primary amine ho e-na le ketane e ka thōko ea masala a N-terminal, re hlokometse NMPylation ea bohlokoa ka ho nkela N3825A le N3825S sebaka.Hoa thahasellisa hore haeba Asn ea bobeli e nkeloa sebaka ke Ala kapa Ser, nsp9 UMPylation e fokotsehile ka matla (ka makhetlo a fetang 10), ha ho nkeloa sebaka ha Ala libakeng tsa 3, 4, le 6 ho na le phello e itekanetseng feela ho nsp9 UMPylation (Setšoantšo sa 2). ) .5C).Liphetho tse tšoanang li ile tsa fumanoa ho sebelisoa ATP, CTP kapa GTP ( appendix SI, Figure S8).Ka kakaretso, lintlha tsena li bontša karolo ea bohlokoa ea N2826 (boemo ba 2 ho nsp9) ho nsp9 NMPylation.
Bakeng sa ho fumana bopaki bo eketsehileng ba khokahano ea ts'ebetso lipakeng tsa N-terminus ea nsp9 le NMPylation, re entse tatellano e mengata ea tatellano (MSA) ea tatellano ea nsp9 ea lelapa la Coronavirus (e fapaneng lipakeng tsa 104 le 113 masala) (SI Appendix, Figure. S6).Ka kakaretso, ho mefuta e 47 (e tsebahalang le e behang) ea meloko e 5 ea Lelapa le lenyenyane la Orthocoronavirinae e tšoaetsang liphoofolo tse anyesang, linonyana le lihahabi tse fapaneng, ke masala a 8 feela ka kakaretso a fumanoeng a sa fetohe.Liphetoho tse pharaletseng ka ho fetisisa, tse kenyelletsang ho tlosoa le ho kenngoa, li ile tsa hlokomeloa lipotolohong pakeng tsa likarolo tsa bobeli tsa sebopeho sa nsp9, joalokaha ho khethiloe ke lithuto tsa pele tsa meralo (26 ??28).Ho ile ha fumanoa mesaletsa e mehlano e sa fetoheng ho β strand le α helix ea karolo ea C-terminal ea nsp9.Masala a mararo a sa fetoheng a etsa moelelo oa NNE oa N terminus ea nsp9.Ho senotsoe hore Asn ea bobeli ea motif ena ke eona feela masala a sa fetoheng, ao hape a arolelanoang ke khopolo-taba ea nsp9 ea coronavirus ea frog e amanang le hole, 'me e emela mefuta ea Microhyla letovirus 1 lelapeng le ka tlase la Letovirinae la Alphaletovirus.Paballo ea masala a likarolo tsa sebopeho sa bobeli sa nsp9 e ka lekanyetsoa ka menahano ea sebopeho ho boloka thepa e tlamang ea RNA kapa e menahaneng.Leha ho le joalo, monahano ona ha o bonahale o sebetsa ho paballo ea NNE, 'me pele ho phuputso ena, mofuta oa lithibelo tse fokotsang phapang ea tatellano ea tripeptide e ne e patiloe ka ho feletseng.
Bakeng sa ho tseba bohlokoa ba paballo ea nsp9-NMPylation le NNE ho ikatisang ha coronavirus, re hlahisitse liphetoho tsa HCoV-229E, tse nkang sebaka se le seng kapa habeli tsa masala a nsp9 N-terminal, se bontšang hore nsp9 NMPylation e kotsi in vitro.Pele re qala, re leka ho araba potso ea hore na tse ling (haufi le nsp8|9 cleavage site) li ama tšebetso ea proteolytic sebakeng sa C-terminal pp1a.Sehlopha sa li-polyprotein tsa nsp7-11 tse nang le li-substitutes tse tsamaellanang ho N-terminus ea nsp9 li ile tsa hlahisoa ka E. coli 'me tsa khaoloa ka recombinant Mpro.Proteolytic cleavage ea libaka tse 'ne (ho kenyeletsoa sebaka sa nsp9 flanking) ha e amehe haholo ke li-substitutes tse hlahisitsoeng (SI appendix, Figure S9), ntle le liphetoho tsa sebopeho liprotheine tsena tse sitisang Mpro-mediated nsp8|9 cleavage (Kapa tse ling) websaeteng.
Lisele tsa Huh-7 li ile tsa fetisoa ka HCoV-229E RNA ea bolelele ba genome, e kenyelletsang Ala kapa Ser substitutes ho NNE tripeptides (N3825, N3826, le E3827) sebakeng sa nsp9 N terminus, ho bonts'a hore liphetoho tse ngata li bolaea.Re khonne ho pholosa kokoana-hloko ka ho fetola Ser kapa Ala ea N-terminal Asn (N2835A kapa N2835S), empa re ile ra hlōleha ho khutlisa kokoana-hloko ka liphetoho tse ling tse le 'ngoe le tse peli ka tatellano ea NNE (N3826A, N3826S, NN3825 / 6AA, NN3825/6SS) , E3827A) (Setšoantšo sa 5D).
Liphetho tsena li bonts'a hore phetisetso ea li-coronavirus ho setso sa 'mele e thibetsoe (e ts'oanang kapa e ts'oanang), e fokotsa phetoho ea tlhaho ea libaka tsa NMPylation' meleng, le ho ts'ehetsa karolo ea bohlokoa ea karabelo ena bophelong ba li-coronavirus.
Sehlopha sa ho qetela sa liteko, re hlahisitse C-terminal His6 e ngotsoeng SARS-CoV-2 nsp12 le nsp9, le mefuta e 'meli e fetotsoeng ea nsp12 ho E. coli.Libaka tse sebetsang tsa libaka tsa libaka tsa NiRAN le RdRp li ne li le ka ho latellana Sebelisa Ala ho e-na le hoo (Setšoantšo sa 6A le sehlomathiso sa SI, Letlapa la S2).K4465 ho SARS-CoV-2 nsp12 e tsamaisana le K4135 ho HCoV-229E (SI Sehlomathiso, Figure S2), e bonahetseng e hlokahala bakeng sa ts'ebetso ea NiRAN le phetisetso ea HCoV-229E (Setšoantšo sa 3D le E).Masala ana a boetse a lumellana le masala a arterial virus EAV nsp9 K94, ao pele a neng a bontšitsoe hore aa hlokahala bakeng sa NiRAN self-UMPylation / self-GMPylation (16).Joalo ka ha ho bonts'itsoe ho Setšoantšo sa 6B, SARS-CoV-2 nsp12 e na le ts'ebetso ea phetisetso ea UMP e sebelisa nsp9 joalo ka substrate, ha nsp12_K4465A e fetotseng sebaka se sebetsang e sa sebetse.Ho kenngoa habeli ka tatellano ea sebopeho sa SDD ea RdRp motif C ha e ame mosebetsi oa phetisetso ea UMP (Setšoantšo sa 6B), e bonts'a hore ts'ebetso ea RdRp ha e na phello e tobileng ho nsp9 UMPylation.Lintlha tse tšoanang li ile tsa fumanoa ho sebelisoa CTP, GTP le ATP (sehlomathiso sa SI, Figure S10).Ka bokhuts'oane, lintlha tsena li bonts'a hore NiRAN-mediated nsp9 NMPylation e na le ts'ebetso e hlokolosi ho li-coronavirus tse emelang mefuta e fapaneng ea leloko la orthocoronavirus subfamily.
SARS-CoV-2 nsp12-mediated NMPylation ea nsp9.(A) Coomassie e silafalitse gel ea SDS-polyacrylamide e bontšang protheine e kopantsoeng e sebelisitsoeng tekong ea NMPylation.Joalo ka taolo, ho ile ha sebelisoa protheine e fetotsoeng e nang le sebaka se sebetsang sebakeng sa NiRAN (K4465A) le sebaka sa RdRp (DD5152/3AA) sa SARS-CoV-2 nsp12.Nomoro ea masalla e ipapisitse le boemo ho pp1ab.(B) Autoradiograph ea ho lemoha UMPylation ho sebelisa nsp9-His6 le [α-32P] UTP e le substrate ea nsp12-His6 (mofuta oa hlaha [wt] le mutant).Boima ba molek'hule (ka kilodaltons) ea protheine e ngotsoeng e bontšoa ka ho le letšehali.
Libaka tsa NiRAN hangata li bolokiloe ho Nidovirales (16), ho bonts'a hore li susumetsa karabelo ea enzymatic ea bohlokoa bakeng sa phetisetso ea Nidovirus.Thutong ena, re khonne ho paka hore sebaka sa NiRAN sa coronavirus se fetisetsa NMP (e hlahisitsoeng ho tloha NTP) ho ea ho nsp9, protheine e makatsang ea RNA e amanang le ho ikatisa ha vaerase (26 ?? 29), ho e tseba e le sepheo sa tlhaho le molekane oa coronavirus RTC.
Sebaka sa NiRAN se arolelana mekhoa e meraro ea tatellano (AN, BN, le CN), e nang le palo e fokolang haholo ea masala a bolokiloeng malapeng ohle ka tatellano ea monophyletic empa e fapane haholo ea Nidovirales (8, 16).Liphuputso tsa morao-rao li bontšitse hore li amana ka mokhoa o hlophisitsoeng le lelapa le sa tloaelehang la liprotheine tse kang tsa protein kinase, tseo qalong li neng li bitsoa lelapa la SelO (17, 19, 22, 30, 31).Liprotheine tse amanang le SelO li na le mameno a kinase, empa ha li na masala a 'maloa a bolokiloeng a sebetsang ho classical kinases (22, 32).Ho ipapisitsoe le maikutlo a morao-rao a limolek'hule tsa ATP tse tlamelletsoeng sebakeng se sebetsang le ho tsitsisoa ke litšebelisano tse itseng, SelO e ile ea nahanoa 'me hamorao ea tiisoa hore e fetisetsa AMP (ho e-na le phosphate) ho protheine substrate (22), ha protheine e 'ngoe ea baktheria ea SelO-joaloka YdiU e na le. haufinyane tjena e bonts'itsoe ho matlafatsa sehokelo se kopaneng sa UMP ho Tyr le masala a hae a liprotheine tse fapaneng (33).
Bakeng sa ho netefatsa le ho holisa ponelopele ea masala a sebaka se sebetsang sa "coronavirus" ea NiRAN, re sebelisitse mekhoa ea biochemical le ea reverse genetics ho etsa tlhahlobo ea phetoho ho coronavirus nsp12 (setšoantšo sa 3D le E le SI, Setšoantšo sa S3 le tafole) S1â S4).Lintlha li bonts'a hore ho nkeloa sebaka ha HCoV-229E K4135, R4178 le D4280 ka Ala ho felisa ts'ebetso ea phetisetso ea NMP ka har'a vitro le ho ikatisa ha kokoana-hloko ho setso sa lisele (setšoantšo sa 3D le E le li-appendices tsa SI, Figure S3), li tšehetsa boteng ba tsona ho NTP γ-phosphate. ( K4135, R4178) le ho hokahanya ha li-ion tsa tšepe tsa sebaka se sebetsang (D4280).Phetisetso ea E4145A ea Glu e baballetsoeng mofuteng oa kokoana-hloko ea sehlaha ea nonyana e boletsoeng esale pele e tla tsitsisa boemo ba K4135 (17) e bontšitsoe ho felisa ho ikatisa ha kokoana-hloko, empa ho makatsang ke hore mosebetsi o ile oa bolokoa ka in vitro NMPylation assay (Figure 3D le E and Sehlomathiso sa SI, Setšoantšo sa S3 Le Litafole S1–S4).Ho ile ha hlokomeloa ka tsela e tšoanang ha ho kenngoa sebaka se lumellanang le YdiU homolog ea Salmonella typhimurium (E130A) (33).Ha li kopane, lintlha tsena li tšehetsa tšebetso ea taolo ea masala ana a bolokiloeng ho e-na le ts'ebetso ea matla.
Ho khutlisa masala a Phe a bolokiloeng (F4281A) ka har'a mefuta e mengata ea nestovirus sebakeng sa HCoV-229E NiRAN (8) ho felletse ka phokotso ea ts'ebetso ea NMPylation in vitro le phokotseho e kholo ea ho ikatisa ha vaerase moetlong oa lisele (Figure 3D, E le SI) sehlomathiso, Setšoantšo sa S3).Lintlha li tsamaellana le tšebetso ea bohlokoa ea taolo ea masala ana, joalo ka mesaletsa ea DFG motif Phe e bontšitsoeng pejana.Ho protheine kinases ea khale, ke karolo ea loop e tlamang ea Mg2 + mme e thusa ho bokella le ho laola mokokotlo???E hlokehang bakeng sa ts'ebetso e sebetsang ea catalytic (32, 34).Ho kenya sebaka sa Ala le Arg bakeng sa masala a K4116 (ho preAN motif), ka ho latellana, ho felisitse katiso ea vaerase, joalo ka ha ho ne ho lebelletsoe, ho bile le litlamorao tse fapaneng mabapi le ts'ebetso ea NMP transferase in vitro, ho ipapisitse le ketane ea lehlakore ea amino acid e hlahisitsoeng (Setšoantšo sa 3D le E le lihlomathiso tsa SI. , Setšoantšo sa S3).Lintlha tse sebetsang li lumellana le boitsebiso ba mohaho, bo bontšang hore masala ana a thehile tšebelisano le ATP phosphate (17).Sebakeng sa NiRAN sa malapa a mang a kokwana-hloko ea sehlaha, boemo ba HCoV-229E pp1a/pp1ab K4116 bo lula ke Lys, Arg kapa His (8), e leng se bontšang hore thibelo ea ts'ebetso ea masala ana a khethehileng e fokotsehile.Ho nkela sebaka sa D4188A le D4283A ho felisa kapa ho fokotsa ka matla mosebetsi oa enzyme le ho felisa ho ikatisa ha kokoana-hloko (Setšoantšo sa 3).Masalla ana a mabeli a bolokiloe ho bongata (empa e seng kaofela) livaerase tse lutseng sehlaheng (8), ho bonts'a ts'ebetso ea bohlokoa e khethehileng ea lelapa empa mohlomong e seng ea ts'ebetso.Liphetoho tsa Ala tsa masala a mang a 'maloa a Lys le Asp (K4113A, D4180A, D4197A le D4273A) tse sa bolokoang ho Coronaviridae kapa malapa a mang a Nestioviridae (8) a sebelisitsoe e le taolo.Joalokaha ho lebeletsoe, li-substitutes tsena li ka mamelloa haholo, ka ho fokotseha ho fokolang ha mosebetsi oa enzyme le ho pheta-pheta kokoana-hloko maemong a mang (Setšoantšo sa 3 le sehlomathiso sa SI, Setšoantšo sa S3).Ka kakaretso, data ea coronavirus mutagenesis e lumellana haholo le "self-GMP" le data ea morao-rao ea liphatsa tsa lefutso ea EAV NiRAN-RdRp (16), eo ho eona EAV nsp9 (coronavirus nsp12 ortholog) masala a K94 (e tsamaellanang le HCoV- 229E K4135) mesebetsi ea bohlokoa), R124 (e tsamaellanang le R4178), D132 (e tsamaellanang le D4188), D165 (e tsamaellanang le D4280), F166 (e tsamaellanang le F4281).Ntle le moo, data ea HCoV-229E mutagenesis e lumellana ebile e atolosoa ho tsoa ho data e tlalehiloeng pele ea SARS-CoV reverse genetics (16), e ts'oana haholo le e hlokometsoeng bakeng sa CN motif e tsamaellanang ea Phe-to-Ala mutant SARS-CoV_nsp12 phenotype e hlalositsoe -F219A le HCoV-229E_F4281A (Setšoantšo sa 3 D le E le sehlomathiso sa SI, Setšoantšo sa S3 le Lethathamo la S1-S4).
Ha ho bapisoa le li-orthologs tsa EAV (16), tse nang le khetho e hlakileng bakeng sa UTP le GTP (karabelong ea boits'oaro ba NMPylation), boithuto ba rona bo bonts'a hore sebaka sa coronavirus sa NiRAN (se emetsoeng ke HCoV-229E le SARS-CoV-2) se ka sebetsa hantle. e fetiselitsoe Li-NMP tse 'nè, le hoja ho na le khetho e fokolang bakeng sa UMP (Figure 1 le 3).Sebopeho se batlang se le tlase haholo sa substrate e ikhethileng ea NTP e lumellana le sebopeho se sa tsoa tlalehoa sa SARS-CoV-2 nsp7/8/12/13, moo ADP-Mg2+ e tlamellang sebakeng se sebetsang sa NiRAN, empa eseng le Karolo ea adenine. ea ho thehoa ha litšebelisano tse khethehileng (17).Thutong ea rona, mofuta oa nucleotide o sebelisitsoeng ho NMPylation reaction ha o na phello e fapaneng mosebetsing oa protheine e fetotsoeng (SI Appendix, Figure S3), e bontšang hore ha ho le e 'ngoe ea masala ana e amanang haufi-ufi le ho tlama ha nucleobase e itseng.Motheo oa sebopeho le bohlokoa ba likokoana-hloko ba likhetho tse fapaneng tsa NTP tsa co-substrate tse hlokometsoeng libakeng tsa NiRAN tsa coronavirus le likokoana-hloko tsa arterial li ntse li lokela ho ithutoa;e ka ba 'nete kapa e ka ba ka lebaka la mefokolo ea lithuto tsa bona tse fapaneng.Hajoale, ho ke ke ha qheleloa ka thoko hore ts'ebetso e ka bang teng ea NMPylator ea "arterial virus" domain name ea NiRAN (ha e bapisoa le ts'ebetso ea "self-NMPylation" e neng e tsejoa pele) e na le khetho e fapaneng ea co-substrate, ho nahanoa hore ho tšoana ho teng lipakeng tsa arterial le coronavirus. Sebaka sa NiRAN se moeling oa sona.Bapisa ka tatelano (16).Ha ho bapisoa le pseudokinase SelO, e sebelisang Mg2 + e le cofactor, mosebetsi oa coronavirus le arterial virus NiRAN e itšetlehile ka Mn2 + (16) (Figure 3C le SI appendix, Figure S1).Ho itšetleha ka Mn2 + le khetho e totobetseng bakeng sa UTP ke tšobotsi e sa tloaelehang ea liprotheine tsa NMPylators, 'me e sa tsoa tiisoa ka protheine ea YdiU ea Salmonella typhimurium, e etsang hore ho be le protheine e tsitsitseng e itšetlehileng ka Mn2 + UMPylation ho sireletsa lisele ho tloha khatellong ea khatello ea kelello Cell ATP letamo. 33).
Ho tšoana ho sa tsoa hlalosoa lipakeng tsa "coronavirus" ea NiRAN domain le cellular protein kinases (17, 19) e fana ka tšehetso e eketsehileng bakeng sa bokhoni ba NiRAN ba ho hokahanya NMP le liprotheine tse ling tseo re li tlalehileng thutong ena.Re tsepamisitse patlo ea rona bakeng sa lipheo tsa NiRAN tse ka bang teng ho liprotheine tse kentsoeng ke HCoV-229E ORF1a, tse tsejoang ka ho thusa ka kotloloho kapa ka tsela e sa tobang replicase ea RTC's ORF1b-encoded (12, 35).Liteko tsa rona li fana ka bopaki bo tiileng bakeng sa NMPylation e sebetsang le e tobileng ea nsp9 (Setšoantšo sa 2).Haeba protheine e lebisitsoeng e sebelisoa ka ho feteletseng ha molar e leng 8 ho makhetlo a 10 ho feta ea enzyme (nsp12), ho netefatsoa hore nsp9 e feletseng (mono) NMPized (Setšoantšo sa 4).Re ile ra etsa qeto ea hore tšebelisano pakeng tsa nsp12 le nsp9 ke ea nakoana 'me e ke ke ea theha mohaho o tsitsitseng o nang le nsp9 (ha ho se na li-subunits tse ling tsa RTC).Qeto ena e tšehetsoa ke lithuto tsa tšebelisano ea protheine ho SARS-CoV proteome (35).Tlhahlobo ea MS e khethile amine ea mantlha ea masala a N-terminal ea nsp9 e le sebaka sa NMPylation (SI sehlomathiso, Figure S5).Sebopeho sa bond ea phosphoramidate le sehlopha sa amino sa N-terminal se khetholla mosebetsi oa NMPylation oa NiRAN-mediated ho tloha Pseudomonas syringae SelO-mediated AMPylation reaction, e leng se susumetsang ho thehoa ha O-linked AMP ho Ser, Thr, kapa Tyr masala a Peptide adduct ( 22), le S. typhimurium YdiU e theha O-linked (le Tyr) le N-linked (with His) peptide-UMP adducts.Lintlha tse fokolang tse fumanehang ho lelapa la SelO la liprotheine li bontša hore litho tsa lelapa lena le leholo la protheine li fapane haholo ka ho thehoa ha li-adducts tsa peptide-NMP.Ena ke tlhokomeliso e khahlisang e lokeloang ke boithuto bo tsoelang pele.
Lintlha tse fumanoeng thutong ena li entse hore re nahane hore NMPylation ea nsp9 e hloka N-terminus ea mahala.Boemong ba ho ikatisa ha vaerase, sena se tla fanoa ke ho petsoha ha proteolytic sebakeng sa ts'ebetso sa nsp8|nsp9 sebakeng sa replicase polyprotein pp1a e lipakeng tsa Mpro le pp1ab.Ho li-coronavirus tse ngata, phapang lipakeng tsa sebaka sena se ikhethileng (VKLQ|NNEI ho HCoV-229E) le libaka tse ling kaofela tsa coronavirus Mpro cleavage ke Asn (ho fapana le masala a mang a manyane, joalo ka Ala, Ser Kapa Gly) a nka P1â???Sebaka (36).Lintlha tsa peptide cleavage tse fumanoeng liphuputsong tsa pele li bonts'itse hore katleho ea cleavage ea sebaka sa nsp8|nsp9 e ne e le tlase ho feta ea libaka tse ling, e bontšang hore 1) sebaka sena se khethehileng se ka ba le karolo ea taolo ts'ebetsong e hlophisitsoeng ka nako ea C-terminal. pp1a sebaka, kapa 2) a Karolo ea Nsp9 N-terminus e khethehileng e bolokiloeng ho ikatisang kokoana-hloko (37).Lintlha tsa rona (Setšoantšo sa 5A) se bonts'itse hore mofuta oa recombinant oa nsp9 o jereng tatellano ea 'nete ea N-terminal e ne e atlehile NMPized ke nsp12.Tatelano ea N-terminal flanking e ile ea tlosoa ke factor Xa (nsp9-His6; SI sehlomathiso, Tafole S1) kapa Mpro-mediated cleavage (nsp7-11-His6; Figure 5A le SI sehlomathiso, Table S1).Habohlokoa, selelekela sa nsp9 se nang le nsp7-11-His6 se sa khaotsoang se bontšitse khanyetso ho NMPylation ea nsp12, e tsamaellanang le data ea rona, e bonts'ang hore nsp9-NMP adduct e thehiloe ka N-terminal primary amine ( SI sehlomathiso, Figure S5) .Ho fumana kutloisiso e tebileng ea boleng ba substrate ea NiRAN, re ile ra shebana le masala a haufi a N-terminal a nsp9.Ha ho se na liprotheine tse ling, li fetoha ka mokhoa o hlophisitsoeng, li thibela hore li se ke tsa fumanoa ka mokhoa o sa ngolisoeng oa nsp9 (26 28, 38), e bontšang phapang ea bona e fokolang ea tlhaho Sena se bakoa ke tatellano ea bohlokoa-e tobileng (eseng sebopeho sa bobeli se amanang) mosebetsi oa sekhechana sa nsp9 N-terminal.Li-substitution tsa Ala tsa masala a bolokiloeng sebakeng sena (Lipalo tsa 5C le D le sehlomathiso sa SI, Setšoantšo sa S8) se senola hore N3826 e bohlokoa bakeng sa nsp9 NMPylation in vitro, athe N3825A le E3827A tse ling li lebisa ho fokotseha ha NMPylation, athe M3829A le P38 do substitutes .Ho hlakile hore e ama nsp9 NMPylation.Leha ho nkela sebaka sa N-terminal Asn (N3825A, N3825S) ho na le phello e itekanetseng feela ho nsp9 NMPylation le katiso ea kokoana-hloko ho cell culture (Figure 5C le D), ho hlakoloa ha tatellano ea masala a Asn ho dipeptide ea N-terminal 3825-NN. e ipakile e le E bolaeang likokoana-hloko, e bontšang hore masala a Asn a hlokahala pele ho masalla a mang ho N-terminus, haholo-holo Asn, le hoja ho bonahala eka ho nkeloa sebaka ha masalla a tšoanang ho ka mamelloa ka mokhoa o itseng (Setšoantšo sa 5B, C, le D).Re etsa qeto ea hore 3825-NN dipeptide, haholo-holo masala a bolokiloeng le a bohlokoa a N3826 ka har'a "coronavirus" (SI sehlomathiso, Figure S6), e tiisa ho tlama le ho tsamaisana ho nepahetseng ha nsp9 N-terminus sebakeng se sebetsang sa NiRAN.
Ho kenya sebaka sa Ala (E3827A) bakeng sa Glu e bolokiloeng ea malapa ohle a manyenyane e boloka nsp9 NMPylation in vitro empa e bolaea likokoana-hloko tsa setso sa lisele (setšoantšo sa 5C le D), se bontšang mosebetsi o eketsehileng oa masalla ana, mohlala, litšebelisanong tsa bohlokoa (NMPylated kapa e sa fetoloang). ) nsp9 N-terminus le lintlha tse ling tse amanang le ho ikatisa ha kokoana-hloko.Liphetoho tsa Nsp9 ha lia ama ts'ebetso ea proteolytic ea nsp9 kapa nsps efe kapa efe e haufi (39) (SI Appendix, Figure S9), e bonts'a hore phenotypes tse bolaeang tsa liphetoho tse 'maloa tsa nsp9 tse hlokometsoeng ha li bakoe ke ho se sebetse ha C proteolytic process-terminal pp1a. .
Lintlha tse ka holimo li fana ka bopaki ba hore ka mor'a phekolo ea Mpro-mediated ea nsp8|9 cleavage site ho pp1a/pp1ab, N-terminus ea nsp9 e ka ba UMPylated (kapa ea fetoloa ka mokhoa o itseng le NMP e 'ngoe).Ntle le moo, paballo e ntle haholo ea N-terminus ea nsp9 (ho kenyeletsoa bonngoe le masala a Asn a sa fetoheng lelapeng la coronavirus) le lintlha tsa liphatsa tsa lefutso tse fumanoeng thutong ena (Lipalo 3E le 5D) li entse hore re fihlele qeto ea hore NMPylation e hlalositsoeng ea nsp9. e amana le baeloji mme e bohlokoa bakeng sa ho ikatisa ha coronavirus.Liphello tsa ts'ebetso ea phetoho ena li ntse li lokela ho ithutoa, ka mohlala, mabapi le ts'ebetso e tlamang ea RNA (2628) e hlalositsoeng pele (e sa tobang) (e sa tobang).N-terminal NMPylation e ka boela ea ama tšebelisano ea nsp9 le liprotheine kapa li-RNA substrates kapa ho thehoa ha likopano tse fapaneng tsa mekhahlelo e mene.Tsena li 'nile tsa hlokomeloa lithutong tsa meralo' me li netefalitsoe hore li amana ka mokhoa o sebetsang le phetiso ea coronavirus, le hoja haholo-holo ho ba sieo ha Tabeng ea phetoho ena (26- ââ29, 40).
Leha sepheo sa sepheo sa "coronavirus" sa NiRAN se ntse se hloka ho hlalosoa ka botlalo, lintlha tsa rona li bonts'a hore sepheo sa protheine ea sebaka sa NiRAN sa coronavirus se patisane haholo.Leha paballo ea masala a bohlokoa a sebaka se sebetsang (8, 16) sebakeng sa NiRAN sa malapa ohle a nidovirus a ts'ehetsa ka matla ts'ebetso ea NMPylator e bolokiloeng liprotheine tsena, boitsebahatso ba masala a pokotho a tlamang a sebaka sena. , 'me e ka fapana lipakeng tsa malapa a fapaneng a merero ea Nidovirales.Ka mokhoa o ts'oanang, lipheo tse amehang tsa livaerase tse ling tse lulang sehlaheng ha li e-so tsejoe.E kanna ea ba li-orthologs tse hole tsa nsp9 kapa liprotheine tse ling, hobane tatellano e kantle ho libaka tse hlano tsa replicase tseo ka kakaretso li bolokiloeng ho livaerase tse hahelletsoeng ha li bolokoe haholo (8), ho kenyelletsa le mofuta oa genome lipakeng tsa Mpro le NiRAN, har'a tsona, nsp9 e fumaneha ioronavirus.
Ho phaella moo, hona joale re ke ke ra tlosa monyetla oa hore sebaka sa NiRAN se na le lipakane tse eketsehileng (ho kenyeletsoa le liselefouno).Tabeng ena, ke habohlokoa ho bolela hore li-homologues tsa baktheria protheine ena e hlahang NMPylators (NMPylators) (30, 31) e bonahala e na le "balaoli ba hloahloa"?NMP e hlophisa mefuta e fapaneng ea liprotheine tsa cellular ho laola kapa ho felisa mesebetsi ea bona e tlase, ka hona e bapala karolo lits'ebetsong tse fapaneng tsa tlhaho, joalo ka karabelo ea khatello ea maikutlo ea cellular le redox homeostasis (22, 33).
Thutong ena (Figure 2 le 4 le SI Sehlomathiso, Figures S3 le S5), re khonne ho paka hore nsp12 e fetisitse karolo ea UMP (NMP) sebakeng se le seng (se bolokiloeng) ho nsp9, ha liprotheine tse ling li sa fetoloa e sebelisitsoeng Tlas'a maemo, ho hlalosoa hantle (ho e-na le ho hlephileng) substrate e khethehileng e tšehetsoa.Tumellanong le sena, ha e bapisoa le N-terminal nsp9 NMPylation, ts'ebetso ea NMPylation ea nsp12 e tlaase haholo, ho fumanoa ha eona ho hloka nako e telele ea ho pepeseha ha autoradiography, 'me ho sebelisoa keketseho ea 10 ea mahloriso a nsp12.Ntle le moo, tlhahlobo ea rona ea MS e hlolehile ho fana ka bopaki ba NMPylation ea nsp12, e fanang ka maikutlo a hore NiRAN domain self-NMPylation ke (ka ho fetisisa) ke ts'ebetso ea bobeli.Leha ho le joalo, hoa lokela ho hlokomeloa hore lithuto tse ling li fane ka bopaki ba pele ba hore boemo ba ho iketsetsa AMPylation ba baktheria NMPylator bo ka laola ts'ebetso ea bona ea NMPylation ho liprotheine tse ling (22, 33).Ka hona, ho hlokahala lipatlisiso tse eketsehileng ho batlisisa liphello tse ka 'nang tsa e-ba teng tsa mesebetsi ea boipheliso ea NMPylation e tlalehiloeng bakeng sa EAV nsp9 (16) le coronavirus nsp12 (phuputso ena), ho kenyelletsa le phello e reriloeng e kang chaperone mabapi le ho menaha ha sebaka sa C-terminal RdRp ( 16)).
Pejana, likhopolo-taba tse 'maloa mabapi le mesebetsi e ka bang teng tlase ho noka ea "nidoviral NiRAN" e kile ea nahanoa, ho kenyeletsoa RNA ligase, RNA -capped guanylate transferase le protheine priming activity (16), empa ha ho le e 'ngoe ea tsona e tsamaellanang le mesebetsi e fumanehang tlase ho noka.Boitsebiso bo fumanoeng libakeng tse latelang ke nako e le 'ngoe ntle le ho etsa maikutlo a eketsehileng.Lintlha tse fumanoeng thutong ena li lumellana haholo le (empa ha li khone ho paka) hore sebaka sa NiRAN se ameha ho qaleng ha motsoako oa RNA o entsoeng ke protheine.Pele ho ne ho lumeloa hore mosebetsi oa sebaka sa NiRAN ho 5 ??²-RNA capping kapa RNA ligation reaction ha e amehe ke tsena le Tšehetso ea lintlha tse ling.Ka hona, mohlala, sebaka se sebetsang sa NiRAN se nkoa se kenyelletsa Asp e bolokiloeng e le setsi se akaretsang (D252 ho Pseudomonas syringae SelO; D4271 ho HCoV-229E pp1ab; D208 ho SARS-CoV-2 nsp12) (SI Sehlomathiso, setšoantšo sa 2 ).S2) (17, 22, 33), ha catalysis ho RNA ligase e itšetlehileng ka ATP le RNA capping enzyme e etsoa ke covalent enzyme-(lysyl-N)-NMP intermediate, e kenyeletsang masala a Lys a sa Fetohang ( 41).Ntle le moo, tatellano e ikhethang e ipapisitseng le tatellano ea "coronavirus" ea NiRAN bakeng sa lipehelo tsa protheine tse bolokiloeng le boits'oaro bo khutsitseng bakeng sa li-co-substrates tsa NTP (e khetha UTP) e hanyetsana le enzyme ea NiRAN-mediated capping kapa RNA ligase-like mesebetsi.
Ho hlakile hore ho hlokahala mosebetsi o mongata o eketsehileng ho netefatsa, 'me, haeba ho netefalitsoe, ho hlakisa karolo e ka bang teng ea nsp9-UMP (nsp9-NMP) ho motsoako oa RNA o hlahisitsoeng ke protheine, o tla hokahanya tse 'maloa tse khahlisang empa (ho fihlela joale) litlaleho tse tlalehiloeng pele. .Maikutlo a ikhethileng.Mohlala, ho fihletsoe qeto ea hore pheletso ea RNA e mpe ea coronavirus e qala ka khoele ea oligo(U) (42, 43).Tlhokomeliso ena e lumellana le khopolo ea hore motsoako oa RNA e mpe-strand e qalisoa ka ho tlama mofuta oa UMPylated oa nsp9 ho poly(A) mohatla (li-triggers), tse ka khothalletsoang ke RNA ea eona e tlamang Ketsahalo le / kapa tšebelisano le protheine e 'ngoe ea RTC.Karolo ea UMP e fanoeng ke nsp9 e ka sebelisoa e le "primer" bakeng sa oligouridylation ea nsp7/8/nsp12, ho sebelisoa mohatla oa 3??²-poly(A) ho genomic RNA kapa tatellano e 'ngoe ea oligo (A) e sebetsa e le template, e tšoanang le mochine o thehiloeng bakeng sa protheine ea picornavirus VPg (44).Ho thoe'ng haeba tlhahiso e le "non-normative"????Ho qalisoa ha motsoako oa (protein-induced) negative-strand RNA ho fana ka sehokelo ho litebello, ho bonts'a hore coronavirus negative-strand RNA e na le UMP (ho e-na le UTP) qetellong ea eona (42), e nkoang e bontša hore nucleic acid Dicer e phunya pheletso ea phosphorylated ke endonuclease e sa tsejoeng ea uridine.Haeba e netefalitsoe, ts'ebetso ena ea nucleic acid hydrolytic e ka thusa ho lokolla mofuta oa oligomeric UMPylated oa nsp9 ho tloha pheletsong ea 5 ² ea mothapo o mobe o qalang.Karolo e ka bang teng ea nsp9 ho protheine priming e boetse e tsamaellana le lithuto tsa morao-rao tsa liphatsa tsa lefutso, tse bonts'itseng hore nsp9 (le nsp8) e sebelisana ka mokhoa o nyarosang le ka ho khetheha le karolo e bolokiloeng ea cis-acting RNA haufi le pheletso ea 3 ea lefutso la coronavirus.45).Ho latela tlaleho ena, litebello tsena tse fetileng joale li ka hlahlojoa hape le ho atolosoa ka lipatlisiso tse ling.
Ka bokhuts'oane, lintlha tsa rona li ile tsa etsa qeto ea ts'ebetso e ikhethileng ea tag ea enzyme e amanang le vaerase e hokahantsoeng le RdRp ho N-terminus.Ho coronavirus, ts'ebetso ena e sa tsoa sibolloa ea UMPylator/NMPylator ea NiRAN-mediated e sebelisetsoa ho itšetleha ka Mn2+ le masala a haufi le Asn le ho etsa hore ho thehoe maqhama (a matla a tlase) a phosphoramidate le N-terminal primary amine.Ka Mpro-mediated cleavage sebakeng sa nsp8|9 cleavage, sepheo sa nsp9 se ka sebelisoa bakeng sa NMPylation, e bonts'a kopano e sebetsang lipakeng tsa protease le domain ea NiRAN, e fetelang ho RdRp.Paballo ea masala a bohlokoa sebakeng sa ts'ebetso sa Nsp12 NiRAN le sepheo sa nsp9, ho kopantsoe le data e fumanoeng ho tsoa ho li-coronavirus tse peli ho kenyeletsoa SARS-CoV-2, e fana ka bopaki bo matla ba hore nsp9 NMPylation ke likarolo tsa coronavirus Conservative le tsona ke mohato oa bohlokoa phetisong ea vaerase.Lintlha tse fumanehang li re lebisa ho fihlela qeto ea hore karolo e ikhethileng ea mofuta oa NMPylated oa nsp9 ho motsoako oa RNA o hlahisitsoeng ke protheine ke boemo bo loketseng bakeng sa coronavirus le livaerase tse ling tse lulang sehlaheng, mme NiRAN e kanna ea lebisa liprotheine tse ling tse sa tsejoeng.Laola kokoana-hloko.Tšebelisano ea moamoheli.Haeba ho netefalitsoe, ho kenya letsoho ha liprotheine ho motsoako oa varal RNA ho tla eketsa tatellano ea tatellano ea libaka tsa Mpro/3CLpro le RdRp lipakeng tsa "coronavirus" e fumanoeng pele le "picornavirus-like supergroup" (9), e seng e kopantsoe ho Pisonivirites e sa tsoa thehoa. 46) sehlopheng.
Lintlha tsa rona li boetse li bontša hore mesebetsi ea enzyme ea mantlha, e khethang le e bolokang tlhokomelo e tšoailoeng thutong ena e ka sebelisoa e le sepheo sa litlhare tse thibelang kokoana-hloko.Metsoako e sitisang ho tlama (le phetoho e latelang) ea nsp9 N-terminus sebakeng se sebetsang sa NiRAN e ka ntlafatsoa hore e be litlhare tse thibelang likokoana-hloko tse sebetsang hantle le tse fapaneng, tse loketseng kalafo ea li-coronavirus tsa liphoofolo le tsa batho ho tsoa ho mefuta e fapaneng ea mafu. , ho kenyeletsoa SARS-CoV-2 le Middle East Respiratory Syndrome Coronavirus.
Tatelano ea kh'outu ea protheine ea coronavirus e hlahisitsoeng thutong ena e matlafalitsoe ke RT-PCR e sebelisa RNA e ikhethileng ho Huh-7 e tšoaelitsoeng ke HCoV-229E kapa Vero E6 e tšoaelitsoeng ke SARS-CoV-2, mme ea kenngoa ho sebelisoa mekhoa e tloaelehileng ea cloning.pMAL-c2 (New England Biological Laboratory) kapa pASK3-Ub-CHis6 (47) vector ea polelo (SI Appendix, Tables S1 le S2).Liphetoho tse le 'ngoe tsa codon li ile tsa hlahisoa ke mutagenesis e tsamaisoang ke sebaka sa PCR (48).E le ho hlahisa protheine ea fusion ea MBP, lisele tsa E. coli TB1 li ile tsa fetoloa ka mokhoa o nepahetseng oa pMAL-c2 oa plasmid construct (SI sehlomathiso, Letlapa la S1).Protheine ea fusion e ile ea hloekisoa ka amylose affinity chromatography 'me e kopantsoe le factor Xa.Kamora moo, protheine ea C-terminal His6-tagged e ile ea hloekisoa ke Ni-immobilized metal affinity chromatography (Ni-IMAC) joalo ka ha ho hlalositsoe pejana (49).Ho hlahisa protheine ea fusion ea ubiquitin, lisele tsa E. coli TB1 li sebelisitse pASK3-Ub-CHIs6 e loketseng ea ho haha plasmid (SI Appendix, Tables S1 le S2) le pCGI plasmid DNA encoding ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Phetoho (47).Protheine ea C-terminal His6-tagged coronavirus e ile ea hloekisoa joalo ka ha ho hlalositsoe pejana (50).
Teko ea self-NMPylation ea HCoV-229E nsp12-His6 e entsoe joalo ka ha ho hlalositsoe ho EAV nsp9 (16).Ka bokhutšoanyane, nsp12-His6 (0.5 µM) e na le 50 mM 4-(2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES)-KOH, pH 8.0, 5 mM dithiothreitol ( DTT), 6 mM MnCl2, 25 µM buffer NTP e boletsoeng le 0.17 µM li tsamaellana le [α32-P]NTP (3,000 Ci/mmol; Hartmann Analytic) ka 30 °C ka metsotso e 30.Litlhahlobong tse ling kaofela (tse tloaelehileng) tsa NMPylation tsa nsp12-mediated nsp9 NMPylation, maemo a karabelo a lokisoa ka tsela e latelang: nsp12-His6 (0.05 µM) le nsp9-His6 (4 µM) boteng ba 50 mM HEPES-KOH (pH 8.0) ), 5 mM DTT, 1 mM MnCl2, 25 µM e bonts'itseng NTP, le 0.17 µM e tsamaellanang [α32-P]NTP.Ka mor'a ho kenya metsotso e 10 ka 30 ° C, sampole ea karabelo e ile ea tsoakoa le SDS-PAGE sampuli buffer: 62.5 mM tris(hydroxymethyl)aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol le 0.005% bromophenol putsoa.Protheine e entsoe ka ho futhumatsa ka 90 ° C ka metsotso e 5 mme e arotsoe ke 12% SDS-PAGE.Gelisi e tsitsitsoe ebile e silafalitsoe ka tharollo ea Coomassie Brilliant Blue (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), e silafalitsoe, 'me e pepesitsoe skrineng sa litšoantšo sa phosphorescent ka lihora tse 20 (ho bona nsp12 ho tsoa ho NMPylation) kapa (boholo) 2 Lihora (ho hlahloba nsp9 NMPylation).Setšoantšo sa Leholiotsoana 9200 (GE Healthcare) se ne se sebelisetsoa ho hlahloba skrine 'me ImageJ e ne e sebelisetsoa ho hlahloba matla a pontšo.
Bakeng sa tlhahlobo ea MS, 1 µM nsp12-His6 le 10 µM nsp9 (ntle le hexahistidine tag) li ile tsa sebelisoa tlhahlobisong ea NMPylation (SI sehlomathiso, Letlapa la S1) le ho eketseha ha khatello ea 500 µM UTP le GTP li ile tsa sebelisoa.Ho ipapisitse le mohopolo oa bona le boleng ba protheine e lebelletsoeng, sistimi ea Waters ACQUITY H-Class HPLC e nang le kholomo ea MassPrep (Metsi) e ne e sebelisetsoa ho tlosa 1 ho 10 µL ea tharollo ea protheine e kentsoeng inthaneteng.Protheine e hlakotsoeng e tšeloa mohloling oa ion oa electrospray oa Synapt G2Si mass spectrometer (Metsi) ka sekhahla se latelang sa buffer A (metsi/0.05% formic acid) le buffer B (acetonitrile/0.045% formic acid), 'me mocheso oa kholomo ke 60 ° C le sekhahla sa phallo ea 0.1 mL/min: elution isocratically le 5% A bakeng sa metsotso e 2, e ntan'o ba gradient ea mola ho 95% B nakong ea metsotso e 8, 'me u boloke 95% B bakeng sa metsotso e meng e 4.
Li-ion tse ntle tse nang le boima ba 500 ho isa ho 5000 m/z lia fumanoa.Glu-fibrinopeptide B e lekantsoe metsotsoana e meng le e meng e 45 bakeng sa tokiso ea ho hoholeha ha boima ka boiketsetso.Sebelisa software ea sesebelisoa sa MassLynx e nang le katoloso ea MaxEnt1 ho hlakola sebatli se tloaelehileng kamora ho theola motheo le ho thella.
UMPylated HCoV-229E nsp9 e ile ea chekoa ka ho kenyelletsa trypsin e fetotsoeng ka tatellano (Serva) 'me ea kenngoa bosiung ba 37 °C.Kholomo e potolohang ea Chromabond C18WP (karolo ea 730522; Macherey-Nagel) e ne e sebelisetsoa ho tlosa letsoai le ho tsepamisa maikutlo ho li-peptide.Qetellong, peptide e ile ea qhibiliha ka 25 µL ea metsi, e nang le 5% acetonitrile le 0.1% formic acid.
Mehlala e ile ea hlahlojoa ke MS ka ho sebelisa Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Sistimi ea ho qetela ea nanoâ HPLC (Dionex), e nang le pheletso e tloaelehileng e kentsoeng 50 cm??75 μm C18 RP kholomo e nang le lifaha tsa makenete tse 2.4 μm (Dr. Albin Maisch High Performance LC GmbH) Hokela ho mass spectrometer inthaneteng ka mohloli oa Proxeon nanospray;kenya 6 µL ea tharollo ea tšilo ea trypsin ka har'a bophara ba 300 µm kahare ×??1 cm C18 PepMap kholomo ea pele ea mahloriso (Thermo Scientific).Ho sebelisa metsi / 0.05% formic acid e le solvent, sampole e ile ea ts'oaroa ka bo eona 'me ea tlosoa letsoai ka sekhahla sa phallo ea 6 µL/min.
Li-gradients tse latelang tsa metsi / 0.05% formic acid (solvent A) le 80% acetonitrile / 0.045% formic acid (solvent B) li ne li sebelisetsoa ho finyella karohano ea li-peptide tsa tryptic ka tekanyo ea phallo ea 300 nL / min: 4% B bakeng sa Metsotso e 5, ebe 30 A linear gradient ho 45% B ka mor'a metsotso e seng mekae, le keketseho ea mola ho 95% solvent B nakong ea metsotso e 5.Hokela kholomo ea chromatographic ho nano-emitter ea tšepe e sa hloekang (Proxeon), 'me u fafatse leseli ka ho toba ho capillary e futhumetseng ea mass spectrometer u sebelisa bokhoni ba 2,300 V. Tlhahlobo ea lipatlisiso e nang le qeto ea 60,000 ho Orbitrap mass analyzer e amahanngoa. bonyane ka lintlha tse tharo tsa lintlha tsa MS/MS, tse sa kenyelletsoeng ka mokhoa o matla bakeng sa metsotsoana ea 30, ho sebelisa linear ion trap collision induced dissociation kapa matla a phahameng a ho thulana ha matla a kopantsoeng le ho lemoha orbitrap, Qeto ke 7,500.
Nako ea poso: Aug-03-2021