Ihe mgbagwoju anya ntụgharị-Coronavirus: NMPylation dị mkpa na nhọrọ nke NiRAN-RdRp subunits na saịtị echekwara na nsp9

Peter Sarnow deziri, Ụlọ Akwụkwọ Ọgwụ Mahadum Stanford, Mahadum Stanford, California, kwadoro na Disemba 25, 2020 (nyocha na Ọktọba 25, 2020)

Anyị na-akọ na mmekọrịta dị n'etiti subunits na mmegharị nke coronaviruses-transcription complexes, nke dị mkpa maka mmụgharị na ichekwa evolushọn.Anyị nyere ihe akaebe na ngalaba NiRAN jikọtara ya na nsp12 nwere nucleoside monophosphate (NMP) transferase ọrụ na trans, wee chọpụta nsp9 (protein njikọ RNA) dị ka ebumnuche ya.NiRAN na-eme ka njikọta nke akụkụ NMP na njedebe amino nsp9 echekwara na mmeghachi omume dabere na ion Mn2+ yana ihe fọdụrụ Asn echekwara.Achọpụtara na ọrụ NiRAN na nsp9 NMPylation dị mkpa maka mmegharị coronavirus.Ihe data ahụ na-enye anyị ohere ijikọ ọrụ a nke akara nrịbama nje nje nested na nlebanya gara aga n'echiche bụ na mmalite nke njikọ RNA na klas nke nje RNA na-arụ ọrụ ma na-agbanwe agbanwe.

A na-ejikọta RNA polymerase RNA polymerase (RdRps) nke Nidovirales (Coronaviridae, Arterioviridae, na ezinụlọ 12 ndị ọzọ) na ngalaba amino-terminal (N-terminal) na protein na-abụghị structural (nsp) wepụtara na polyprotein, nke a na-akpọ NiRAN. 1ab bụ nke mejuputara isi protease viral (Mpro).Na mbụ, a na-akọ ọrụ GMPylation/UMPylation nke NiRAN-RdRp nsp nsp n'onwe ya, a na-atụkwa aro ka ọ na-ewepụta oge maka ịnyefe nucleoside monophosphate (NMP) na nje (nke a na-amaghị ugbu a) na / ma ọ bụ cell bioopolymerization Things.N'ebe a, anyị na-egosi na coronavirus (Human Coronavirus [HCoV] -229E na Ọrịa siri ike iku ume Coronavirus 2) nsp12 (NiRAN-RdRp) nwere ọrụ NMPylation nke Mn2+ dabere na nsp9, nke sitere na nsp9 site na nhazi nke Mpro-mediated nsp9 After N-terminal flanking nsps ka ewepụtara proteolytically, phosphoramidate na-ejikọta ya na amine nke mbụ (N3825) na N-terminal nke nsp9.Uridine triphosphate bụ nucleotide kacha amasị na mmeghachi omume a, mana adenosine triphosphate, guanosine triphosphate, na cytidine triphosphate bụkwa ihe mgbakwunye kwesịrị ekwesị.Ọmụmụ mgbanwe site na iji recombinant coronavirus nsp9 na nsp12 proteins na genetically engineered HCoV-229E mutant kpebiri ihe fọdụrụ dị mkpa maka nsp9 NMPylation nke NiRAN na mgbasa ozi nje na mmegharị nje na omenala cell.Data ahụ kwadoro amụma nke NiRAN na-arụ ọrụ na saịtị ma kpebie ọrụ dị mkpa nke nsp9 N3826 residues na nsp9 NMPylation na nje virus na vitro.Ihe fọdụrụ a bụ akụkụ nke usoro N-terminal NNE tripeptide echekwara wee gosi na ọ bụ naanị ihe fọdụrụ na nsp9 na homologs ya na ezinụlọ coronavirus.Ọmụmụ ihe a na-enye ntọala siri ike maka ọmụmụ ihe na-arụ ọrụ nke NMPylation nke nje ndị ọzọ a na-akwụ ụgwọ ma na-atụ aro ihe ndị nwere ike ime maka mmepe nke ọgwụ nje.

Nidovirales nti-stranded RNA nje na-ebute ọtụtụ vertebrates na invertebrates (1, 2).Iwu a gụnyere ezinụlọ 14 (3), nke a mụọla ezinụlọ Coronavirus nke ọma n'ime afọ 20 gara aga.N'oge ahụ, atọ zoonotic coronaviruse si n'anụmanụ pụta ma bute nnukwu ntiwapụ nke ọrịa iku ume na ụmụ mmadụ.Gụnyere ọrịa na-efe efe na-adịgide adịgide nke ọrịa na-efe efe siri ike kpatara.Ọrịa iku ume Coronavirus 2 (SARS-CoV-2) (4âââ7).Nidoviruses na-ekekọrịta otu genome nzukọ, yana akụkụ nke membrane-bound replication-transcription complex (RTC) ka etinyere na 5-?²-terminal ụzọ abụọ n'ụzọ atọ na isi akụkụ nhazi nke nje nje, yana ụfọdụ ngwa. .Protein, etinyere na njedebe 3'² nke atọ nke genome (1).Ewezuga otu ezinụlọ nke nje virus planarian (Monoviridae) (8), nje virus niile akwu na-etinye koodu RTC n'ime nnukwu oghere ọgụgụ abụọ mepere emepe (ORF) ORF1a na ORF1b, nke a sụgharịrị site na genomic RNA nke.ORF1a na-etinye koodu polyprotein (pp) 1a, yana ORF1a na ORF1b jikọtara pp1ab.Site na ntinye aka nke isi protease (Mpro) nke ORF1a debere, ma pp1a na pp1ab na-ahazi proteolytically n'ime ụdị protein dị iche iche na-abụghị usoro (nsps), nke a makwaara dị ka 3CLpro, n'ihi na ọ nwere homology na 3Cpro nke picornavirus ( 9).A na-eche na nsps ndị a na-agbakọta n'ime nnukwu RTC dị ike, na-eme ka njikọ nke genomic RNA (mmegharị) na nhazi nke RNA subgenomic (transcription), na-ejikwa ya na-ahazi okwu nke ORF dị na ala nke ORF1b (10? ? ?12).

Isi RTC na-agụnye RNA-based RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) na ọtụtụ enzymes nhazi RNA, nke etinyere na ORF1b na n'ime ezinụlọ coronavirus Ọ nwere nsp12-nsp16 na nsp9-nsp12 n'ime ezinụlọ Arterioviridae (lee ntụaka 10ââ 12).RdRp na HEL1 na-anọchi anya ngalaba abụọ (otu ụzọ n'ụzọ ise) nke nje akwu nnụnụ ahụ ma nwee homology n'etiti nje RNA ndị ọzọ.Ekwenyere na ọ ga-enyere aka n'ụdị isi ndị ọzọ, gụnyere ọtụtụ obere nsps ewepụtara na mpaghara carboxy-terminal (C-terminal) nke pp1a, mgbada Mpro (coronavirus nsp5 na nje arterial nsp4, n'otu n'otu).Ha nwere oke nchekwa nchekwa ezinaụlọ yana mmemme dị iche iche (elele na ntụnye aka 10ââ12).

N'oge na-adịbeghị anya, a chọtara ngalaba nwere njirimara motif usoro pụrụ iche na amino terminus (N-terminus) dị n'akụkụ RdRp na nje niile akwụ ụgwọ, mana ọ nweghị nje RNA ọzọ (16).Dabere na ọnọdụ ya na nucleotide transferase (nucleoside monophosphate [NMP] transferase), a na-akpọ ngalaba a NiRAN (Nestvirus RdRp metụtara nucleotide transferase).Njikọ abụọ-ngalaba nke NiRAN-RdRp mejupụtara nsp12 na ezinụlọ Coronaviridae na nsp9 na ezinụlọ Arterioviridae, na ndị ọzọ nestoviridae, NiRAN-RdRp na-atụ anya ka ahapụ ya dị ka nsp nọọrọ onwe ya site na polyprotein viral.Na coronavirus, ngalaba NiRAN nwere ihe fọdụrụ 1/450 ma jikọọ ya na ngalaba C-terminal RdRp site na mpaghara njikọ (16?19).Na Equine Arteritis Virus (EAV) (Arteriviridae), recombinant nsp9 na-egosi Mn2+ ion-based (self) UMPylation and GMPylation services, nke na-adabere na usoro atọ echekwara na nestovirus, AN, BN na CN ihe fọdụrụ na usoro.Ebe N na-anọchi anya NiRAN) (16).Mfe N-terminal nke ebumnuche ndị a bụ ebumnobi preAN na-adịchaghị mkpa.A na-echekwa ụfọdụ n'ime ihe ndị a na protein kinases nwere njikọ dị anya, ebe egosiri na ha na-etinye aka na nucleoside triphosphate (NTP) njikọ na ọrụ catalytic (20, 21).N'ikwekọ na nlebanya a, enwere ike ikpokọta ọtụtụ ihe fọdụrụ na saịtị na-arụ ọrụ na pseudokinase SelO sitere na sirinji Pseudomonas na SARS-CoV-2 nsp7/8/12/13 supercomplex ebipụtara na nso nso a.Ihe fọdụrụ nke Coronavirus NiRAN echekwara na microstructure eletrọn.Protein recombinant (17).A na-eche na akwụkwọ (onwe) U/GMPylation ga-emepụta ọnọdụ na-adịru nwa oge iji nyefee NMP na mkpụrụ (nke a na-amaghị ugbu a) (16), na myirịta nhazi dị n'etiti NiRAN na protein kinase (17, 19) ) Ọ bụ echiche na NiRAN na-agbanwe protein ndị ọzọ.

Ọtụtụ atụmatụ, gụnyere ihe pụrụ iche na nke pụrụ iche na-emekọ ihe n'usoro ya na nje ndị a na-akwụ ụgwọ na nkewa nke mkpụrụ ndụ ihe nketa na RdRp, na-eme NiRAN ka ọ bụrụ usoro nhazi usoro nhazi nke ọma maka nje ndị a na-akwụ ụgwọ, bụ nke dị oké mkpa maka mpụta na njirimara ha.Na mbụ, a na-akpọ ọrụ atọ nwere ike ime nke metụtara NiRAN iji hazie ntụgharị asụsụ genome/subgenomic ma ọ bụ ntụgharị/nsụgharị.Mgbe ị na-atụle data dị ụkọ na ezughị ezu dị n'oge ahụ, ọrụ ọ bụla nwere uru na ọghọm ya (16).N'ime nyocha a, anyị bu n'obi ijikọ biochemical na tụgharịa ọmụmụ mkpụrụ ndụ ihe nketa nke coronaviruses na-anọchite anya mkpụrụ ndụ abụọ ahụ, wee tinye nchoputa anyị na nzụlite evolushọn nke mmụgharị eke sitere na ezinụlọ coronavirus, iji nweta nghọta na mpaghara a dị omimi.Anyị na-akọ na nnukwu ọganihu na nghọta nke NiRAN site na njirimara nke ebumpụta ụwa na RTC, nke (n'ime echiche atọ dịnụ) na-enye aka na ọrụ nke ngalaba a n'ịmalite njikọ nke nje virus RNA.Nchọpụta a na-emepekwa ohere maka ọrụ ndị ọzọ nke NiRAN na interface nje virus.

Iji mara njirimara enzymatic nke ngalaba NiRAN metụtara nje corona virus nsp12, anyị mepụtara ụdị recombinant nke coronavirus mmadụ 229E (HCoV-229E) nsp12 na E. coli, yana mkpado His6 na C-terminus, wee jikọta ya. protein nwere [α32-P] Tinye ya na NTP n'ihu MnCl2 dị ka akọwara na Ngwa na Ụzọ.Nyocha nke ngwaahịa mmeghachi omume gosipụtara ọnụnọ nke protein akpọrọ redio na-akwaga nsp12 (106 kDa), na-egosi na coronavirus nsp12 na-eme ka nguzobe nke protein-NMP covalent, nke ọma kpụrụ ya na uridine monophosphate (UMP) (Figure 1A) na B).Ntụle ọnụọgụgụ gosiri na atụnyere nucleotides ndị ọzọ, ike mgbaama nke ntinye aka UMP mụbara site na ugboro 2 ruo 3 (Njirimara 1C).Ihe omuma a dakọtara na amụma mbufe NMP nke ngalaba NiRAN nke coronavirus (16), mana na-egosi na mmasị nucleotide nke ngalaba NiRAN nke coronavirus na nje akwara dị iche.

Ọrụ NMPylation nke HCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) ka etinyere ya na [α-32P] NTP n'ihu 6 mM MnCl2 maka nkeji 30 (lee Ngwa na Ụzọ maka nkọwa).SDS-PAGE kewapụrụ ngwaahịa mmeghachi omume ahụ wee jiri na-acha anụnụ anụnụ Coomassie mee ya.(B) A na-ahụta protein nke akpọrọ redio site na onyonyo phosphorous.A na-egosipụta ọnọdụ nsp12-His6 na protein molecular mass markers (na kilodaltons) na A na B. (C) Achọpụtara ike nke mgbaàmà redioaktivu (pụtara ± SEM) site na nyocha atọ onwe ha.* P≤0.05.Ike mgbama (pasent) metụtara UTP.

Ọ bụ ezie na egosipụtara na ọrụ enzyme metụtara NiRAN dị mkpa maka mmegharị nke EAV na SARS-CoV na omenala cell (16), ekpebibeghị ọrụ NiRAN akọwapụtara na ebumnuche ndị nwere ike.Myirịta nhazi a na-akọ na nso nso a n'etiti NiRAN na ezinụlọ nke protein nwere protein kinase-dị ka mpịachi (17, 22) kpaliri anyị ịnwale echiche na NiRAN na-eme ka NMPylation nke protein ndị ọzọ.Anyị mepụtara usoro ebumnuche nwere ike ime, gụnyere protein ndị na-abụghị usoro nke HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), nke ọ bụla nwere mkpado C-terminal His6 (SI appendix, Table S1), na tinye protein ndị a na [α32-P] uridine triphosphate ([α32-P] UTP) na ọnụnọ ma ọ bụ enweghị nsp12.Serum albumin bovine na protein fusion MBP-LacZa emepụtara na E. coli jere ozi dị ka njikwa (Njirimara 2A, ụzọ 1 ruo 7).A tụlere protein nke akpọrọ redio site na sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) na autoradiography, wee chọpụta na enwere mgbaàmà redioaktivu siri ike na mmeghachi omume nwere nsp12 na nsp9.Ọnọdụ nke mgbama ahụ kwekọrọ na mkpokọta molekụla nke nsp9, na-egosi nsp12-mediated UMPylation nke nsp9 (Njirimara 2B, egwu 7).Ọ dịghị protein ule ọzọ ahụrụ ka ọ bụ UMPylated, nke mere ka anyị kwubie na nsp9 bụ kpọmkwem mkpụrụ nke nsp12.N'ikwekọ na data onwe-NMPylation nke egosiri na eserese 1, nsp12 nwere ike ịnyefe NMP anọ na nsp9, ọ bụ ezie na arụmọrụ dị iche, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Foto).3 A na B).N'okpuru ọnọdụ ndị a na-eji na nyocha a (mechie mmeghachi omume na oge ikpughe, belata ntinye nke nsp12; ihe na usoro), enweghi ike ịchọta onwe-NMPylation nke nsp12 (tụlee Figure 2B, lane 7, and Figure 1B), nke gosipụtara na ọ dị irè (Na ọtụtụ okirikiri) UMP si na nsp12 gaa nsp9.Ọrụ nnyefe UMP chọrọ ọnụnọ Mn2+ ion, dị ka egosiri na Figure 3C, ebe ọ bụ naanị obere ọrụ UMP transferase ka a hụrụ n'ihu Mg2+, ọ nweghịkwa ọrụ n'ihu cations abụọ ọzọ a nwalere.Enwetara data yiri nke ahụ na nyocha NMPylation nwere cytidine triphosphate (CTP), guanosine triphosphate (GTP), na adenosine triphosphate (ATP) (SI appendix, Figure S1).

HCoV-229E nsp12-mediated UMPylation nke nsp9.A na-eji usoro ntinye protein (gụnyere bovine serum albumin, MBP-lacZα, na usoro HCoV-229E nsps nke akara C-terminal His6 nke ORF1a debere) iji nyochaa ọrụ UMPylation nke HCoV-229E nsp12-His6⁺-mediated protein.Tinye protein na [α-32P] UTP maka nkeji 10 na-anọghị (A) ma ọ bụ ọnụnọ (B) nke nsp12 dị ka akọwara na ihe na ụzọ.N'elu A na B, a na-egosi SDS-polyacrylamide gel nke nwere Coomassie Brilliant Blue, na n'okpuru A na B, a na-egosipụta autoradiogram kwekọrọ.A na-enye ọnọdụ nke ihe nrịbama molekul protein (na kilodaltons) n'aka ekpe.A na-egosikwa ọnọdụ nsp12-His6 (B, n'elu) na mgbaàmà redioactive hụrụ n'oge ntinye nke nsp12-His6 na nsp9-His6 (B, lane 7), nke na-egosi na [α-32P] UMP na nsp9-His6. (12.9 kDa), nke ahụghị maka protein ndị ọzọ nwalere.

HCoV-229E NiRAN na-agbasa biochemical na njirimara virological nke nsp9 NMPylation.(A na B) Ọrụ nke nucleotide co-substrate eji na mmeghachi omume.Nsp12-His6 na nsp9-His6 na-agwakọta ma tinye ya na ọnụnọ nke dị iche iche [α-32P] NTP na ọkọlọtọ NMPylation assay.(A, n'elu) Coomassie-nsp9-His6 nke SDS-PAGE kewapụrụ.(A, ala) Autoradiograph nke otu mpaghara nke gel.(B) Ọrụ ikwu (pụtara ± SEM) na ọnụnọ nke nucleotide cofactor na-ekpebi site na nnwale atọ onwe ha.* P≤0.05.(C) Ọrụ nke ion ígwè.Egosipụtara bụ ule NMPylation ọkọlọtọ n'ihu [α-32P] UTP na ion metal dị iche iche, nke ọ bụla nwere ntinye nke 1 mM.Na C, n'elu, a na-egosi Coomassie ntụpọ nsp9-His6, na na C, na ala, a na-egosi autoradiography kwekọrọ.Ogo nke protein akpọrọ (na kilodaltons) ka egosiri n'aka ekpe nke A na C. (D) Ụdị mutant nke HCoV-229E nsp12-His6 na-ebu nnọchi amino acid akọwapụtara dị na [α-32P] UTP, dịka akọwara. na Akụrụngwa na Ụzọ.Nsp9-His6 nke a na-akpọ redio nke emepụtara na mmeghachi omume NMPylation na-achọpụta site na foto phosphorylation (D, n'elu).A na-egosipụta ọrụ ndị ikwu na-atụnyere protein ụdị anụ ọhịa (wt) na D, a na-ewerekwa ala dị ka nkezi (± SEM) site na nnwale nnwere onwe atọ.Asterisks na-egosi ngbanwe nke ihe fọdụrụ na-echekwaghị.(E) Nje Virus nke dị n'ọdịbendị nke sel p1 nwetara awa 24 ka e butere ọrịa site na nyocha plaque.Egosiputa ngbanwe codon na ngalaba NiRAN nke injinia HCoV-229E mutant (ọnụọgụ fọdụrụ dabere na ọnọdụ ha na pp1ab).Ejiri nsp12_DD4823/4AA mutant saịtị RdRp na-enweghị mmeghari-eji dị ka njikwa.

Iji nweta nghọta miri emi nke saịtị na-arụ ọrụ nke NiRAN wee chọpụta ihe ndị fọdụrụ metụtara ọrụ nke nsp9-kpọmkwem NMP transferase, anyị mere mmụgharị nyocha, nke anyị na-edochi conservative residues na NiRAN AN, BN na CN motifs ( 16) Ọ bụ Ala (SI mgbakwunye, eserese S2).Na mgbakwunye, a tụlere mmetụta nke mgbanwe mgbanwe Arg-to-Lys ma ọ bụ Lys-to-Arg n'ọnọdụ abụọ.Dị ka njikwa (na-adịghị mma), ihe fọdụrụ na-adịghị ma ọ bụ nke na-adịghị echekwa na ngalaba NiRAN nke coronaviruses na nje ndị ọzọ akwu na-anọchi anya Ala. Dochie K4116A (na motif preAN), K4135A (AN), R4178A (BN), D4188A (motif). BN) na D4280A (CN) na-ebelata ma ọ bụ ọbụna wepụ nsp9 NMPylation site na nsp12, ebe protein ndị nwere mgbanwe mgbanwe (R4178K) , K4116R) na-ejigide 60% na 80% nke ọrụ ha, nke na-egosi na izu ike nke mgbochi n'akụkụ ha. ụdọ nwere mmetụta physicochemically (Foto 3D).Dochie ọtụtụ ihe ndị ọzọ echekwara E4145A, D4273A, F4281A na D4283A adịchaghị emerụ ahụ, yana nsp9 UMPylation na-ebelata obere ntakịrị.Enwetara nsonaazụ yiri nke ahụ na nsp9 NMPylation mmeghachi omume metụtara NTP ndị ọzọ (N'ihe atụ 3D na SI appendix, Figure S3), na-akwado na mmetụta a hụrụ na kpọmkwem amino acid nnọchi na-adabereghị na ụdị nucleotide co-substrate eji.Ọzọ, anyị nwalere mmetụta nwere ike nke nsp12 ndị a nọchiri anya n'ịmegharị nke coronaviruses na omenala cell.Iji mezuo nke a, anyị jiri ndebiri DNA (cDNA) dabara adaba dabara adaba na nje virus vaccinia recombinant (23, 24) iji degharịa mkpụrụ ndụ 5-7.Titration nke ụmụ nje na-efe efe nke emepụtara na mkpụrụ ndụ ndị a gosiri na ọtụtụ mkpụrụ ndụ HCoV-229E NiRAN agaghị ekwe omume (Njirimara 3E).Otu ìgwè nke mutant viral na-enweghị ike ime ihe na-agụnye ihe ndị ọzọ egosiri iji kpochapụ ma ọ bụ belata ọrụ NMP transferase na vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), ma enwere ụzọ abụọ ọzọ (K4116R, E48045A) . % edobere?Ọrụ NMPylation in vitro ha na-egosi na a na-etinye ihe mgbochi ndị ọzọ.N'otu aka ahụ, mmụgharị abụọ ọzọ (R4178K, F4281A) nke butere mbelata agafeghị oke na ọrụ NiRAN's in vitro NMPylation rụpụtara nje dị ndụ, agbanyeghị, nje ndị a belatara nke ukwuu site na mmegharị.N'ikwekọ na data ọrụ in vitro nke egosiri na eserese 3D, na-edochi ihe fọdụrụ anọ ndị ọzọ na-echekwaghị na coronavirus na/ma ọ bụ nje ndị ọzọ akwụghị ụgwọ (K4113A, D4180A, D4197A, D4273A) (8, 16) mepụtara nje ndị nwere ike ime nwa, n'agbanyeghị na ha nwere. Titer dị ntakịrị belatara ma e jiri ya tụnyere nje ụdị anụ ọhịa (Foto 3E).

Iji mụọ ma NiRAN-mediated NMP transferase ọrụ dabere na ngalaba RdRp na-arụ ọrụ, ihe fọdụrụnụ Asp abụọ echekwara na-etinye aka na nhazi nke ion metal divalent (11) na RdRp motif C bụ nke Ala dochie ya. ya nsp9 NMPylation ọrụ, na-egosi na nsp12-mediated in vitro nsp9 NMPylation ọrụ anaghị achọ ọrụ polymerase (SI Mgbakwunye, Figure S4).

Ka emechara nsp9-kpọmkwem NMP transferase ọrụ maka nsp12, anyị gbalịrị ịkọwapụta NMP-nsp9 adduct site na mass spectrometry (MS).Ụdị ụdị protein zuru oke nke recombinant HCoV-229E nsp9 gosipụtara ọnụ ọgụgụ kasị elu na 12,045 Da (Onyonyo 4A).Mgbakwunye nke nsp12 agbanweghị àgwà nke nsp9, na-egosi na nsp12 na nsp9 agaghị emepụta ihe mgbagwoju anya n'okpuru ọnọdụ ejiri (denaturation) (Figure 4A).N'ihu UTP na GTP, nleba anya nke mmeghachi omume nke nwere nsp9 na nsp12 gosipụtara n'otu n'otu gosipụtara na protein protein nke UTP kpaliri 306 Da, na protein protein nke GTP kpaliri 345 Da, na-egosi na nsp9 molecule ọ bụla na-ejikọta UMP ma ọ bụ GMP. (Foto nke 4) C na D).A na-eche na ike achọrọ maka nsp9 NMPylation nke NiRAN na-esite na NTP hydrolysis na ntọhapụ pyrophosphate.Ọ bụ ezie na ejiri nsp9 (ntụgharị) okpukpu iri karịa nsp12 (enzyme) mee ihe na mmeghachi omume a, ọ fọrọ nke nta ka ọ bụrụ NMPylation nke nsp9 zuru ezu, na-egosi na mmekọrịta dị n'etiti nsp12 na nsp9 dị mkpụmkpụ, na nsp12 nwere ike NMPylate ọzọ nsp9. in vitro molekul.

Otu NMPylation nke nsp9 n'ihu nsp12 na UTP ma ọ bụ GTP.Egosipụtara bụ ụdịdị ụdịdị dị iche iche nke protein protein zuru oke nke HCoV-229E nsp9 (SI appendix, Tebụl S1) (AD).(A) nsp9 naanị, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 n'ihu UTP, (D) nsp9 + nsp12-His6 n'ihu GTP.

Iji chọpụta ihe fọdụrụ nsp9 UMPylated site nsp12, nsp9-UMP ka ejiri trypsin kechie.Ekewapụrụ peptides ndị a sitere na nano-high performance liquid chromatography (HPLC) wee nyochaa site na tandem mass spectrometry (MS/MS) n'ịntanetị.Nyocha data site na iji ngwugwu ngwanrọ Byonic (Protein Metrics) gosiri UMPylation nke amino acid N-terminal.Eji aka gosi nke a.Ụdị ihe dị iche iche nke tandem nke peptide precursor [UMP] NNEIMPGK (SI appendix, Figure S5A) kpughere mpempe akwụkwọ na 421 m/z, na-egosi na UMP na-ejikọta ya na 1 nke nsp9.

Na N-terminus nke nsp9, a na-echekwa Asn n'etiti ndị òtù Orthocoronavirinae (SI appendix, Figure S6).Ọ bụ ezie na anyị kwenyere na N-terminal primary amine nitrogen bụ ihe kacha anabata maka UMP, anyị kpebiri inwetakwu ihe akaebe nke njikọ NMP na N-terminal.N'ihi nke a, ihe na-abụghị NMPylated na NMPylated N-terminal peptide nsp9 sachara nke HPLC nwetara na ọnụnọ nke acetone na sodium cyanoborohydride.N'okpuru ọnọdụ ndị a, ọ bụ naanị amines praịmarị n'efu nwere ike iji propyl (25) gbanwee.N-terminal nsp9-enwetara peptide nwere usoro NNEIMPGK nwere amines abụọ bụ isi, otu na N-terminus nke Asn na nke ọzọ n'akụkụ agbụ Lys na C-terminus.Ya mere, enwere ike iwebata otu propyl na nsọtụ abụọ ahụ.Chromatogram ion ewepụtara nke peptides na-abụghị NMPylated ka egosiri na mgbakwunye SI, eserese S5B.Dị ka a tụrụ anya, N-terminal na C-terminal (mono) propylated (SI appendix, Figure S5B, elu lane) na peptides dipropylated (SI appendix, Figure S5B, ala ala).Ụkpụrụ a na-agbanwe site na iji NMPylated N-terminal peptide nke nsp9.N'okwu a, ọ bụ naanị C-terminal propylated peptides ka a ga-amata, mana N-terminal propylated peptides na dipropylated peptides adịghị amata (SI Appendix, Figure S5C), na-egosi na ebufela UMP na N-terminal primary amine Iji gbochie nke a. otu site n'ime mgbanwe.

Na-esote, anyị na-eji Ala ma ọ bụ Ser dochie anya ma ọ bụ hichapụ ihe fọdụrụ echekwara na N-terminus nke nsp9 iji kọwaa ihe mgbochi ndị akọwapụtara.Dabere na data MS anyị na-egosi na NiRAN na-emepụta nsp9-NMP ntinye ya na amine bụ isi nke N-terminal residue nke nsp9, anyị chere na nsp9 NMPylation chọrọ protease master viral (Mpro, nsp5) ịhapụ nsp9 N-terminal site na polyproteins nke mbụ ya.Iji nwalee echiche a, anyị mepụtara protein precursor nsp7-11 nwere nsp9 na E. coli ma mee nyocha NMPylation ọkọlọtọ n'ihu [α-32P] UTP (ihe na ụzọ).Dị ka e gosiri na foto 5A (ụzọ 3), ihe nsp7-11 precursor adịghị ebipụ ya na nsp12.N'ụzọ dị iche, ọ bụrụ nsp7-11 na-agbaji site recombinant nsp5 ịhapụ nsp9 (na nsps ndị ọzọ) site na precursor, a na-achọpụta protein nke redio nke na-akwaga nsp9, na-akwado nkwubi okwu anyị na NiRAN na N-Nhọrọ nke covalent nsp9-NMP na-akwado. .Amine isi njedebe nke N-terminal Asn (ọnọdụ 3825 na pp1a/pp1ab).A na-akwadokwa nkwubi okwu a site na nnwale site na iji nsp9 construct, nke nwere otu ma ọ bụ abụọ ọzọ fọdụrụnụ na N-terminus.N'okwu abụọ ahụ, UMPylation nke nsp9 na-eme ka NiRAN kagburu (Ngbakwunye SI, Figure S7).Ọzọ, anyị mepụtara protein nke nwere otu ma ọ bụ abụọ Asn residues ehichapụ na 3825-NNEIMPK-3832 peptide usoro na N-terminal nke nsp9.N'okwu abụọ ahụ, nsp9 UMPylation kwụsịrị kpamkpam (Njirimara 5B), na-enye ihe akaebe ọzọ na ezigbo nsp9 N-terminus na-arụ ọrụ dị ka onye na-anabata NMP.

Nhazi proteolytic nke nsp9 na ọrụ nke N-terminal residues na nsp12-mediated UMPylation.(A) nsp9 UMPylation chọrọ nsp9 N-terminal efu.Nsp7-11-His6 bu ụzọ tinye ya na 30 Celsius C na nchekwa nchọpụta NMPylation nwere UTP na ọnụnọ ma ọ bụ enweghị recombinant Mpro (nsp5-His6).Mgbe awa 3 gachara, malite nyocha NMPylation site n'ịgbakwunye nsp12-His6 dị ka akọwara na ngwa na ụzọ.A na-eji mmeghachi omume nwere nsp5-His6 (ụzọ 1) na nsp9-His6 (ụzọ 2) dị ka njikwa.Mgbe nkeji 10 gasịrị, a kwụsịrị mmeghachi omume ahụ ma kewapụ ngwakọta mmeghachi omume site na SDS-PAGE.Ejiri Coomassie Brilliant Blue (A, n'elu) metọọ protein ahụ.Nsp7-11-His6 precursor na ngwaahịa ahazi nke sitere na nsp5-His6 mgbasa ozi na-egosi n'aka nri.Biko mara (n'ihi obere nha) na nsp7 na nsp11-His6 adịghị achọpụta na gel a, na mmeghachi omume na-agbakwunyere nsp5-His6 (ụzọ 1 na 4; ọnọdụ nke nsp5-His6 na-egosi site na gburugburu siri ike) ma ọ bụ nsp9-His6 (Lane 2) nwere obere ego nke MBP (gosipụtara site na okirikiri mepere emepe) dị ka adịghị ọcha fọdụrụnụ n'ihi na e gosipụtara ha dị ka protein fusion MBP (SI appendix, Table S1).(B) Nsp9-His6 dị iche iche enweghị otu ma ọ bụ abụọ N-terminal Asn residues (ọnụọgụ fọdụrụ dị ka ọnọdụ dị na pp1a / pp1ab) ma sachaa ma tinye ya na nsp12-His6 na [α-32P] UTP.B, SDS-PAGE nwere ntụpọ na Coomassie ka egosiri n'elu, B, a na-egosipụta autoradiograph kwekọrọ na ala.A na-egosi ọnọdụ nke nrịbama arọ molekụla (na kilodaltons) n'aka ekpe.(C) HCoV-229E nsp9-His6 N-terminal echekwabara ka ejiri Ala ma ọ bụ Ser dochie ya, ma otu protein ahụ ka ejiri mee ihe na nsp12-His6 mgbasa ozi UMPylation.SDS-PAGE kewapụrụ ngwaahịa mmeghachi omume ahụ wee jiri Coomassie Brilliant Blue mee ya (C, n'elu), yana nsp9-His6 nke akpọrọ redio bụ nke phosphorescence imaging (C, etiti).N'iji protein ụdị anụ ọhịa (wt) dị ka ntụaka (nke edobere na 100%), a gbakọrọ ọrụ NMPylation (pụtara ± SEM) site na nnwale nnwere onwe atọ.(D) Nje Virus nke dị n'ọdịbendị sel p1 nke mkpụrụ ndụ Huh-7 nwere ụdị anụ ọhịa HCoV-229E nwere mkpụrụ ndụ anụ ọhịa HCoV-229E, yana mutant na-ebu nnọchi amino acid ahapụtara na nsp9 bụ ihe nyocha plaque kpebiri.Ejiri RdRp motif C okpukpu abụọ DD4823/4AA na-enweghị mmeghari ka ejiri mee njikwa na-adịghị mma.

A na-echekwa N-terminus nke nsp9 (karịsịa ọkwa 1, 2, 3, na 6) n'etiti ndị otu ezinụlọ Orthocoronavirinae (Agbakwunye SI, Figure S6).Iji mụọ ọrụ ga-ekwe omume nke ihe ndị a na nsp12-mediated nsp9 NMPylation, abụọ Asn residues na N-terminus nke nsp9 dochie ya na Ala ma ọ bụ Ser (naanị ma ọ bụ na ngwakọta).Tụnyere ụdị nsp9 anụ ọhịa, dochie N3825 na Ala ma ọ bụ Ser rụpụtara ihe karịrị mbelata okpukpu abụọ na nsp12-mediated UMPylation (Figure 5C).N'ikwekọ na nkwubi okwu anyị na NMPylation na-eme na N-terminal primary amine kama n'akụkụ akụkụ nke N-terminal residue, anyị hụrụ NMPylation dị ịrịba ama na ndochi nke N3825A na N3825S.N'ụzọ na-akpali mmasị, ọ bụrụ na Ala ma ọ bụ Ser dochie Asn nke abụọ, nsp9 UMPylation na-ebelata ike (karịa ugboro 10), ebe nnọchi Ala na ọnọdụ 3, 4, na 6 nwere naanị mmetụta na-agafeghị oke na nsp9 UMPylation (Nyocha 2). ) .5C).Enwetara nsonaazụ yiri nke ahụ site na iji ATP, CTP ma ọ bụ GTP (Agbakwunye SI, Figure S8).Na mkpokọta, data ndị a na-egosi isi ọrụ nke N2826 (ọnọdụ 2 na nsp9) na nsp9 NMPylation.

Iji nwetakwuo ihe akaebe nke njikọ ọrụ dị n'etiti N-terminus nke nsp9 na NMPylation, anyị mere nhazi ọtụtụ usoro (MSA) nke usoro nsp9 nke ezinụlọ Coronavirus (dị iche n'etiti 104 na 113 residues) (SI Mgbakwunye, Foto) S6).Na mkpokọta, n'ime ụdị 47 (mara na ntinye) nke ụdị 5 nke ezinụlọ Orthocoronavirinae nke na-efe efe dị iche iche mammals, nnụnụ na ndị na-akpụ akpụ, naanị 8 residues n'ozuzu ka achọtara na-agbanwe agbanwe.A hụrụ mgbanwe kachasị ukwuu, gụnyere nhichapụ na ntinye, na okirikiri n'etiti ihe nhazi nke abụọ nke nsp9, dị ka ihe ọmụmụ nhazi gara aga kpebiri (26 ??28).Achọpụtara ihe fọdụrụ na-adịghị agbanwe agbanwe na β strand na α helix nke akụkụ C-terminal nke nsp9.Ihe fọdụrụ atọ na-agbanwe agbanwe mebere NNE motif nke N njedebe nke nsp9.A na-ekpughe na Asn nke abụọ nke motif a bụ naanị ihe fọdụrụ na-adịghị agbanwe agbanwe, nke a na-ekerịtakwa nsp9 nke hypothetical nsp9 nke frog coronavirus dị anya, ma na-anọchite anya ụdị Microhyla letovirus 1 na ezinụlọ Letovirinae nke Alphaletovirus.Enwere ike ịhazi nchekwa nke ihe ndị fọdụrụ na nsp9 ihe nhazi nke abụọ site na nleba anya nhazi iji nọgide na-enwe mpịachi ma ọ bụ njirimara njide RNA mara.Otú ọ dị, echiche a adịghị ka ọ na-emetụta nchekwa nke NNE, na tupu ọmụmụ ihe a, ọdịdị nke ihe mgbochi na-egbochi mgbanwe nke usoro tripeptide bụ kpamkpam.

Iji chọpụta mkpa nsp9-NMPylation na nchekwa NNE dị na mmegharị coronavirus, anyị mepụtara mutants HCoV-229E, nke na-ebu otu ma ọ bụ okpukpu abụọ nke nsp9 N-terminal residues, na-egosi na nsp9 NMPylation na-emerụ ahụ na vitro.Tupu anyị amalite, anyị na-agbalị ịza ajụjụ a ma ndị a nnọchi (nso nsp8|9 cleavage saịtị) na-emetụta na proteolytic nhazi nke C-terminal pp1a mpaghara.Otu nsp7-11 polyprotein na-ewu nke nwere ngbanwe kwekọrọ na N-terminus nke nsp9 ka emepụtara na E. coli wee bechapụ ya na recombinant Mpro.Mkpochapu proteolytic nke saịtị anọ ahụ (gụnyere saịtị nsp9 flanking) adịghị emetụta nke ukwuu site na ntinye ọ bụla ewepụtara (SI appendix, Figure S9), ewezuga mgbanwe nhazi na protein ndị a na-egbochi Mpro-mediated nsp8 | 9 cleavage (Ma ọ bụ ihe ọzọ) webụsaịtị.

Ejiri mkpụrụ ndụ genome-ogologo HCoV-229E RNA gbanwee mkpụrụ ndụ Huh-7, na-edobe nnọchi Ala ma ọ bụ Ser na NNE tripeptides echekwara (N3825, N3826, na E3827) na njedebe nsp9 N, na-egosi na ọtụtụ n'ime mgbanwe ndị a na-egbu egbu.Anyị nwere ike ịzọpụta nje a site na dochie Ser ma ọ bụ Ala nke N-terminal Asn (N2835A ma ọ bụ N2835S), mana ọ naghị agbake nje ahụ na mgbanwe ndị ọzọ na otu abụọ na usoro NNE (N3826A, N3826S, NN3825/6AA, NN3825/6SS), E3827A) (Ọgụgụ 5D).

Nsonaazụ ndị a na-egosi na mmechi nke coronaviruses na omenala anụ ahụ bụ amachibidoro (otu ma ọ bụ ihe yiri ya), na-amachi mmụgharị sitere n'okike nke saịtị nsp9 NMPylation n'ime ahụ, yana ịkwado ọrụ dị mkpa nke nzaghachi a na usoro ndụ nke coronaviruses.

N'ime nnwale ikpeazụ nke ikpeazụ, anyị mepụtara C-terminal His6 akpọrọ SARS-CoV-2 nsp12 na nsp9, yana ụdị mutant abụọ nke nsp12 na E. coli.Ihe fọdụrụ na saịtị ndị nọ na ngalaba NiRAN na RdRp bụ otu n'ime ha Jiri Ala kama (ọgụgụ 6A na SI mgbakwunye, Tebụl S2).K4465 na SARS-CoV-2 nsp12 kwekọrọ na K4135 na HCoV-229E (SI Mgbakwunye, Figure S2), nke gosipụtara na achọrọ maka ọrụ NiRAN na mmegharị HCoV-229E (Onyonyo 3D na E).Ihe fọdụrụ a dabara na nje akwara akwara EAV nsp9 K94 residue, nke egosiri na mbụ dị mkpa maka NiRAN self-UMPylation/self-GMPylation (16).Dị ka egosiri na eserese 6B, SARS-CoV-2 nsp12 nwere ọrụ nnyefe UMP site na iji nsp9 dị ka mkpụrụ, ebe nsp12_K4465A mutant saịtị na-adịghị arụ ọrụ.Ngbanwe okpukpu abụọ n'ime usoro njirimara SDD nke RdRp motif C anaghị emetụta ọrụ nnyefe UMP (Ọnụ ọgụgụ 6B), na-egosi na ọrụ RdRp enweghị mmetụta kpọmkwem na nsp9 UMPylation.Enwetara data yiri nke ahụ site na iji CTP, GTP na ATP (Agbakwunye SI, Figure S10).Na nchịkọta, data ndị a na-egosi na nsp9 NMPylation na-eme NiRAN nwere ọrụ nchekwa na coronaviruses na-anọchite anya ụdị dị iche iche nke ezinụlọ orthocoronavirus.

SARS-CoV-2 nsp12-mediated NMPylation nke nsp9.(A) Coomassie nwere ntụpọ SDS-polyacrylamide gel na-egosi protein recombinant ejiri na ule NMPylation.Dịka njikwa, ejiri protein mutant nwere nnọchi saịtị na-arụ ọrụ na ngalaba NiRAN (K4465A) na ngalaba RdRp (DD5152/3AA) nke SARS-CoV-2 nsp12.Nọmba fọdụrụ dabere na ọnọdụ na pp1ab.(B) Autoradiograph nke nchọpụta UMPylation site na iji nsp9-His6 na [α-32P] UTP dị ka mkpụrụ nke nsp12-His6 (ụdị ọhịa [wt] na mutant).A na-egosi oke molekụla (na kilodaltons) nke protein akpọrọ n'aka ekpe.

A na-echekwa ngalaba NiRAN na Nidovirales (16), na-egosi na ha na-eme ka mmeghachi omume enzymatic dị mkpa maka mmegharị Nidovirus.N'ime ọmụmụ ihe a, anyị nwere ike igosi na ngalaba NiRAN nke coronavirus na-ebufe NMP (nke sitere na NTP) gaa nsp9, protein dị omimi RNA na-ejikọta na nje virus (26 ?? 29), iji chọpụta ya dị ka ebumnobi ebumpụta ụwa na Onye mmekọ nke coronavirus RTC.

Ngalaba NiRAN na-ekerịta motifs usoro atọ (AN, BN, na CN), nke nwere ọnụ ọgụgụ dị nta nke ihe fọdụrụ nke echekwara na ezinụlọ niile na monophyletic mana dị iche iche Nidovirales usoro (8, 16).Nnyocha e mere n'oge na-adịbeghị anya egosiwo na ha na-ejikọta ya na ezinụlọ na-enweghị atụ nke protein kinase-dị ka protein, nke a na-akpọbu ezinụlọ SelO (17, 19, 22, 30, 31).Protein ndị metụtara SelO nwere mpịaji kinase, mana enweghị ọtụtụ saịtị na-arụ ọrụ echekwara na kinases oge gboo (22, 32).Dabere na ntụgharị ntụgharị nke mkpụrụ ndụ ATP na-ejikọta na saịtị na-arụ ọrụ ma kwụsie ike site na mmekọrịta dị iche iche, SelO bụ hypothesized wee kwenye na ọ ga-ebufe AMP (kama phosphate) na mkpụrụ protein (22), ebe protein SelO ọzọ yiri protein YdiU nwere. N'oge na-adịbeghị anya, egosiri na ọ ga-eme ka njikọ nke UMP na Tyr na ihe fọdụrụ ya dị iche iche nke protein protein dị iche iche (33).

Iji gosi na ịgbasa amụma nke fọdụrụ na saịtị putative na-arụ ọrụ nke ngalaba coronavirus NiRAN, anyị na-eji biochemical na ntụgharị usoro mkpụrụ ndụ ihe nketa iji mee nyocha mgbanwe na coronavirus nsp12 (Ọnyocha 3D na E na SI mgbakwunye, Figure S3 na tebụl) S1â S4).Ihe omuma a gosiputara na ngbanwe nke HCoV-229E K4135, R4178 na D4280 na Ala na-ewepu in vitro NMP transferase ọrụ na nje mmeghari na omenala cell (Onyonyo 3D na E na SI appendices, Figure S3), na-akwado ọnụnọ ha na NTP γ-phosphate. (K4135, R4178) na nhazi nke ion metal saịtị na-arụ ọrụ (D4280).Mgbanwe E4145A nke Glu echekwara na nso nso nke nje akwu nnụnụ buru amụma na ọ ga-eme ka ọnọdụ K4135 (17) kwụsie ike ka egosipụtara iji wepụ nje virus, ma ọ bụ ihe ijuanya na a na-edobe ọrụ ahụ na in vitro NMPylation assay (Onyonyo 3D na E na SI mgbakwunye, onyonyo S3 na Tebụlụ S1–S4).Emere nlebanya yiri nke ahụ mgbe ewebatara ngbanwe kwekọrọ na YdiU homolog nke Salmonella typhimurium (E130A) (33).Ejikọtara ọnụ, data ndị a na-akwado ọrụ nhazi nke ihe fọdụrụ echekwara karia ọrụ catalytic.

Dochie ihe fọdụrụ nke Phe (F4281A) echekwara n'ime oke nestovirus na ngalaba HCoV-229E NiRAN (8) butere mbelata ọrụ NMPylation na vitro yana mbelata dị ukwuu na mmegharị nje na omenala cell (Ọnyocha 3D, E na SI) ihe odide ntụkwasị, foto S3).Data ahụ dabara na ọrụ nhazi dị mkpa nke ihe fọdụrụ a, dị ka DFG motif Phe residue homologous nke egosiri na mbụ.N'ime protein kinase nke oge gboo, ọ bụ akụkụ nke Mg2+ na-ejikọ akaghị ma na-enyere aka ịgbakọta na ịhazi ọkpụkpụ azụ???Achọrọ maka ọrụ catalytic dị irè (32, 34).Dochie Ala na Arg maka ihe fọdụrụ na K4116 (na preAN motif), n'otu n'otu, kpochapụ nje virus na, dị ka a tụrụ anya ya, nwere mmetụta dị iche iche na ọrụ NMP transferase na vitro, dabere na amino acid n'akụkụ agbụ nke ewepụtara (Nyocha 3D na E na SI ngwa ngwa. , Foto S3).Ihe omuma a na-arụ ọrụ kwekọrọ na ozi nhazi, na-egosi na ihe fọdụrụ a emeela ka mmekọrịta ya na ATP phosphate (17).Na ngalaba NiRAN nke ezinụlọ nje ndị ọzọ akwụ ụgwọ, ọnọdụ nke HCoV-229E pp1a/pp1ab K4116 bụ Lys, Arg ma ọ bụ Ya (8), na-egosi na mmachi arụ ọrụ nke ihe fọdụrụ a kapịrị ọnụ adịla jụụ.Ndochi nke D4188A na D4283A na-ewepụ ma ọ bụ na-ebelata ọrụ enzyme ma wepụ nje virus (Fig 3).A na-echekwa ihe abụọ ndị a n'ọtụtụ (ma ọ bụghị ihe niile) nje a na-akwụ ụgwọ (8), na-egosi ọrụ dị mkpa akọwapụtara nke ezinụlọ mana ikekwe na-abụghị ọrụ catalytic.Mgbanwe Ala nke ọtụtụ ndị ọzọ fọdụrụ Lys na Asp (K4113A, D4180A, D4197A na D4273A) ndị na-adịghị echekwabara na Coronaviridae ma ọ bụ ndị ọzọ Nestioviridae ezinụlọ (8) ka ejiri dị ka njikwa.Dị ka a tụrụ anya, ngbanwe ndị a bụ nke a na-anabata nke ọma, na-enwe ntakịrị mbelata ọrụ enzyme na mmegharị nje n'ọnọdụ ụfọdụ (Foto 3 na SI appendix, Figure S3).N'ozuzu, coronavirus mutagenesis data kwekọrọ na GMP nke onwe ya na ntụgharị data mkpụrụ ndụ ihe nketa nke EAV NiRAN-RdRp (16), nke EAV nsp9 (coronavirus nsp12 ortholog) fọdụrụnụ K94 (dakọtara na HCoV-229E K4135) ọrụ dị mkpa). R124 (nke kwekọrọ na R4178), D132 (dabere na D4188), D165 (dabere na D4280), F166 (dabere na F4281).Na mgbakwunye, data HCoV-229E mutagenesis kwekọrọ na gbasaa site na SARS-CoV reverse genetics data akọwara na mbụ (16), dị ka nke a hụrụ maka CN motif Phe-to-Ala mutant SARS-CoV_nsp12. phenotype kọwara -F219A na HCoV-229E_F4281A (Njirimara 3 D na E na SI mgbakwunye, eserese S3 na tebụl S1-S4).

E jiri ya tụnyere EAV orthologs (16), nke nwere mmasị doro anya maka UTP na GTP (na mmeghachi omume NMPylation nke onwe), ọmụmụ anyị na-egosi na ngalaba coronavirus NiRAN (nke HCoV-229E na SARS-CoV-2 nọchiri anya ya) nwere ike ịrụ ọrụ nke ọma. bufere NMP anọ niile, n'agbanyeghị na enwere ntakịrị mmasị maka UMP (Figure 1 na 3).Nkọwapụta dịtụ ala nke otu NTP ngalaba-substrate kwekọrọ na usoro SARS-CoV-2 nsp7/8/12/13 a kọrọ na nso nso a, nke ADP-Mg2+ jikọtara na saịtị NiRAN na-arụ ọrụ, mana ọ bụghị na akụkụ adenine. nke guzobe nke kpọmkwem mmekọrịta (17).N'ime ọmụmụ ihe anyị, ụdị nucleotide ejiri mee ihe na mmeghachi omume NMPylation enweghị mmetụta dị iche iche na ọrụ nke protein mutant (SI Appendix, Figure S3), na-egosi na ọ dịghị nke ọ bụla n'ime ihe ndị a nwere njikọ chiri anya na njikọ nke otu nucleobase.A ka ga-amụ ihe ndabere nhazi na ihe nwere ike ime ihe dị iche iche nke NTP na-ahọrọ n'ime ngalaba NiRAN nke coronaviruses na nje akwara;ha nwere ike ịbụ eziokwu ma ọ bụ nwere ike ịbụ n'ihi njedebe nke ọmụmụ ha.Ka ọ dị ugbu a, enweghị ike iwepụ na ọrụ NMPylator nwere ike nke ngalaba nje akwara akwara NiRAN (ma e jiri ya tụnyere ọrụ NMPylation nke a na-amabu na mbụ) nwere mmasị dị iche iche, na-eburu n'uche na myirịta dị n'etiti akwara na coronavirus. Ngalaba NiRAN dị na njedebe ya.Tụlee usoro dabere na (16).E jiri ya tụnyere pseudokinase SelO, nke na-eji Mg2+ dị ka onye na-arụkọ ọrụ ọnụ, ọrụ nke coronavirus na arterial virus NiRAN dabeere na Mn2+ (16) (Ọnọdụ 3C na SI mgbakwunye, Figure S1).Ndabere Mn2 + na mmasị doro anya maka UTP bụ ihe pụrụ iche nke protein NMPylators, na ekwenyela na nso nso a na protein YdiU nke Salmonella typhimurium, nke na-eme ka Mn2 + na-adabere na protein chaperone UMPylation iji chebe mkpụrụ ndụ site na nrụgide cell ATP ọdọ mmiri. 33).

Myirịta nhazi nke akọwara n'oge na-adịbeghị anya n'etiti ngalaba coronavirus NiRAN na protein protein kinases (17, 19) na-enye nkwado ọzọ maka ikike NiRAN ijikọ njikọ NMP na protein ndị ọzọ anyị kọrọ na ọmụmụ a.Anyị lekwasịrị anya na nchọ anyị maka ebumnuche NiRAN nwere ike ime na protein ndị HCoV-229E ORF1a kpuchiri, nke amaara na ọ na-enyere aka n'ụzọ ozugbo ma ọ bụ na-edobe RTC's ORF1b-encoded oyiri (12, 35).Nnwale anyị na-enye ihe akaebe zuru oke maka NMPylation dị irè yana kpọmkwem nke nsp9 (Nyocha 2).Ọ bụrụ na a na-eji protein e lekwasịrị anya na ngafe molar nke dị 8 ruo 10 ugboro dị elu karịa nke enzyme (nsp12), a kwadoro na nsp9 bụ kpamkpam (mono) NMPized (Nyocha 4).Anyị kwubiri na mmekọrịta dị n'etiti nsp12 na nsp9 adịghị adịte aka na ọ gaghị etolite mgbagwoju anya na nsp9 (na enweghị ndị ọzọ RTC subunits).A kwadoro nkwubi okwu a site na ọmụmụ mmekọrịta protein na SARS-CoV proteome (35).Nyocha MS mere ka amine isi nke N-terminal residue nke nsp9 dị ka saịtị NMPylation (SI appendix, Figure S5).Nhazi nke njikọ phosphoramidate na N-terminal amino group na-ekewa ọrụ NMPylation nke NiRAN na-arụ ọrụ site na Pseudomonas syringae SelO-mediated AMPylation reaction, nke na-eme ka e guzobe O-njikọ AMP na Ser, Thr, ma ọ bụ Tyr residues Peptide adduct ( 22), na S. typhimurium YdiU ụdị O-njikọ (ya na Tyr) na N-njikọ (na Ya) peptide-UMP adducts.Ozi pere mpe dị na ezinụlọ SelO nke protein na-egosi na ndị otu ezinụlọ protein a buru ibu dị iche iche na nhazi nke peptide-NMP adducts.Nke a bụ nlebanya na-adọrọ mmasị nke kwesịrị ịmụkwu ihe.

Ihe data enwetara n'ọmụmụ ihe a mere ka anyị chepụta na NMPylation nke nsp9 chọrọ N-terminus n'efu.N'ihe gbasara mmụgharị nje virus, a ga-enye nke a site na mgbawa proteolytic nke saịtị nhazi nsp8|nsp9 dị na polyprotein pp1a nke Mpro na pp1ab na-agbaziri.N'ọtụtụ coronaviruses, ọdịiche dị n'etiti saịtị a kpọmkwem (VKLQ | NNEI na HCoV-229E) na saịtị ndị ọzọ coronavirus Mpro cleavage bụ Asn (kama karịa obere ihe fọdụrụ, dị ka Ala, Ser ma ọ bụ Gly) bi P1â???Ebe (36).Ihe omuma nke peptide cleavage enwetara na omumu ihe omumu gosiputara na nsp8|nsp9 nke nsp9 na-adighi nma karia nke saịtị ndi ozo, na-egosi na 1) saịtị a nwere ike inwe oru nhazi na nhazi nhazi oge nke C-terminal. pp1a mpaghara, ma ọ bụ 2) ọrụ nke nsp9 N-terminus pụrụ iche echekwara na mmegharị nje (37).Ihe omuma anyi (Njirimara 5A) gosiri na recombinant ụdị nsp9 na-ebu ezigbo N-terminal usoro bụ NMPized nke ọma site nsp12.E wepụrụ usoro flanking N-terminal site na ihe Xa (nsp9-His6; SI appendix, Table S1) ma ọ bụ Mpro-mediated cleavage (nsp7-11-His6; Figure 5A na SI appendix, Table S1).N'ụzọ dị mkpa, nsp9-nwere precursor nsp7-11-His6 nke na-adịghị ahụkebe gosipụtara nguzogide NMPylation nke nsp12, nke kwekọrọ na data anyị, na-egosi na nsp9-NMP na-emepụta site na N-terminal primary amine (SI appendix, Figure S5) .Iji nweta nghọta miri emi nke nkọwa mkpụrụ nke NiRAN, anyị lekwasịrị anya na nsp9 dị n'akụkụ N-terminal.Na enweghị protein ndị ọzọ, ha na-agbanwe n'ụzọ nhazi, na-egbochi achọpụtara ha n'ụdị nsp9 (26 28, 38), nke na-egosi oke ọdịdị ọdịdị ha nke a bụ n'ihi usoro dị mkpa-kpọmkwem (ọ bụghị usoro nke abụọ metụtara) ọrụ nke nsp9 N-terminal iberibe.Mgbanwe Ala nke ihe fọdụrụ echekwara na mpaghara a (Njirimara 5C na D na SI mgbakwunye, eserese S8) na-ekpughe na N3826 dị mkpa maka nsp9 NMPylation in vitro, ebe N3825A na E3827A ngbanwe na-eduga n'ibelata NMPylation, ebe M3829A na P3830A dochie anya. .O doro anya na emetụta nsp9 NMPylation.Ọ bụ ezie na ngbanwe nke N-terminal Asn (N3825A, N3825S) nwere naanị mmetụta na-agafeghị oke na nsp9 NMPylation na nje virus na omenala cell (Njirimara 5C na D), ihichapụ nke usoro ihe fọdụrụ nke Asn na N-terminal 3825-NN dipeptide. egosiputara na ọ bụ nje na-egbu egbu, na-egosi na achọrọ otu ihe fọdụrụ Asn tupu ihe fọdụrụ na N-terminus, ọkachamma Asn, n'agbanyeghị na ọ dị ka enwere ike ịnabata ihe ngbanwe nke ihe ndị yiri ya (Njirimara 5B, C, na D).Anyị kwubiri na 3825-NN dipeptide, karịsịa ihe nchekwa N3826 dị mkpa na nke dị n'ime mpaghara coronavirus (SI appendix, Figure S6), na-eme ka njide ziri ezi na nhazi nke nsp9 N-terminus na saịtị ọrụ nke NiRAN.

Dochie Ala (E3827A) maka Glu echekwara nke ezinụlọ niile na-ejigide nsp9 NMPylation na vitro mana ọ na-egbu nje na omenala cell (Njirimara 5C na D), na-egosi ọrụ ọzọ nke ihe fọdụrụ a, dịka ọmụmaatụ, na mmekọrịta isi (NMPylated ma ọ bụ agbanweghị agbanwe). ) nsp9 N-terminus na ihe ndị ọzọ na-etinye aka na mmegharị nje.Mgbanwe Nsp9 emetụtaghị usoro proteolytic nke nsp9 ma ọ bụ nsps ọ bụla dị n'akụkụ (39) (SI Mgbakwunye, Figure S9), na-egosi na phenotypes na-egbu egbu nke ọtụtụ nsp9 mutations hụrụ emeghị ka dysregulation nke C proteolytic usoro-terminal pp1a mpaghara. .

Ihe data a dị n'elu na-enye ihe akaebe na mgbe ọgwụgwọ Mpro-mediated nke nsp8|9 na-ehichapụ saịtị na pp1a/pp1ab, N-terminus nke nsp9 nwere ike ịbụ UMPylated (ma ọ bụ jiri NMP ọzọ gbanwee akụkụ ụfọdụ).Na mgbakwunye, nchekwa dị mma nke N-terminus nke nsp9 (gụnyere ihe fọdụrụ Asn na-agbanwe agbanwe na ezinụlọ coronavirus) yana data ntụgharị mkpụrụ ndụ ihe nketa enwetara n'ọmụmụ ihe a (Njirimara 3E na 5D) mere ka anyị kwubie na nsp9 NMPylation akọwapụtara. bụ ihe metụtara ndụ yana ihe dị mkpa maka mmegharị coronavirus.A ka ga-amụ nsonaazụ na-arụ ọrụ nke mgbanwe a, dịka ọmụmaatụ, gbasara nke akọwara na mbụ (na-abụghị nke akọwapụtara) nsp9 (ụdị na-agbanwebeghị) ọrụ njikọ RNA (2628).N-terminal NMPylation nwekwara ike imetụta mmekọrịta nke nsp9 na protein ma ọ bụ RNA substrates ma ọ bụ guzobe mgbakọ dị iche iche anọ.Achọpụtara ihe ndị a n'ọmụmụ ihe nhazi wee gosi na ha na-arụ ọrụ na mmegharị coronavirus, ọ bụ ezie na ọ kachasị na enweghị N'ọnọdụ mgbanwe a (26-ââ29, 40).

Ọ bụ ezie na ebumnuche ebumnuche nke ngalaba coronavirus NiRAN ka kwesịrị ka akọwapụta nke ọma, data anyị na-egosi na ebumnuche ebumnuche protein nke ngalaba coronavirus NiRAN dị warara.Ọ bụ ezie na nchekwa nke isi ihe fọdụrụ na saịtị (8, 16) na ngalaba NiRAN nke ezinụlọ nidovirus niile na-akwadosi ike ọrụ nke NMPylator echedoro protein ndị a, njirimara nke ihe fọdụrụ n'akpa uwe na-ejikọta mkpụrụ nke ngalaba a Nchekwa na nchekwa ga-anọgide na-egosipụta. , ma nwee ike ịdị iche n'etiti ezinaụlọ dị iche iche nke ebumnuche Nidovirales.N'otu aka ahụ, a ka ekpebibebeghị ebumnuche dị mkpa nke nje ndị ọzọ akwu.Ha nwere ike ịbụ orthologs dịpụrụ adịpụ nke nsp9 ma ọ bụ protein ndị ọzọ, n'ihi na usoro ndị dị n'èzí ngalaba replicate ise nke a na-echekwakarị na nje ndị a na-echekwa adịghị echekwa (8), gụnyere genome array n'etiti Mpro na NiRAN, N'ime ha, nsp9 dị na coronavirus.

Na mgbakwunye, anyị enweghị ike iwepụ ugbu a na ngalaba NiRAN nwere ebumnuche ndị ọzọ (gụnyere cellular).N'okwu a, ọ bara uru ịkọ na homologues nje na protein a na-apụta NMPylators (NMPylators) (30, 31) yiri ka ọ nwere "ndị nchịkwa nchịkwa"?NMP na-ahazi ụdị protein cellular dị iche iche iji chịkwaa ma ọ bụ kpochapụ ihe omume ha na-agbada, si otú ahụ na-ekere òkè n'ọtụtụ usoro ndu, dị ka nzaghachi nrụgide cellular na redox homeostasis (22, 33).

N'ime ọmụmụ ihe a (Figures 2 na 4 na SI Appendix, Figures S3 na S5), anyị nwere ike igosi na nsp12 bufere akụkụ UMP (NMP) n'otu ọnọdụ (echekwara) na nsp9, ebe protein ndị ọzọ adịghị emegharị na eji N'okpuru ọnọdụ ndị a, a na-akwado nkọwapụta nke ọma (kama ịtọghe).N'ikwekọ na nke a, ma e jiri ya tụnyere N-terminal nsp9 NMPylation, nsp12's nke onwe NMPylation ọrụ dị ntakịrị, nchọpụta ya na-achọ ogologo oge ikpughe autoradiography, yana mmụba okpukpu iri na nsp12 ntinye uche na-eji.Na mgbakwunye, nyocha MS anyị enyeghị ihe akaebe maka NMPylation nke nsp12, nke na-egosi na ngalaba NiRAN onwe-NMPylation bụ (nke kacha mma) ọrụ nke abụọ.Agbanyeghị, ekwesịrị iburu n'uche na ọmụmụ ihe ndị ọzọ enyela ihe akaebe izizi na ọnọdụ AMPylation nke nje bacteria NMPylator nwere ike ijikwa ọrụ NMPylation ha na mkpụrụ protein ndị ọzọ (22, 33).Ya mere, a chọkwuru nyocha iji nyochaa mmetụta nwere ike ịrụ ọrụ nke ọrụ NMPylation onwe onye akọọrọ maka EAV nsp9 (16) na coronavirus nsp12 (ihe ọmụmụ a), gụnyere mmetụta chaperone a tụrụ aro na mpịachi nke ngalaba C-terminal RdRp. 16)).

Na mbụ, a tụlere ọtụtụ echiche banyere ọrụ nwere ike ịdaba na mpaghara nidoviral NiRAN, gụnyere RNA ligase, RNA -capped guanylate transferase na protein priming ọrụ (16), ma ọ dịghị nke ọ bụla n'ime ha dakọtara na ọrụ ndị dị na ala.Ozi enwetara n'ọnọdụ ndị a bụ kpọmkwem otu oge n'emeghị echiche ndị ọzọ.Ihe data enwetara n'ọmụmụ ihe a kacha kwekọọ na (ma enweghị ike igosi) na ngalaba NiRAN na-etinye aka na mmalite nke protein-induced RNA njikọ.A kwenyere na mbụ na ọrụ nke ngalaba NiRAN na 5 ??²-RNA capping ma ọ bụ mmeghachi omume RNA ligation anaghị emetụta ndị a yana Nkwado nke data ndị ọzọ.Ya mere, dịka ọmụmaatụ, saịtị NiRAN na-arụ ọrụ ka a na-ewere na ọ gụnyere Asp echekwara dị ka ntọala izugbe (D252 na Pseudomonas syringae SelO; D4271 na HCoV-229E pp1ab; D208 na SARS-CoV-2 nsp12) (SI Ihe mgbakwunye, ọnụ ọgụgụ 2). ).S2) (17, 22, 33), ebe catalysis na ATP-based RNA ligase na RNA capping enzyme na-eme site na covalent enzyme (lysyl-N) -NMP etiti, nke gụnyere ihe fọdụrụ Lys na-abụghị agbanwe ( 41).Na mgbakwunye, nkọwapụta dị ịrịba ama dabere na usoro nke coronavirus NiRAN maka ebumnuche protein echekwara yana ụdị ntụsara ahụ maka ndị mmekọ NTP (na-ahọrọ UTP) na-emegide NiRAN-mediated capping enzyme ma ọ bụ ọrụ RNA ligase.

N'ụzọ doro anya, a chọrọ ọtụtụ ọrụ mgbakwunye iji nyochaa na, ọ bụrụ na egosipụtara ya, kọwapụta na ọrụ nsp9-UMP (nsp9-NMP) nwere ike na njikọ RNA na-ebute protein, nke ga-ejikọta ọtụtụ ihe na-adọrọ mmasị mana (ruo ugbu a) akụkọ ndị a kọrọ na mbụ. .Nlebanya dịpụrụ adịpụ.Dịka ọmụmaatụ, ekpebiela na njedebe nke RNA na-adịghị mma nke coronavirus na-amalite na eriri oligo (U) (42, 43).Nlebanya a kwekọrọ n'echiche bụ na a na-ebute njikọ nke eriri RNA na-adịghị mma site na ijikọta ụdị UMPylated nke nsp9 na ọdụ poly (A) (na-akpalite), nke nwere ike ịkwalite site na njikọ RNA ya Ọrụ na / ma ọ bụ mmekọrịta ya na ya. protein RTC ọzọ.Enwere ike iji akụkụ UMP nke nsp9 nyere dị ka "primer" maka nsp7/8/nsp12-mediated oligouridylation, na-eji ọdụ 3??²-poly(A) na genomic RNA ma ọ bụ oligo ọzọ (A) nwere usoro. na-eje ozi dị ka ndebiri, dị ka usoro e guzobere maka protein picornavirus VPg (44).Gịnị ma ọ bụrụ na atụmatụ a bụ "na-abụghị normative"????Mmalite nke njikọ RNA na-adịghị mma (protein butere) na-enye njikọ na nleba anya, na-egosi na RNA na-adịghị mma nke coronavirus nwere UMP (kama UTP) na njedebe ya (42), nke a na-ewere na ọ na-egosi na nucleic acid Dicer na-agbajie ngwụcha phosphorylated site na endonuclease nke uridine na-amaghị ama.Ọ bụrụ na akwadoro ya, ọrụ hydrolytic nucleic acid a nwere ike inye aka hapụ ụdị oligomeric UMPylated nke nsp9 site na njedebe 5 ² nke eriri na-adịghị mma.Ọrụ enwere ike nke nsp9 na protin priming kwekọrọ na ọmụmụ ihe gbasara mkpụrụ ndụ ihe nketa gara aga, nke egosila na nsp9 (na nsp8) na-emekọrịta nke ọma na kpọmkwem na ihe RNA nke na-eme ihe echekwara na nso njedebe 3 nke genome coronavirus.45).Dị ka akụkọ a si kwuo, ihe ndị a gara aga nwere ike nyochaa ugbu a ma gbasaa site na nyocha ọzọ.

Na nchịkọta, data anyị kpebiri kpọmkwem ọrụ mkpado nje nested nje nwere njikọ na RdRp na N-terminus.Na coronavirus, a na-eji ọrụ UMPylator/NMPylator mgbasa ozi NiRAN achọpụtara ọhụrụ ka a na-eji dabere na Mn2+ na ihe ndị dị n'akụkụ Asn ma mee ka njikọ nke phosphoramidate (ike dị ala) na amine N-terminal isi.Site na Mpro-mediated cleavage na nsp8 | 9 nkwụsịtụ, a pụrụ iji nsp9 lekwasịrị anya maka NMPylation, na-egosi njikọ ọrụ n'etiti protease na ngalaba NiRAN, nke na-agbatị RdRp.Nchekwa nke isi ihe fọdụrụ na nsp12 NiRAN na-arụ ọrụ na saịtị nsp9 na nsp9, yana data enwetara site na coronaviruses abụọ gụnyere SARS-CoV-2, na-enye ihe akaebe siri ike na nsp9 NMPylation bụ njirimara Conservative coronavirus bụkwa isi nzọụkwụ na nje virus.Ihe data dịnụ na-eduga anyị ikwubi na ọrụ a kapịrị ọnụ nke NMPylated ụdị nsp9 na protein-induced RNA njikọ bụ ihe ezi uche dị na ya dị n'ọnọdụ maka coronavirus na nje ndị ọzọ akwu, na NiRAN nwekwara ike gbado anya protein ndị ọzọ na-amaghị.Hazie nje virus.Mmekọrịta ndị ọbịa.Ọ bụrụ na akwadoro, itinye aka na protein primers na njikọ RNA viral ga-abawanye usoro nke ngalaba MPro / 3CLpro na RdRp n'etiti coronavirus nke achọpụtara na mbụ na picornavirus-dị ka supergroup (9), nke ejikọtala ugbu a na Pisonivirites guzobere nso nso a. 46) na ngalaba.

Ihe omuma anyi gosikwara na enwere ike iji ihe di nkpa, nke nhọrọ na ihe nleba anya nke emeputara n'ime ihe omumu a dika ebumnuche maka ogwu mgbochi.Ngwakọta ndị na-egbochi njikọ (na mgbanwe na-esote) nke nsp9 N-terminus echekwara na saịtị na-arụ ọrụ nke NiRAN nwere ike ịmepụta ka ọ bụrụ ọgwụ mgbochi nje dị irè ma dị iche iche, nke kwesịrị ekwesị maka ọgwụgwọ anụmanụ na ụmụ mmadụ coronaviruses sitere na ọrịa dị iche iche (sub) genus. , gụnyere SARS-CoV-2 na Middle East Respiratory Syndrome Coronavirus.

Usoro ntinye nke protein coronavirus emepụtara na ọmụmụ a bụ RT-PCR gbasiri ike site na iji RNA dịpụrụ adịpụ na Huh-7 bu nje HCoV-229E ma ọ bụ Vero E6 butere SARS-CoV-2 wee tinye ya site na iji usoro cloning ọkọlọtọ.pMAL-c2 (New England Biological Laboratory) ma ọ bụ pASK3-Ub-CHis6 (47) vector nkwupụta (SI Ihe Mgbakwunye, Tebụl S1 na S2).Mutagenesis dabere na saịtị PCR webatara ngbanwe otu codon (48).Iji mepụta protein fusion MBP, mkpụrụ ndụ E. coli TB1 gbanwere site na pMAL-c2 plasmid construct kwesịrị ekwesị (SI appendix, Table S1).Ejiri amylose affinity chromatography sachapụ protein ngwakọta ahụ wee kechie ya na Factor Xa.N'ikpeazụ, chromatography Ni-immobilized metal affinity chromatography (Ni-IMAC) sachara protein C-terminal His6-tagged dị ka akọwara na mbụ (49).Iji mepụta protein fusion ubiquitin, mkpụrụ ndụ E. coli TB1 jiri ihe kwesịrị ekwesị pASK3-Ub-CHis6 plasmid construct (SI Appendix, Tables S1 na S2) na pCGI plasmid DNA na-edobe ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Mgbanwe (47).A sachara protein coronavirus nke C-terminal His6 dị ka akọwara na mbụ (50).

Emere nnwale onwe NMPylation nke HCoV-229E nsp12-His6 dịka akọwara na EAV nsp9 (16).Na nkenke, nsp12-His6 (0.5 µM) nwere 50 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) -KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 na µM ihe nchekwa. NTP akọwapụtara na 0.17 µM dakọtara [α32-P] NTP (3,000 Ci / mmol; Hartmann Analytic) na 30 Celsius C maka nkeji 30.N'ihe niile (ọkọlọtọ) NMPylation nyocha nke nsp12-mediated nsp9 NMPylation, a na-edozi ọnọdụ mmeghachi omume dị ka ndị a: nsp12-His6 (0.05 µM) na nsp9-His6 (4 µM) na ọnụnọ 50 mM HEPES-KOH (pH 8.0) ), 5 mM DTT, 1 mM MnCl2, 25 µM gosiri NTP, na 0.17 µM dakọtara [α32-P] NTP.Mgbe incubating maka nkeji 10 na 30 Celsius C, a na-agwakọta ihe mmeghachi omume ahụ na ihe nchekwa SDS-PAGE: 62.5 mM tris (hydroxymethyl) aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol na 0.005% bromophen. acha anụnụ anụnụ.Akwụsịla protein ahụ site na kpo oku na 90 Celsius maka nkeji 5 wee kewaa ya na 12% SDS-PAGE.A na-edozi gel ahụ ma jiri Coomassie Brilliant Blue solution mee ihe (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), agbachapụ, ma kpughee ya na ihuenyo foto phosphorescent maka awa 20 (iji chọpụta nsp12 sitere na NMPylation) ma ọ bụ (kachasị) awa 2 (iji nyochaa nsp9 NMPylation).A na-eji ihe onyonyo Typhoon 9200 (GE Healthcare) nyochaa ihuenyo wee jiri ImageJ nyochaa ike mgbama ahụ.

Maka nyocha MS, 1 µM nsp12-His6 na 10 µM nsp9 (na-enweghị mkpado hexahistidine) ka ejiri NMPylation analysis (SI appendix, Table S1) na mmụba nke 500 µM UTP na GTP na-eji.Dabere na ntinye uche ha na ogo protein a na-atụ anya ya, a na-eji sistemu Waters ACQUITY H-Class HPLC nke nwere kọlụm MassPrep (Mmiri) mebie 1 ruo 10 µL nke ngwọta protein echekwara n'ịntanetị.A na-agbanye protein ahụ mebiri emebi n'ime isi iyi nke Synapt G2Si mass spectrometer (Waters) site na gradient nke buffer A (mmiri / 0.05% formic acid) na ihe nchekwa B (acetonitrile / 0.045% formic acid), na kọlụm okpomọkụ bụ. 60 Celsius C na ọnụego erute nke 0.1 ml / min: elution dị iche iche na 5% A maka nkeji 2, wee bụrụ gradient linear ruo 95% B n'ime nkeji 8, ma nọgide na-enwe 95% B maka nkeji 4 ọzọ.

A na-achọpụta ion ndị dị mma nwere oke oke nke 500 ruo 5000 m/z.A na-atụ Glu-fibrinopeptide B kwa sekọnd iri anọ na ise maka mgbazi mkpagharị akpaka akpaka.Jiri sọftụwia ngwa MassLynx nwere ndọtị MaxEnt1 iji mebie nkezi ụdịdị dị iche iche mgbe i wepụrụ ntọala ntọala na ire ụtọ.

A gbarie UMPylated HCoV-229E nsp9 site n'ịgbakwunye usoro-ọkwa gbanwetụrụ trypsin (Serva) wee tinye ya n'otu abalị na 37 Celsius.A na-eji kọlụm atụgharị Chromabond C18WP (nọmba akụkụ 730522; Macherey-Nagel) were desa na itinye uche na peptides.N'ikpeazụ, a gbazere peptide na 25 µL mmiri, nke nwere 5% acetonitrile na 0.1% formic acid.

MS nyochara ihe atụ ndị a site na iji Orbitrap Velos Pro mass spectrometer (Thermo Scientific).The kacha nanoâ HPLC usoro (Dionex), onwem na a omenala ọgwụgwụ-ndokwasa 50 cm ??75 μm C18 RP kọlụm juru n'ọnụ na 2.4 μm magnetik beads (Dr. Albin Maisch High Performance LC GmbH) Jikọọ na mass spectrometer online site na Proxeon nanospray isi iyi;gbanye 6µL nke ngwọta mgbaze trypsin n'ime dayameta ime 300 µm × ??1 cm C18 PepMap kọlụm tupu itinye uche (Thermo Scientific).N'iji mmiri/0.05% formic acid dị ka ihe mgbaze, a na-ejide ihe nlele ahụ na-akpaghị aka wee desalinated na ọsọ ọsọ nke 6 µL / min.

A na-eji gradients ndị na-esonụ nke mmiri / 0.05% formic acid (ihe mgbaze A) na 80% acetonitrile / 0.045% formic acid (ihe mgbaze B) iji nweta nkewa nke peptides tryptic na ọsọ ọsọ nke 300 nL / min: 4% B maka. Nkeji 5, wee 30 A linear gradient ruo 45% B n'ime nkeji nkeji, yana mmụba ahịrị ruo 95% mgbaze B n'ime nkeji ise.Jikọọ kọlụm chromatographic na igwe anaghị agba nchara nano-emitter (Proxeon), wee fesa eluent ozugbo na capillary na-ekpo ọkụ nke mass spectrometer na-eji ikike nke 2,300 V. A na-ejikọta nyocha nyocha na mkpebi nke 60,000 na Orbitrap mass analyzer. nwere opekata mpe data atọ MS/MS nyocha, ewepụrụ n'ike n'ike maka sekọnd 30, na-eji nkukota ọnyà linear ion induced dissociation ma ọ bụ nkukota ike dị elu jikọtara ya na nchọpụta orbitrap, mkpebi ahụ bụ 7,500.