Hoʻoponopono ʻia e Peter Sarnow, Stanford University School of Medicine, Stanford University, Kaleponi, apono ʻia ma Dekemaba 25, 2020 (nānā ʻia ma ʻOkakopa 25, 2020)
Hōʻike mākou i ka pilina ma waena o nā subunits i ka replication o nā coronaviruses-transcription complexes, he mea nui ia no ka hana hou ʻana a me ka mālama ʻana.Hāʻawi mākou i nā hōʻike e pili ana i ka domain NiRAN e pili ana me nsp12 he nucleoside monophosphate (NMP) transferase hana ma trans, a ʻike ʻia ka nsp9 (kahi RNA binding protein) i kāna pahuhopu.Hoʻopili ʻo NiRAN i ka hoʻopili covalent o ka moiety NMP i ka nsp9 amino terminal i mālama ʻia i loko o kahi pane e hilinaʻi ana i nā ion Mn2+ a me nā koena Asn i mālama ʻia.Ua ʻike ʻia he mea nui ka hana NiRAN a me nsp9 NMPylation no ka hana hou coronavirus.ʻAe ka ʻikepili iā mākou e hoʻopili i kēia hana o ka mea hoʻomaʻamaʻa enzyme virus nested i nā ʻike mua i loko o ke kuhiakau i ka hoʻomaka ʻana o ka RNA synthesis i loko o kahi papa o nā maʻi RNA he hana a me ka evolutionarily kūlike.
Hoʻopili ʻia ka RNA-dependent RNA polymerase (RdRps) o Nidovirales (Coronaviridae, Arterioviridae, a me 12 ʻohana ʻē aʻe) i ka domain amino-terminal (N-terminal) i loko o ka protein non-structural (nsp) i hoʻokuʻu ʻia mai ka polyprotein, i kapa ʻia ʻo NiRAN Hoʻokumu ʻia ʻo 1ab i ka protease nui viral (Mpro).Ma mua, ua hōʻike ʻia ka hana pono GMPylation/UMPylation o ka arterial virus NiRAN-RdRp nsp, a ua manaʻo ʻia e hana i kahi transient no ka hoʻoili ʻana o ka nucleoside monophosphate (NMP) i ka mea maʻi (ʻike ʻole ʻia) a / a i ʻole nā mea biopolymerization cell.Maʻaneʻi, hōʻike mākou i ka coronavirus (Human Coronavirus [HCoV]-229E a me Severe Acute Respiratory Syndrome Coronavirus 2) nsp12 (NiRAN-RdRp) he Mn2+-dependent NMPylation activity, i loaʻa mai nsp9 ma o ka hoʻokumu ʻana o Mpro-mediated nsp9 After. ka N-terminal flanking nsps ua hoʻokuʻu proteolytically, ua hoʻopaʻa ʻia ka phosphoramidate i ka amine mua (N3825) ma ka N-terminal o nsp9.ʻO ka uridine triphosphate ka nucleotide i makemake ʻia i kēia pane ʻana, akā ʻo ka adenosine triphosphate, guanosine triphosphate, a me ka cytidine triphosphate he mau co-substrates kūpono.ʻO nā haʻawina mutation e hoʻohana ana i ka recombinant coronavirus nsp9 a me nsp12 proteins a me ka genetically engineered HCoV-229E mutants i hoʻoholo i nā koena e pono ai no NiRAN-mediated nsp9 NMPylation a me ka hoʻopiʻi hou ʻana i ka moʻomeheu cell.Ua hōʻoia ka ʻikepili i ka wānana o nā koena o ka pūnaewele ikaika o NiRAN a ua hoʻoholo i ke kuleana koʻikoʻi o nā koena nsp9 N3826 i nsp9 NMPylation a me ka replication virus in vitro.ʻO kēia koena he ʻāpana o ka N-terminal NNE tripeptide sequence a hōʻike ʻia ʻo ia wale nō ke koena invariant o nsp9 a me kāna mau homologs i ka ʻohana coronavirus.Hāʻawi kēia haʻawina i kahi kumu paʻa no ka noiʻi hana o ka hana NMPylation o nā maʻi maʻi pūnana ʻē aʻe a hoʻolālā i nā pahuhopu hiki ke hoʻomohala i nā lāʻau antiviral.
Hoʻopilikia ʻo Nidovirales positive-stranded RNA virus i nā ʻano vertebrates a me invertebrates (1, 2).Aia ke kauoha i kēia manawa he 14 mau ʻohana (3), kahi i aʻo nui ʻia ai ka ʻohana Coronavirus i nā makahiki he 20 i hala.I kēlā manawa, ua puka mai ʻekolu zoonotic coronaviruses mai nā pūʻali holoholona a ua hōʻeha nui i nā maʻi hanu nui i nā kānaka.Me nā maʻi maʻi hoʻomau i hoʻokumu ʻia e nā maʻi maʻi maʻi koʻikoʻi.Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (4âââ7).Hoʻokaʻawale nā Nidoviruses i kahi hui genome maʻamau, a ua hoʻopili ʻia ka subunit o ka membrane-bound replication-transcription complex (RTC) i loko o ka 5-?²-terminal ʻelua hapakolu a me ka subunit kūkulu nui o ka mea maʻi virus, a me kekahi mau mea pono. .Protein, hoʻopaʻa ʻia i ka 3??² hope kolu o ka genome (1).Koe wale no hoʻokahi ʻohana o nā maʻi hoʻomaʻi planarian (Monoviridae) (8), hoʻopaʻa ʻia nā maʻi maʻi pūnana a pau i nā subunits RTC i ʻelua mau papa heluhelu hamama (ORF) ORF1a a me ORF1b, i unuhi ʻia mai ka genomic RNA o.Hoʻopili ʻo ORF1a i ka polyprotein (pp) 1a, a me ORF1a a me ORF1b e hoʻopili pū i ka pp1ab.Me ke komo ʻana o ka protease nui (Mpro) i hoʻopaʻa ʻia e ORF1a, ua hana ʻia ʻo pp1a a me pp1ab i kahi ʻano o nā protein non-structural (nsps), i ʻike ʻia ʻo 3CLpro, no ka mea he homology me ka 3Cpro o ka picornavirus ( 9).Manaʻo ʻia kēia mau nsps e hui pū ʻia i loko o kahi RTC ikaika nui, hoʻokaʻawale i ka synthesis o genomic RNA (replication) a me kahi pūʻulu o ka subgenomic RNA (transcription), a hoʻohana ʻia e hoʻonohonoho i ka ʻōlelo o ka ORF aia ma lalo o ORF1b (10? ? ?12).
Loaʻa ka RTC nui i ka RNA-dependent RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) a me kekahi mau enzymes processing RNA, i hoʻopili nui ʻia ma ORF1b a i ka ʻohana coronavirus Loaʻa iā ia ka nsp12-nsp16 a nsp9-nsp12 ma ka ʻohana Arterioviridae (e nānā i ka helu 10ââ 12).Hōʻike ʻo RdRp a me HEL1 i ʻelua (hoʻokahi hapalima) mau kikowaena mālama ʻia o ka pūnana manu a loaʻa ka homology ma waena o nā maʻi RNA ʻē aʻe.Manaʻo ʻia ke kōkua ʻia ʻo Core replicase e nā ʻāpana ʻē aʻe, me kekahi mau nsps liʻiliʻi i hoʻokuʻu ʻia mai ka ʻāpana carboxy-terminal (C-terminal) o pp1a, i lalo o Mpro (coronavirus nsp5 a me arterial virus nsp4, kēlā me kēia).Loaʻa iā lākou ka palekana pili ʻohana a me nā hana like ʻole (nānā ʻia ma ka reference 10ââ12).
I kēia mau lā, ua loaʻa kahi kikowaena me nā hiʻohiʻona kumu kaʻina kūʻokoʻa ma ka terminal amino (N-terminus) e pili ana me RdRp i nā maʻi maʻi pūnana a pau, akā ʻaʻohe mea maʻi RNA ʻē aʻe (16).Ma muli o kona wahi a me ka hana nucleotide transferase (nucleoside monophosphate [NMP] transferase), ua kapa ʻia kēia inoa ʻo NiRAN (Nestvirus RdRp-related nucleotide transferase).ʻO ka hui ʻelua-domain o NiRAN-RdRp ka nsp12 i ka ʻohana Coronaviridae a me ka nsp9 i ka ʻohana Arterioviridae, a ma nā nestoviridae ʻē aʻe, manaʻo ʻia e hoʻokuʻu ʻia ʻo NiRAN-RdRp ma ke ʻano he nsp kūʻokoʻa mai ka polyprotein viral.I loko o ka coronavirus, aia i loko o ka waihona NiRAN ??1/450 koena a ua pili i ka C-terminal RdRp domain ma o ka linker region (16?19).Ma ka Equine Arteritis Virus (EAV) (Artiviridae), hōʻike ka recombinant nsp9 i ka Mn2+ ion-dependent (self) UMPylation a me nā hana GMPylation, e hilinaʻi ana i ʻekolu mau kumu hoʻonohonoho i mālama ʻia i ka nestovirus, AN, BN a me CN ʻO nā koena i ke kaʻina.Kahi ʻo N no NiRAN) (16).ʻO ka ʻaoʻao N-terminal o kēia mau kumu he kumu hoʻohaʻahaʻa preAN.Mālama ʻia kekahi o kēia mau koena i loko o nā kinases protein pili mamao, kahi i hōʻike ʻia ai lākou e pili ana i ka nucleoside triphosphate (NTP) paʻa a me ka hana catalytic (20, 21).E like me kēia nānā ʻana, hiki ke hōʻuluʻulu ʻia kekahi mau koena pūnaewele ikaika i ka pseudokinase SelO mai Pseudomonas syringae me ka SARS-CoV-2 nsp7/8/12/13 supercomplex.Mālama ʻia nā koena NiRAN Coronavirus i hoʻopaʻa ʻia i ka microstructure electron.ʻO ka protein recombinant (17).Manaʻo ʻia ʻo ka U / GMPylation i kākau ʻia e hoʻopuka i kahi kūlana transient e hoʻololi i ka NMP i ka substrate (16), a me ke ʻano like o ka NiRAN a me ka protein kinase (17, 19) ) ʻO ke kuhiakau Hoʻololi ʻo NiRAN i nā protein ʻē aʻe.
Nui nā hiʻohiʻona, me kāna hui ʻōnaehana kū hoʻokahi a kū hoʻokahi me nā maʻi maʻi nested a me ka hoʻokaʻawale genetic mai RdRp, e hana ʻo NiRAN i kahi enzyme hoʻoponopono kī kūpono no nā maʻi maʻi pūnana, he mea koʻikoʻi i ko lākou puka ʻana a me ka ʻike.Ma mua, ua kāhea ʻia ʻekolu mau hana e pili ana iā NiRAN e hoʻoponopono i ka unuhi genome/subgenomic a i ʻole replication/transcription.I ka noʻonoʻo ʻana i ka ʻikepili liʻiliʻi a piha ʻole i loaʻa i kēlā manawa, loaʻa i kēlā me kēia hana kona mau pono a me nā hemahema (16).Ma kēia noiʻi, manaʻo mākou e hoʻohui i ka biochemical a me ka hoʻohuli ʻana i nā haʻawina hoʻohālikelike o nā coronaviruses e hōʻike ana i nā ʻano ʻelua, a kau i kā mākou ʻike i ke ʻano evolutionary o ka mutation kūlohelohe o ka ʻohana coronavirus, i loaʻa ai ka ʻike i kēia aupuni Pohihihi.Hōʻike mākou i nā holomua nui i ka hoʻomaopopo ʻana i ka NiRAN ma o ka ʻike ʻana i nā pahuhopu kūlohelohe i RTC, ʻo ia (ma waena o nā kuhiakau ʻekolu i loaʻa) kōkua i ke kuleana o kēia kahua i ka hoʻomaka ʻana i ka synthesis o ka RNA virus nested.Ke wehe nei kēia noiʻi i nā hiki no nā hana ʻē aʻe o NiRAN ma ka interface host virus.
I mea e ʻike ai i nā waiwai enzymatic o ka puni honua NiRAN pili i ka corona virus nsp12, ua hana mākou i kahi ʻano recombinant o ke kanaka coronavirus 229E (HCoV-229E) nsp12 ma E. coli, me kahi hōʻailona His6 ma ka C-terminus, a hoʻohui i ka pūmua me [α32-P ] Hoʻomoʻa pū me ka NTP i mua o MnCl2 e like me ia i wehewehe ʻia ma nā Mea Hana a me nā Hana.ʻO ka nānā ʻana o ka huahana hopena i hōʻike ʻia i ka hele ʻana o kahi protein radiolabeled e neʻe pū ana me ka nsp12 (106 kDa), e hōʻike ana i ka coronavirus nsp12 e hoʻokumu i ka hoʻokumu ʻana o nā covalent protein-NMP adducts, i hana ʻia me ka uridine monophosphate (UMP) (Figure 1A) A. B).Ua hōʻike ʻia ka loiloi quantitative i hoʻohālikelike ʻia me nā nucleotides ʻē aʻe, ua hoʻonui ʻia ka ikaika o ka hōʻailona o ka hoʻohui ʻana o UMP e 2 a 3 mau manawa (Figure 1C).Ua kūlike kēia ʻikepili me ka hana wānana NMP transferase o ka domain NiRAN o ka coronavirus (16), akā e hōʻike ana he ʻokoʻa nā koho nucleotide o ka domain NiRAN o ka coronavirus a me ka maʻi arterial.
Hana NMPylation pono'ī o HCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) ua hoʻokomo ʻia me ka [α-32P] NTP i koho ʻia ma ke alo o 6 mM MnCl2 no 30 mau minuke (e nānā i nā Mea Hana a me nā Hana no nā kikoʻī).Ua hoʻokaʻawale ʻia nā huahana hopena e SDS-PAGE a hoʻopaʻa ʻia me Coomassie brilliant blue.(B) ʻIke ʻia ka protein radiolabeled e ke kiʻi phosphorous.Hōʻike ʻia nā kūlana o ka nsp12-His6 a me ka protein molecular mass markers (i nā kilodaltons) ma A a me B. (C) Ua hoʻoholo ʻia ka ikaika o ka hōʻailona radioactive (mean ± SEM) mai ʻekolu mau hoʻokolohua kūʻokoʻa.*P≤0.05.ʻO ka ikaika hōʻailona (pakeneka) pili i ka UTP.
ʻOiai ua hōʻike ʻia nā hana enzyme e pili ana iā NiRAN he mea pono no ka hoʻopiʻi ʻana o EAV a me SARS-CoV i ka moʻomeheu cell (16), ʻaʻole i hoʻoholo ʻia ka hana kikoʻī a me nā pahuhopu hiki ke hoʻoholo.ʻO ka ʻano like ʻole i hōʻike ʻia ma waena o NiRAN a me kahi ʻohana o nā protein me ka protein kinase-like folds (17, 22) ua hoʻoikaika iā mākou e hoʻāʻo i ke kuhiakau e hoʻopiʻi ʻo NiRAN i ka NMPylation o nā protein ʻē aʻe.Ua hoʻokumu mākou i kahi pūʻulu o nā pahuhopu homologous hiki ke loaʻa, me nā protein non-structural i hoʻopili ʻia e HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), i kēlā me kēia me kahi C-terminal His6 tag (SI appendix, Table S1), A e hoʻomoʻa i kēia mau protein me [α32-P] uridine triphosphate ([α32-P] UTP) i mua a i ʻole ka nsp12.ʻO Bovine serum albumin a me MBP-LacZα fusion protein i hana ʻia ma E. coli i lawelawe ma ke ʻano he mana (Figure 2A, nā ala 1 a hiki i 7).Ua kālailai ʻia ka protein radiolabeled e ka sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) a me ka autoradiography, a ua ʻike ʻia aia kahi hōʻailona radioactive ikaika i loko o ka hopena i loaʻa ka nsp12 a me ka nsp9.ʻO ke kūlana o ka hōʻailona e pili ana i ka molecular mass o nsp9, e hōʻike ana i ka nsp12-mediated UMPylation o nsp9 (Figure 2B, track 7).ʻAʻole i ʻike ʻia nā protein hoʻāʻo ʻē aʻe i UMPylated, i alakaʻi iā mākou e hoʻoholo i ka nsp9 he substrate kikoʻī o nsp12.Kūlike me ka ʻikepili self-NMPylation i hōʻike ʻia ma ka Figure 1, hiki i ka nsp12 ke hoʻololi i nā NMP ʻehā i nsp9, ʻoiai he ʻokoʻa ka pono, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Kiʻi).3 A a me B).Ma lalo o nā kūlana i hoʻohana ʻia i kēia assay (hōʻemi i ka hopena a me ka manawa hōʻike, hoʻemi i ka neʻe ʻana o nsp12; nā mea hana a me nā ʻano hana), ʻaʻole hiki ke ʻike ʻia ka NMPylation ponoʻī o nsp12 (hoʻohālikelike i ke kiʻi 2B, alahele 7, a me ka Figure 1B), ka mea. ua hōʻoia i ka maikaʻi (A me nā pōʻai he nui) Ua neʻe ʻo UMP mai nsp12 a i nsp9.Pono ka hana UMP transferase i ke alo o nā ion Mn2+, e like me ka mea i hōʻike ʻia ma ka Figure 3C, ʻoiai ʻo ka liʻiliʻi liʻiliʻi o ka hana transferase UMP i ʻike ʻia ma ke alo o Mg2+, a ʻaʻohe hana i mua o nā cations divalent ʻelua i hoʻāʻo ʻia.Ua loaʻa nā ʻikepili like i loko o nā hōʻike NMPylation e loaʻa ana ka cytidine triphosphate (CTP), guanosine triphosphate (GTP), a me adenosine triphosphate (ATP) (SI appendix, Figure S1).
HCoV-229E nsp12-hoʻopili UMPylation o nsp9.Ua hoʻohana ʻia kahi ʻano o nā substrates protein (me ka bovine serum albumin, MBP-lacZα, a me kahi moʻo o HCoV-229E nsps i hōʻailona ʻia me C-terminal His6 i hoʻopili ʻia e ORF1a) no ka loiloi ʻana i ka hana UMPylation o HCoV-229E nsp12-His6⁺-mediated. pūmua.Hoʻomoʻa i ka protein me ka [α-32P] UTP no 10 mau minuke i ka wā ʻole (A) a i ʻole ke alo (B) o nsp12 e like me ka wehewehe ʻana i nā mea a me nā ʻano hana.Ma ka piko o A a me B, hōʻike ʻia ka gel SDS-polyacrylamide i hoʻopaʻa ʻia me Coomassie Brilliant Blue, a ma lalo o A a me B, hōʻike ʻia nā autoradiograms pili.Hāʻawi ʻia ke kūlana o ka mākaʻikaʻi molecular protein (ma kilodaltons) ma ka hema.ʻO ke kūlana o nsp12-His6 (B, luna) a me ka hōʻailona radioactive i ʻike ʻia i ka wā o ka incubation o nsp12-His6 me nsp9-His6 (B, lane 7) i hōʻike ʻia, e hōʻike ana i ka [α-32P]UMP i nsp9-His6 (12.9 kDa), ʻaʻole i nānā ʻia no nā protein ʻē aʻe i hoʻāʻo ʻia.
HCoV-229E NiRAN i hoʻopili ʻia i ka biochemical a me ka virological characterization o nsp9 NMPylation.(A a me B) Ke kuleana o ka nucleotide co-substrate i hoʻohana ʻia i ka hopena.Nsp12-His6 a me nsp9-His6 ua hui pū ʻia a hoʻoulu ʻia i mua o nā ʻano like ʻole [α-32P] NTP i ka hōʻike NMPylation maʻamau.(A, luna) Coomassie-stained nsp9-His6 i hoʻokaʻawale ʻia e SDS-PAGE.(A, lalo) Autoradiograph o ka wahi like o ka gel.(B) Hoʻoholo ʻia ka hana pili (mean ± SEM) i mua o ka cofactor nucleotide i koho ʻia mai ʻekolu mau hoʻokolohua kūʻokoʻa.*P≤0.05.(C) Ke kuleana o nā ion metala.Hōʻike ʻia ka hōʻike NMPylation maʻamau ma ke alo o [α-32P] UTP a me nā ion metala ʻē aʻe, kēlā me kēia me ka manaʻo o 1 mM.Ma C, ka luna, ua hōʻike ʻia ʻo Coomassie stained nsp9-His6, a ma C, ka lalo, hōʻike ʻia ka autoradiography pili.Hōʻike ʻia ka nui o ka protein i hōʻailona ʻia (ma kilodaltons) ma ka hema o A a me C. (D) ʻO ke ʻano mutant o HCoV-229E nsp12-His6 e lawe ana i ka hoʻololi ʻana i ka amino acid i ʻōlelo ʻia ma [α-32P]UTP, e like me ka mea i wehewehe ʻia. ma na Mea Hana a me na Hana.Ua ʻike ʻia ka radiolabeled nsp9-His6 i hana ʻia i ka hopena NMPylation e ka phosphorylation imaging (D, luna).Hōʻike ʻia ka hana pili i hoʻohālikelike ʻia me ka protein ʻano ʻāhiu (wt) ma D, a lawe ʻia ka lalo ma ke ʻano he awelika (± SEM) mai ʻekolu hoʻokolohua Kūʻokoʻa.Hōʻike nā asterisk i nā pani o nā koena i mālama ʻole ʻia.(E) Loaʻa ka helu virus ma ka moʻomeheu supernatant o nā cell p1 i 24 mau hola ma hope o ka hoʻoholo ʻia ʻana o ka maʻi e ka plaque assay.Hōʻike ʻia nā hoʻololi codon ma ka domain NiRAN o ka mutant HCoV-229E i hana ʻia (ua hoʻokumu ʻia ka helu koena ma ko lākou kūlana ma pp1ab).Ua hoʻohana ʻia ka replication-deficient RdRp active site mutant nsp12_DD4823/4AA ma ke ʻano he mana.
No ka loaʻa ʻana o ka ʻike hohonu o ka pūnaewele ikaika o NiRAN a hoʻoholo i nā koena e pili ana i ka hana o nsp9-specific NMP transferase, ua hana mākou i ka loiloi mutation, kahi i hoʻololi ai mākou i nā koena conservative i nā kumu NiRAN AN, BN a me CN ( 16) ʻO ia ʻo Ala (SI appendix, Kiʻi S2).Eia kekahi, ua loiloi ʻia ka hopena o ka conservative Arg-to-Lys a i ʻole Lys-to-Arg i nā hihia ʻelua.Ma ke ʻano he mana (negative), ua hoʻololi ʻia nā koena i mālama ʻole ʻia a i ʻole ka liʻiliʻi ma ka NiRAN domain o nā coronaviruses a me nā maʻi maʻi pūnana ʻē aʻe me Ala. ʻO BN) a me D4280A (CN) e hoʻemi nui a hoʻopau paha i ka nsp9 NMPylation ma o nsp12, ʻoiai nā protein me nā conservative substitutions (R4178K), K4116R) e mālama i ka 60% a me 80% o kā lākou hana, e hōʻike ana i ka hoʻomaha ʻana o nā kapu ma ko lākou ʻaoʻao. ʻO nā kaulahao he physicochemically sensitive (Figure 3D).ʻO ka hoʻololi ʻana i kekahi mau koena i mālama ʻia ʻo E4145A, D4273A, F4281A a me D4283A ʻoi aku ka liʻiliʻi o ka pōʻino, a ʻo ka nsp9 UMPylation e hōʻemi ʻia.Loaʻa nā hopena like i nā hopena nsp9 NMPylation e pili ana i nā NTP ʻē aʻe (Figure 3D a me SI appendix, Figure S3), e hōʻoia ana he kūʻokoʻa nā hopena i ʻike ʻia ma ka hoʻololi ʻana i ka waikawa amino kikoʻī i ke ʻano o ka nucleotide co-substrate i hoʻohana ʻia.Ma hope aʻe, ua hoʻāʻo mākou i ka hopena hiki ke hoʻololi i kēia nsp12 i ka replication o nā coronaviruses i ka moʻomeheu cell.No kēia hopena, ua hoʻohana mākou i nā ʻano hoʻohālike DNA (cDNA) kūpono i hoʻopili ʻia i loko o ka maʻi maʻi maʻi recombinant (23, 24) no ke kākau ʻana i 5 -7 mau cell.Ua hōʻike ʻia ka hoʻohālikelike ʻana o nā hua maʻi maʻi maʻi i hana ʻia i loko o kēia mau keena i hiki ʻole i ka hapa nui o nā mutants HCoV-229E NiRAN (Figure 3E).ʻO kahi pūʻulu o nā mutants viral hiki ʻole ke loaʻa nā mea ʻē aʻe i hōʻike ʻia e hoʻopau a hōʻemi nui paha i ka hana transferase in vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), akā aia ʻelua mau koho ʻē aʻe (K4116R, E4145A) 8 % mālama ʻia?ʻO kā lākou hana in vitro NMPylation e hōʻike ana e pili ana nā palena hou.Pēlā nō, ʻelua mau hoʻololi ʻē aʻe (R4178K, F4281A) i hoʻohaʻahaʻa haʻahaʻa i ka hana NMPylation in vitro o NiRAN i hana i nā maʻi maʻi ola, akā naʻe, ua hoʻemi nui kēia mau maʻi i nā titers ma o ka hana hou ʻana.Kūlike me ka ʻikepili hana in vitro i hōʻike ʻia ma ke Kiʻi 3D, e pani ana i ʻehā mau koena ʻē aʻe i mālama ʻole ʻia i loko o ka coronavirus a/a i ʻole nā maʻi maʻi pūnana ʻē aʻe (K4113A, D4180A, D4197A, D4273A) (8, 16) i hoʻopuka i nā maʻi maʻi. he ho'ēmi haʻahaʻa haʻahaʻa i hoʻohālikelike ʻia me ka maʻi ʻano hihiu (Figure 3E).
I mea e aʻo ai inā pili ka hana hoʻoili NMP i hoʻololi ʻia e NiRAN i ka domain RdRp ikaika, ua hoʻololi ʻia nā koena Asp ʻelua i mālama ʻia i ka hui ʻana o nā ion metala divalent (11) ma RdRp motif C e Ala. kāna hana nsp9 NMPylation, e hōʻike ana i ka hana nsp12-mediated in vitro nsp9 NMPylation ʻaʻole pono i ka hana polymerase (SI Appendix, Figure S4).
Ma hope o ka hoʻokumu ʻana i ka hana nsp9-specific NMP transferase no nsp12, ua hoʻāʻo mākou e ʻike i ka hoʻohui ʻana o NMP-nsp9 e ka mass spectrometry (MS).Ua hōʻike ʻia ke kiʻekiʻe kiʻekiʻe ma 12,045 Da (Figure 4A).ʻAʻole i hoʻololi ka hoʻohui o nsp12 i ka maikaʻi o nsp9, e hōʻike ana i ka nsp12 a me ka nsp9 ʻaʻole e hana i kahi paʻa paʻa ma lalo o nā kūlana i hoʻohana ʻia (denaturation) (Figure 4A).Ma ke alo o UTP a me GTP, ua hōʻike ʻia ke ana nui o ka hopena i loaʻa ka nsp9 a me ka nsp12 i ka neʻe ʻana o ka nuipa protein o UTP i 306 Da, a ua neʻe ka nui o ka protein o GTP i 345 Da, e hōʻike ana i kēlā me kēia mole nsp9 e hoʻopaʻa i kahi UMP a i ʻole GMP. (Kiʻi 4) C a me D).Manaʻo ʻia ʻo ka ikehu i koi ʻia no ka NiRAN-mediated nsp9 NMPylation mai ka NTP hydrolysis a me ka hoʻokuʻu pyrophosphate.ʻOiai ua hoʻohana ʻia he 10-fold molar excess o nsp9 (target) ma mua o ka nsp12 (enzyme) i kēia pane ʻana, ua ʻike ʻia ka NMPylation kokoke piha o nsp9, e hōʻike ana he pōkole ka pilina ma waena o nsp12 a me nsp9, a hiki i ka nsp12 ke NMPylate hou aku nsp9 in vitro mole.
Hoʻokahi NMPylation o nsp9 i mua o nsp12 a me UTP a i ʻole GTP.Hōʻike ʻia ka deconvoluted piha pūmua nui kikoʻī o HCoV-229E nsp9 (SI appendix, Papa S1) (AD).(A) nsp9 wale nō, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 i mua o UTP, (D) nsp9 + nsp12-His6 i mua o GTP.
No ka hoʻoholo ʻana i nā koena nsp9 UMPylated e nsp12, ua ʻoki ʻia ka nsp9-UMP me trypsin.Ua hoʻokaʻawale ʻia nā peptides e ka nano-high performance liquid chromatography (HPLC) a nānā ʻia e ka tandem mass spectrometry (MS/MS) ma ka pūnaewele.ʻIkepili ʻikepili me ka hoʻohana ʻana i ka pūʻolo polokalamu Byonic (Protein Metrics) i hōʻike i ka UMPylation o ka N-terminal amino acid.Hoʻopaʻa lima ʻia kēia.Ua hōʻike ka tandem mass spectrum o ka precursor peptide [UMP] NNEIMPGK (SI appendix, Figure S5A) i kahi ʻāpana ma 421 m/z, e hōʻike ana ua hoʻopaʻa ʻo UMP i ke koena 1 o nsp9.
Ma ka N-terminus o nsp9, mālama ʻia ʻo Asn i waena o nā lālā o Orthocoronavirinae (SI appendix, Kiʻi S6).ʻOiai ke manaʻoʻiʻo nei mākou ʻo ka N-terminal primary amine nitrogen ka mea ʻae loa no UMP, ua hoʻoholo mākou e kiʻi i nā hōʻike hou aʻe o ka paʻa ʻana o NMP ma ka N-terminal.No kēia kumu, ua loaʻa ka non-NMPylated a me ka NMPylated N-terminal peptide nsp9 i hoʻomaʻemaʻe ʻia e HPLC i ke alo o ka acetone a me ka sodium cyanoborohydride.Ma lalo o kēia mau kūlana, hiki ke hoʻololi ʻia nā amine mua manuahi me propyl (25).ʻO ka N-terminal nsp9-derived peptide me ke kaʻina NNEIMPGK he ʻelua mau amine, hoʻokahi ma ka N-terminus o Asn a ʻo kekahi ma ke kaulahao ʻaoʻao o Lys ma ka C-terminus.No laila, hiki ke hoʻokomo ʻia nā hui propyl ma nā ʻaoʻao ʻelua.Hōʻike ʻia nā chromatograms ion i unuhi ʻia o nā peptides non-NMPylated ma ka appendix SI, Kiʻi S5B.E like me ka mea i manaʻo ʻia, hiki ke ʻike ʻia ka N-terminal a me C-terminal (mono) propylated (SI appendix, Figure S5B, ala kiʻekiʻe) a me nā peptides dipropylated (SI appendix, Figure S5B, ala haʻahaʻa).Hoʻololi kēia hiʻohiʻona me ka hoʻohana ʻana i ka NMPylated N-terminal peptide o nsp9.I kēia hihia, hiki ke ʻike ʻia nā peptides propylated C-terminal wale nō, akā ʻaʻole ʻike ʻia nā peptides N-terminal propylated a me nā peptides dipropylated (SI Appendix, Figure S5C), e hōʻike ana ua hoʻololi ʻia ʻo UMP i ka N-terminal primary amine No ka pale ʻana i kēia. hui mai ka hana ʻana i nā loli.
A laila, hoʻololi mākou (me Ala a i ʻole Ser) a i ʻole e holoi i nā koena i mālama ʻia ma ka N-terminus o nsp9 e wehewehe i nā palena kikoʻī.Ma muli o kā mākou ʻikepili MS e hōʻike ana i ka hana ʻana o NiRAN i kahi hoʻohui nsp9-NMP me ka amine mua o ke koena N-terminal o nsp9, ua manaʻo mākou e koi ana ka nsp9 NMPylation i ka viral master protease (Mpro, nsp5) e hoʻokuʻu i ka nsp9 N-terminal mai. ʻO kāna polyprotein precursor.No ka hoʻāʻo ʻana i kēia kuhiakau, ua hana mākou i kahi protein precursor nsp7-11 i loaʻa i ka nsp9 ma E. coli a hana i kahi hoʻāʻo NMPylation maʻamau i mua o [α-32P] UTP (nā mea a me nā ʻano).E like me ka mea i hōʻike ʻia ma ke Kiʻi 5A (alanui 3), ʻaʻole i hoʻopaʻa ʻia ka nsp7-11 precursor me ka nsp12.I ka hoʻokaʻawale ʻana, inā ʻoki ʻia ka nsp7-11 e ka recombinant nsp5 e hoʻokuʻu i ka nsp9 (a me nā nsps ʻē aʻe) mai ka precursor, ʻike ʻia kahi protein radiolabeled e neʻe ana me nsp9, e hōʻoia ana i kā mākou hopena ʻo NiRAN a me N- Selective formation of covalent nsp9-NMP addducts .ʻO ka amine kumu mua o ka N-terminal Asn (kūlana 3825 ma pp1a/pp1ab).Kākoʻo ʻia kēia hopena e nā hoʻokolohua me ka hoʻohana ʻana i ka hana nsp9, aia i loko o hoʻokahi a ʻelua mau koena hou ma ka N-terminus.Ma nā hihia ʻelua, ua hoʻopau ʻia ka UMPylation o nsp9 i hoʻopili ʻia e NiRAN (SI Appendix, Figure S7).A laila, hana mākou i protein me hoʻokahi a ʻelua paha koena Asn i holoi ʻia mai ka 3825-NNEIMPK-3832 peptide sequence ma ka N-terminal o nsp9.I nā hihia ʻelua, ua paʻa loa ka nsp9 UMPylation (Figure 5B), e hāʻawi ana i nā hōʻike hou e hana ana ka nsp9 N-terminus maoli ma ke ʻano he mea loaʻa NMP.
ʻO ka hana proteolytic o nsp9 a me ke kuleana o nā koena N-terminal ma nsp12-mediated UMPylation.(A) nsp9 UMPylation koi i ka nsp9 N-terminal manuahi.Nsp7-11-His6 ua hoʻokomo mua ʻia ma 30 °C ma NMPylation detection buffer loaʻa ka UTP ma ke alo a i ʻole ka loaʻa ʻole o ka Mpro recombinant (nsp5-His6).Ma hope o 3 mau hola, e hoʻomaka i ka NMPylation assay ma ka hoʻohui ʻana i ka nsp12-His6 e like me ka mea i wehewehe ʻia ma nā Materials and Methods.Ua hoʻohana ʻia ka hopena i loaʻa ka nsp5-His6 (lane 1) a me nsp9-His6 (lane 2) ma ke ʻano he mana.Ma hope o 10 mau minuke, ua hoʻopau ʻia ka hopena a ua hoʻokaʻawale ʻia ka hui ʻana e SDS-PAGE.Ua uhi ʻia ka protein me Coomassie Brilliant Blue (A, luna).Hōʻike ʻia ka Nsp7-11-His6 precursor a me ka huahana i hana ʻia mai ka nsp5-His6 mediated cleavage ma ka ʻākau.E ʻoluʻolu e hoʻomaopopo (ma muli o ko lākou liʻiliʻi liʻiliʻi) ʻaʻole ʻike ʻia ka nsp7 a me ka nsp11-His6 i kēia gel, a ua hoʻohui ʻia ka hopena me nsp5-His6 (lanes 1 a me 4; hōʻike ʻia ke kūlana o nsp5-His6 e kahi pōʻai paʻa) a i ʻole nsp9-His6 (Lane 2) he wahi liʻiliʻi o MBP (i hōʻike ʻia e nā pōʻai hāmama) ma ke ʻano he koena haumia no ka mea ua hōʻike ʻia lākou ma ke ʻano he MBP fusion proteins (SI appendix, Table S1).(B) ʻAʻole i hoʻokahi a i ʻole ʻelua mau koena N-terminal Asn ka ʻano like ʻole Nsp9-His6 (helu helu koena e like me ke kūlana ma pp1a/pp1ab) a ua hoʻomaʻemaʻe ʻia a hoʻoulu ʻia me nsp12-His6 a me [α-32P] UTP.Hōʻike ʻia ʻo B, SDS-PAGE me Coomassie ma luna, B, hōʻike ʻia ka autoradiograph pili ma lalo.Hōʻike ʻia ke kūlana o ka māka kaumaha molekala (ma kilodaltons) ma ka hema.(C) HCoV-229E nsp9-His6 N-terminal conserved koena ua pani 'ia me Ala a i ole Ser, a ua like ka nui o ka protein i hoohana 'ia i ka nsp12-His6 i ho'opili 'ia UMPylation reaction.Ua hoʻokaʻawale ʻia nā huahana hopena e SDS-PAGE a ua hoʻopaʻa ʻia me Coomassie Brilliant Blue (C, luna), a ua ʻike ʻia ka radiolabeled nsp9-His6 e ka phosphorescence imaging (C, middle).Ke hoʻohana nei i ka protein wild-type (wt) ma ke ʻano he kuhikuhi (i hoʻonohonoho ʻia i ka 100%), ua helu ʻia ka hana NMPylation pili (mean ± SEM) mai ʻekolu mau hoʻokolohua kūʻokoʻa.(D) ʻO nā mea hoʻomaʻi maʻi i loko o ka p1 cell culture supernatant o Huh-7 cell i loaʻa i ka HCoV-229E wild-type Huh-7 cell, a me nā mutants e lawe ana i nā mea hoʻololi amino acid i loko o nsp9 i hoʻoholo ʻia e ka plaque assay.Ua hoʻohana ʻia ka motif C double mutant DD4823/4AA ma ke ʻano he mana maikaʻi ʻole.
Mālama ʻia ka N-terminus o nsp9 (ʻoi aku ka nui o nā kūlana 1, 2, 3, a me 6) i waena o nā lālā o ka ʻohana subfamily Orthocoronavirinae (SI appendix, Kiʻi S6).No ke aʻo ʻana i ke kuleana kūpono o kēia mau koena i nsp12-mediated nsp9 NMPylation, ua hoʻololi ʻia ʻelua mau koena Asn ma ka N-terminus o nsp9 me Ala a i ʻole Ser (hoʻokahi a hui pū paha).Ke hoʻohālikelike ʻia me ka nsp9 ʻano ʻāhiu, hoʻololi i ka N3825 me Ala a i ʻole Ser ka hopena i ʻoi aku ma mua o ka hoʻemi ʻelua ʻelua o ka UMPylation-mediated nsp12 (Figure 5C).E like me kā mākou hopena i loaʻa ʻo NMPylation ma ka N-terminal primary amine ma kahi o ke kaulahao ʻaoʻao o ke koena N-terminal, ʻike mākou i ke koena nui o ka NMPylation me ka pani ʻana o N3825A a me N3825S.ʻO ka mea e mahalo ai, inā hoʻololi ʻia ka Asn lua e Ala a i ʻole Ser, ʻoi aku ka ikaika o ka nsp9 UMPylation (ʻoi aku ma mua o 10 mau manawa), aʻo ka hoʻololi ʻana o Ala ma nā kūlana 3, 4, a me 6 he hopena haʻahaʻa wale nō i ka nsp9 UMPylation (Figure 2 ).5C).Loaʻa nā hopena like me ka hoʻohana ʻana i ka ATP, CTP a i ʻole GTP (SI appendix, Figure S8).ʻO ka hui pū ʻana, hōʻike kēia mau ʻikepili i ke kuleana koʻikoʻi o N2826 (kūlana 2 i nsp9) ma nsp9 NMPylation.
I mea e loaʻa ai nā hōʻike hou aʻe o ka pilina hana ma waena o ka N-terminus o nsp9 a me NMPylation, ua hana mākou i kahi alignment sequence lehulehu (MSA) o ke kaʻina nsp9 o ka ʻohana Coronavirus (e like ʻole ana ma waena o 104 a me 113 koena) (SI Appendix, Figure S6 ).ʻO ka huina, ma 47 (ʻike ʻia a i ʻike ʻia) nā ʻano o 5 genera o ka ʻohana subfamily Orthocoronavirinae e hoʻopōʻino ana i nā mammals like ʻole, nā manu, a me nā mea kolo, he 8 wale nō koena i ʻike ʻia he ʻano like ʻole.ʻO nā hoʻololi nui loa, me ka holoi ʻana a me ka hoʻokomo ʻana, ua ʻike ʻia i loko o nā pōʻai ma waena o nā mea hana lua o nsp9, e like me ka hoʻoholo ʻana e nā haʻawina hoʻolālā mua (26 ??28).ʻElima mau koena invariant i loaʻa i ke kaula β a me ka α helix o ka ʻāpana C-terminal o nsp9.ʻEkolu mau koena hoʻololi ʻole ke kumu NNE o ka palena N o nsp9.Ua hōʻike ʻia ʻo ka lua o ka Asn o kēia kumu ʻo ia wale nō ke koena invariant, i kaʻana like ʻia e ka hypothetical nsp9 o ka frog coronavirus pili mamao, a e hōʻike ana i ka Microhyla letovirus 1 ʻano i loko o ka subfamily Letovirinae o Alphaletovirus.Hiki ke hoʻopololei ʻia ka mālama ʻana o nā koena ma nsp9 mau mea hoʻolālā lua e ka noʻonoʻo ʻana i ka hoʻolālā e mālama ai i nā waiwai paʻa RNA i ʻike ʻia.Eia naʻe, ʻaʻole pili kēia manaʻo i ka mālama ʻana i ka NNE, a ma mua o kēia noiʻi ʻana, ua uhi ʻia ke ʻano o nā kaohi e kaupalena ana i ka hoʻololi ʻana o ke kaʻina tripeptide.
I mea e hoʻoholo ai i ke koʻikoʻi o ka nsp9-NMPylation a me ka mālama ʻana i ka NNE i ka hoʻopiʻi coronavirus, ua hana mākou i nā mutants HCoV-229E, e lawe ana i hoʻokahi a pālua paha i nā koena nsp9 N-terminal, e hōʻike ana he mea pōʻino ka nsp9 NMPylation in vitro.Ma mua o ka hoʻomaka ʻana, e hoʻāʻo mākou e pane i ka nīnau inā pili kēia mau pani (kokoke i ka nsp8|9 cleavage site) i ka hana proteolytic o ka C-terminal pp1a māhele.ʻO kahi pūʻulu o nā nsp7-11 polyprotein constructs i loaʻa nā pani kūpono i ka N-terminus o nsp9 i hana ʻia ma E. coli a ʻoki ʻia me ka recombinant Mpro.ʻAʻole i hoʻopilikia nui ʻia ka proteolytic cleavage o nā pae ʻehā (me ka nsp9 flanking site) e nā mea hoʻololi i hoʻokomo ʻia (SI appendix, Figure S9), me ka ʻole o ka hoʻololi ʻana i ka hoʻololi ʻana i kēia mau protein e hoʻopilikia i ka cleavage Mpro-mediated nsp8|9 (A i ʻole) pūnaewele puni honua.
Ua hoʻololi ʻia nā pūnaewele Huh-7 me ka HCoV-229E RNA lōʻihi genome, e hoʻopili ana i nā pani ʻana o Ala a i ʻole Ser i loko o nā tripeptides NNE i mālama ʻia (N3825, N3826, a me E3827) ma ka palena nsp9 N, e hōʻike ana he make ka hapa nui o nā mutation.Ua hiki iā mākou ke hoʻopakele i ka maʻi maʻi ma o ka hoʻololi ʻana i ka Ser a i ʻole ʻAla o ka N-terminal Asn (N2835A a i ʻole N2835S), akā ʻaʻole hiki ke hoʻihoʻi i ka maʻi me nā hoʻololi ʻē aʻe hoʻokahi a pālua i ka kaʻina NNE (N3826A, N3826S, NN3825/6AA, NN3825/6SS), E3827A) (Kiʻi 5D).
Hōʻike kēia mau hopena i ka hoʻopaʻa ʻia ʻana o nā coronaviruses i ka moʻomeheu kikoʻī (like a i ʻole like), ka palena ʻana i ka mutation kūlohelohe o nā kahua nsp9 NMPylation i loko o ke kino, a me ke kākoʻo ʻana i ke kuleana koʻikoʻi o kēia pane i ke ola o nā coronaviruses.
I nā hoʻokolohua hope loa, ua hana mākou i ka C-terminal His6 i kapa ʻia ʻo SARS-CoV-2 nsp12 a me nsp9, a me ʻelua mau ʻano mutant o nsp12 ma E. coli.ʻO nā koena pūnaewele hana ma nā kāʻei kapu NiRAN a me RdRp i hoʻohana ʻia iā Ala ma kahi (Figure 6A a me SI appendix, Papa S2).K4465 ma SARS-CoV-2 nsp12 pili me K4135 ma HCoV-229E (SI Appendix, Kiʻi S2), i hōʻike ʻia e koi ʻia no ka hana NiRAN a me ka hoʻopiʻi ʻana o HCoV-229E (Figure 3D a me E).Hoʻopili pū kēia koena me ka maʻi virus arterial EAV nsp9 K94 koena, i hōʻike mua ʻia he pono no NiRAN self-UMPylation/self-GMPylation (16).E like me ka mea i hōʻike ʻia ma ke Kiʻi 6B, ua loaʻa iā SARS-CoV-2 nsp12 ka hana hoʻoili UMP me ka hoʻohana ʻana i ka nsp9 ma ke ʻano he substrate, aʻo ka nsp12_K4465A mutant kahua ikaika ʻaʻole hana.ʻAʻole pili ka hoʻololi pālua i ke kaʻina hana SDD o RdRp motif C i ka hana UMP transferase (Figure 6B), e hōʻike ana ʻaʻohe hopena pololei o ka hana RdRp i nsp9 UMPylation.Ua loaʻa nā ʻikepili like me ka hoʻohana ʻana iā CTP, GTP a me ATP (SI appendix, Kiʻi S10).I ka hōʻuluʻulu ʻana, hōʻike kēia mau ʻikepili i ka NiRAN-mediated nsp9 NMPylation he hana conservative i nā coronaviruses e hōʻike ana i nā ʻano genera like ʻole o ka subfamily orthocoronavirus.
ʻO SARS-CoV-2 nsp12 i hoʻopili ʻia e NMPylation o nsp9.(A) Coomassie stained SDS-polyacrylamide gel e hōʻike ana i ka protein recombinant i hoʻohana ʻia i ka hoʻāʻo NMPylation.Ma ke ʻano he kaohi, ua hoʻohana ʻia kahi protein mutant me ka hoʻololi ʻana i ka pūnaewele ikaika i ka domain NiRAN (K4465A) a me RdRp domain (DD5152/3AA) o SARS-CoV-2 nsp12.Hoʻokumu ʻia ka helu koena ma ke kūlana ma pp1ab.(B) Autoradiograph of UMPylation detection using nsp9-His6 and [α-32P]UTP as the substrate of nsp12-His6 (wild type [wt] and mutant).Hōʻike ʻia ka nui molekala (i nā kilodaltons) o ka protein i hōʻailona ʻia ma ka hema.
Mālama ʻia nā kāʻei kapu NiRAN ma Nidovirales (16), e hōʻike ana e hoʻolalelale lākou i nā hopena enzymatic pono no ka replication Nidovirus.Ma kēia noiʻi ʻana, ua hiki iā mākou ke hōʻoia i ka hoʻololi ʻana o ka domain NiRAN o ka coronavirus i ka NMP (i hana ʻia mai ka NTP) i nsp9, kahi protein hoʻopaʻa RNA pohihihi i komo i ka replication virus (26 ?? 29), e hoʻoholo ai ma ke ʻano he pahuhopu kūlohelohe a. hoa o ka coronavirus RTC.
Hāʻawi ka domain NiRAN i ʻekolu mau manaʻo kaʻina (AN, BN, a me CN), aia kahi helu liʻiliʻi loa o nā koena i mālama ʻia i nā ʻohana āpau i ka papa Nidovirales monophyletic akā ʻokoʻa loa (8, 16).Ua hōʻike ʻia nā haʻawina hou e pili ana lākou i kahi ʻohana nui ʻole o ka protein kinase-like protein, i kapa mua ʻia ʻo ka ʻohana SelO (17, 19, 22, 30, 31).Loaʻa i nā pūmua pili pili i ka SelO nā kinase, akā nele i nā koena wahi hana i mālama ʻia i nā kinase maʻamau (22, 32).Ma muli o ka hoʻohuli ʻana o nā molekala ATP i hoʻopaʻa ʻia i ka pūnaewele ikaika a hoʻopaʻa ʻia e nā pilina kikoʻī, ua kuhi ʻia ʻo SelO a ma hope iho ua hōʻoia ʻia e hoʻololi i ka AMP (ma kahi o ka phosphate) i ka substrate protein (22), aʻo kekahi pūmua SelO-like YdiU i loaʻa. Ua hōʻike ʻia i kēia manawa e hoʻopili i ka covalent attachment o UMP iā Tyr a me kāna mau koena o nā substrates protein ʻokoʻa (33).
I mea e hōʻoia ai a hoʻonui i ka wānana o nā koena o ka pūnaewele coronavirus o ka domain NiRAN coronavirus, ua hoʻohana mākou i nā ʻano biochemical a me ka hoʻohuli ʻana i nā genetics e hana i ka loiloi mutation ma ka coronavirus nsp12 (Figure 3D a me E a me SI appendix, Figure S3 a me ka papa) S1â S4).Hōʻike ka ʻikepili i ka hoʻololi ʻana o HCoV-229E K4135, R4178 a me D4280 me Ala e hoʻopau i ka hana in vitro NMP transferase a me ka replication virus ma ka moʻomeheu cell (Figure 3D a me E a me SI appendice, Figure S3), e kākoʻo ana i ko lākou hele ʻana ma NTP γ-phosphate. (K4135, R4178) a me ka hoʻohui ʻana o nā ion metala wahi hana (D4280).Ua hōʻike ʻia ka hoʻololi ʻana o E4145A o ka Glu i mālama ʻia i ka laulā o ka pūnana manu i wānana ʻia e hoʻopaʻa i ke kūlana K4135 (17) i hōʻike ʻia e hoʻopau i ka replication viral, akā ʻo ka mea kupanaha, ua paʻa ka hana ma ka in vitro NMPylation assay (Figure 3D a me E a SI appendix, Kii S3 a me na Papa S1–S4).Ua hana ʻia kahi ʻike like i ka wā i hoʻokomo ʻia ai ka hoʻololi kūpono i ka YdiU homolog o Salmonella typhimurium (E130A) (33).Hoʻohui pū ʻia, kākoʻo kēia mau ʻikepili i ka hana hoʻoponopono o kēia koena i mālama ʻia ma mua o ka hana catalytic.
ʻO ka hoʻololi ʻana i ke koena Phe i mālama ʻia (F4281A) i loko o ka laulā o ka nestovirus ma ka domain HCoV-229E NiRAN (8) i hopena i ka emi ʻana o ka hana NMPylation in vitro a me ka emi nui o ka replication virus ma ka moʻomeheu cell (Figure 3D, E a me SI) hookui, Kii S3).Kūlike ka ʻikepili me ka hana hoʻoponopono koʻikoʻi o kēia koena, e like me ka homologous DFG motif Phe koena i hōʻike ʻia ma mua.I loko o ka classical protein kinases, he ʻāpana ia o ka Mg2+ hoʻopaʻa loop a kōkua i ka hōʻuluʻulu a hoʻoponopono i ka iwi kuamoʻo???Pono no ka hana catalytic pono (32, 34).ʻO ka hoʻololi ʻana iā Ala a me Arg no nā koena K4116 (ma ke kumu preAN), i hoʻopau ʻia ka hoʻopiʻi viral a, e like me ka mea i manaʻo ʻia, ua loaʻa nā hopena like ʻole i ka hana NMP transferase in vitro, ma muli o ke kaulahao ʻaoʻao amino acid i hoʻokomo ʻia (Figure 3D A me E a me SI appendice. , Kii S3).Kūlike ka ʻikepili hana me ka ʻike hoʻolālā, e hōʻike ana ua hoʻokumu kēia koena i kahi pilina me ATP phosphate (17).Ma ke kahua NiRAN o nā ʻohana virus nested ʻē aʻe, noho ʻia ke kūlana o HCoV-229E pp1a/pp1ab K4116 e Lys, Arg a i ʻole His (8), e hōʻike ana ua hoʻomaha ʻia ka palena hana o kēia koena kikoʻī.ʻO ka hoʻololi ʻana o D4188A a me D4283A e hoʻopau a hōʻemi ikaika i ka hana enzyme a hoʻopau i ka hoʻopili hou ʻana i nā maʻi (Figure 3).Mālama ʻia kēia mau koena ʻelua i ka hapa nui (akā ʻaʻole nā mea āpau) nested virus (8), e hōʻike ana i kahi hana koʻikoʻi ʻohana akā ʻaʻole catalytic paha.Ua hoʻohana ʻia nā pani ʻala o nā koena Lys a me Asp ʻē aʻe (K4113A, D4180A, D4197A a me D4273A) ʻaʻole i mālama ʻia i loko o ka Coronaviridae a i ʻole nā ʻohana Nestioviridae ʻē aʻe (8) i hoʻohana ʻia ma ke ʻano he mana.E like me ka mea i manaʻo ʻia, hiki ke ʻae ʻia kēia mau pani, me ka emi iki o ka hana enzyme a me ka hoʻopiʻi viral i kekahi mau hihia (Figure 3 a me SI appendix, Figure S3).Ma ke ʻano holoʻokoʻa, kūlike loa ka ʻikepili coronavirus mutagenesis me ka GMP ponoʻī a hoʻohuli i ka ʻikepili genetics o EAV NiRAN-RdRp (16), kahi e hana nui ai ʻo EAV nsp9 (coronavirus nsp12 ortholog) K94 (e pili ana me HCoV-229E K4135), R124 (pili me R4178), D132 (pili me D4188), D165 (pili me D4280), F166 (pili me F4281).Eia kekahi, ua kūlike ka ʻikepili mutagenesis HCoV-229E me ka hoʻonui ʻia mai ka ʻike i hōʻike mua ʻia ʻo SARS-CoV reverse genetics data (16), e like me nā mea i ʻike ʻia no ka motif CN pili Phe-to-Ala mutant SARS-CoV_nsp12 The phenotype wehewehe -F219A a me HCoV-229E_F4281A (Figure 3 D a me E a me SI appendix, Kii S3 a me ka Papa S1-S4).
Ke hoʻohālikelike ʻia me nā EAV orthologs (16), nona ka makemake maopopo no ka UTP a me ka GTP (i ka hopena NMPylation ponoʻī), hōʻike kā mākou haʻawina e hiki ke hana maikaʻi ʻia ka domain NiRAN coronavirus (i hōʻike ʻia e HCoV-229E a me SARS-CoV-2). ua hoʻoili ʻia nā NMP ʻehā, ʻoiai aia kahi makemake liʻiliʻi no UMP (Nā Kiʻi 1 a me 3).ʻO ka haʻahaʻa haʻahaʻa haʻahaʻa o ka NTP co-substrate kikoʻī e kūlike me ka SARS-CoV-2 nsp7/8/12/13 supercomposite structure i hōʻike ʻia nei, kahi i hoʻopaʻa ʻia ai ʻo ADP-Mg2+ i ka pūnaewele ikaika o NiRAN, akā ʻaʻole me ka ʻāpana adenine. o ka hoʻokumuʻana i nā pilina kūikawā (17).I loko o kā mākou noiʻi ʻana, ʻaʻohe ʻano o ka nucleotide i hoʻohana ʻia i ka hopena NMPylation i ka hana o ka protein mutant (SI Appendix, Figure S3), e hōʻike ana ʻaʻohe o kēia mau koena i pili i ka paʻa ʻana o kahi nucleobase kikoʻī.Ke aʻo ʻia nei ke kumu hoʻokumu a me ke ʻano koʻikoʻi olaola o nā makemake co-substrate ʻē aʻe i ʻike ʻia ma nā kāʻei NiRAN o nā coronaviruses a me nā maʻi maʻi arterial;ʻoiaʻiʻo paha lākou a ma muli paha o nā palena o kā lākou mau haʻawina.I kēia manawa, ʻaʻole hiki ke hoʻoholo ʻia he ʻokoʻa ka makemake co-substrate ka hana NMPylator o ka maʻi virus arterial domain NiRAN (hoʻohālikelike ʻia me ka hana NMPylation ponoʻī i hōʻike mua ʻia) i kahi makemake co-substrate ʻē aʻe, me ka noʻonoʻo ʻana i ka like o ka arterial a me ka coronavirus. Aia ka waihona NiRAN i kona palena.Hoʻohālikelike ma muli o ke kaʻina (16).Hoʻohālikelike ʻia me ka pseudokinase SelO, e hoʻohana ana i ka Mg2+ ma ke ʻano he cofactor, pili ka hana o ka coronavirus a me ka maʻi arterial NiRAN i Mn2+ (16) (Figure 3C a me SI appendix, Figure S1).ʻO ka hilinaʻi Mn2+ a me ka makemake ʻike no ka UTP he hiʻohiʻona ʻokoʻa ia o ka pūmua NMPylators, a ua hōʻoia hou ʻia i loko o ka protein YdiU o Salmonella typhimurium, ka mea e hoʻolalelale i ka Mn2 +-dependent protein chaperone UMPylation e pale i nā cell mai ke kaumaha induction Cell ATP pool ( 33).
ʻO ke ʻano like ʻole i wehewehe ʻia ma waena o ka domain coronavirus NiRAN a me nā kinases protein cellular (17, 19) e hāʻawi i ke kākoʻo hou no ka hiki o NiRAN ke hoʻopili covalently i ka NMP i nā protein ʻē aʻe a mākou i hōʻike ai ma kēia noiʻi.Ua kālele mākou i kā mākou ʻimi ʻana i nā pahuhopu NiRAN hiki i nā protein i hoʻopaʻa ʻia e HCoV-229E ORF1a, i ʻike ʻia e kōkua pololei a ʻole ʻole paha i ka RTC's ORF1b-encoded replicase (12, 35).Hāʻawi kā mākou mau hoʻokolohua i nā hōʻike paʻa no ka NMPylation pono a kikoʻī o nsp9 (Figure 2).Inā hoʻohana ʻia ka protein i hoʻopaʻa ʻia i loko o kahi molar oi aku he 8 a 10 mau manawa kiʻekiʻe ma mua o ka enzyme (nsp12), ua hōʻoia ʻia ʻo nsp9 ua piha (mono) NMPized (Figure 4).Ua hoʻoholo mākou he pōkole ka pilina ma waena o nsp12 a me nsp9 a ʻaʻole ia e hana i kahi paʻa paʻa me nsp9 (i ka nele o nā ʻāpana RTC ʻē aʻe).Kākoʻo ʻia kēia hopena e ka noiʻi ʻana i ka pilina protein ma ka SARS-CoV proteome (35).Ua ʻike ʻia ka ʻike ʻana o MS i ka amine mua o ke koena N-terminal o nsp9 e like me ka pūnaewele NMPylation (SI appendix, Figure S5).ʻO ka hoʻokumu ʻana o ka paʻa phosphoramidate a me ka pūʻulu amino N-terminal e hoʻokaʻawale i ka hana NMPylation i hoʻopili ʻia e NiRAN mai ka Pseudomonas syringae SelO-mediated AMPylation reaction, ka mea e hoʻolalelale i ka hoʻokumu ʻana o O-linked AMP ma Ser, Thr, a i ʻole Tyr koena Peptide adduct ( 22), a me S. typhimurium YdiU i hoʻohui i ka O-linked (me Tyr) a me ka N-linked (me kāna) peptide-UMP hoʻohui.ʻO ka ʻike liʻiliʻi i loaʻa ma ka ʻohana SelO o nā protein e hōʻike ana he ʻokoʻa nā lālā o kēia ʻohana protein nui i ka hoʻokumu ʻana i nā mea hoʻohui peptide-NMP.He ʻike hoihoi kēia e pono ai ke aʻo hou ʻia.
ʻO ka ʻikepili i loaʻa i kēia haʻawina i alakaʻi iā mākou i ke kuhiakau e koi ana ka NMPylation o nsp9 i kahi N-terminus manuahi.Ma ka pōʻaiapili o ka hoʻopiʻi viral, e hāʻawi ʻia kēia e ka proteolytic cleavage o ka nsp8|nsp9 kahua hana i loko o ka replicase polyprotein pp1a i hoʻopili ʻia e Mpro a me pp1ab.I ka hapanui o nā coronaviruses, ʻo ka ʻokoʻa ma waena o kēia pūnaewele kikoʻī (VKLQ|NNEI ma HCoV-229E) a me nā wahi ʻē aʻe coronavirus Mpro cleavage ʻo Asn (ma mua o kahi koena liʻiliʻi, e like me Ala, Ser Or Gly) noho P1â???Wahi (36).Ua hōʻike ʻia ka ʻikepili cleavage peptide i loaʻa i nā haʻawina mua i ka haʻahaʻa o ka cleavage pono o ka nsp8|nsp9 pūnaewele ma mua o nā pūnaewele ʻē aʻe, e hōʻike ana he 1) hiki i kēia pūnaewele kikoʻī ke kuleana hoʻoponopono i ka hoʻoili ʻana i ka manawa kūpono o ka C-terminal. ʻāpana pp1a, a i ʻole 2) a ʻO ke kuleana o ka nsp9 N-terminus i mālama ʻia i ka hoʻopili hou ʻana i ka maʻi virus (37).Ua hōʻike ʻia kā mākou ʻikepili (Figure 5A) i ke ʻano recombinant o nsp9 e lawe ana i ke kaʻina N-terminal maoli i hana pono ʻia e nsp12.Ua wehe ʻia ka ʻaoʻao ʻaoʻao o N-terminal e ka helu Xa (nsp9-His6; SI appendix, Table S1) a i ʻole Mpro-mediated cleavage (nsp7-11-His6; Figure 5A a me SI appendix, Table S1).ʻO ka mea nui, ʻo ka precursor nsp9 i ʻoki ʻole ʻia i ka nsp7-11-His6 i hōʻike i ke kūʻē ʻana i ka NMPylation o nsp12, i kūlike me kā mākou ʻikepili, e hōʻike ana ua hoʻokumu ʻia ka hoʻohui nsp9-NMP ma o ka N-terminal primary amine (SI appendix, Figure S5) .No ka loaʻa ʻana o ka ʻike hohonu o ka kikoʻī o ka substrate NiRAN, ua kālele mākou i nā koena N-terminal e pili ana i nsp9.I ka loaʻa ʻole o nā protein ʻē aʻe, hiki ke hoʻololi ʻia lākou, e pale ana iā lākou mai ka ʻike ʻia ʻana ma ke ʻano unlabeled o nsp9 (26 28, 38), e hōʻike ana i kā lākou ʻokoʻa kūlohelohe ʻokoʻa ʻO kēia ma muli o ke kaʻina koʻikoʻi-specific (ʻaʻole pili i ke ʻano lua). hana o ka nsp9 N-terminal hakina.ʻO nā pani ala o nā koena i mālama ʻia ma kēia māhele (Nā kiʻi 5C a me D a me SI appendix, Figure S8) e hōʻike ana he mea nui ka N3826 no ka nsp9 NMPylation in vitro, aʻo nā pani N3825A a me E3827A e alakaʻi i ka emi ʻana o ka hoʻololi ʻana o ka M3829A a me ka P3830A. .E pili ana i ka nsp9 NMPylation.ʻOiai ʻo ka hoʻololi ʻana o ka N-terminal Asn (N3825A, N3825S) he hopena maʻalahi wale nō i ka nsp9 NMPylation a me ka hoʻopiʻi ʻana i nā maʻi i loko o ka moʻomeheu cell (Figure 5C a me D), ʻo ka holoi ʻana i kahi kaʻina Asn koena mai ka N-terminal 3825-NN dipeptide Ua hōʻike ʻia he mea make ia i nā maʻi maʻi, e hōʻike ana e koi ʻia kahi koena Asn ma mua o kahi koena ʻē aʻe ma ka N-terminus, ʻoi aku ka maikaʻi o Asn, ʻoiai me he mea lā hiki ke ʻae ʻia ke hoʻololi ʻana i nā koena like (Figure 5B, C, a me D).Manaʻo mākou ʻo ka 3825-NN dipeptide, ʻoi aku ka nui o ke koena N3826 i mālama ʻia a koʻikoʻi i loko o ka pae coronavirus (SI appendix, Kiʻi S6), e hōʻoia i ka hoʻopaʻa ʻana a me ka hoʻonohonoho pono ʻana o ka nsp9 N-terminus i ka pūnaewele ikaika o NiRAN.
ʻO ka hoʻololi ʻana iā Ala (E3827A) no ka Glu i mālama ʻia o nā ʻohana āpau e mālama i ka nsp9 NMPylation in vitro akā make i nā maʻi maʻi i loko o ka moʻomeheu cell (Figure 5C a me D), e hōʻike ana i ka hana hou o kēia koena, no ka laʻana, i nā pilina koʻikoʻi (NMPylated a i hoʻololi ʻole ʻia. ) nsp9 N-terminus a me nā mea ʻē aʻe i pili i ka hoʻopili hou ʻana i ka maʻi virus.ʻAʻole pili ka hoʻololi ʻana o Nsp9 i ke kaʻina hana proteolytic o nsp9 a i ʻole kekahi nsps pili (39) (SI Appendix, Figure S9), e hōʻike ana i nā phenotypes lethal o kekahi mau mutations nsp9 i nānā ʻia ʻaʻole i kumu ʻia e ka dysregulation o ka C proteolytic process-terminal pp1a wahi. .
Hōʻike ka ʻikepili i luna ma hope o ka mālama ʻana i ka Mpro-mediated o ka nsp8|9 cleavage site ma pp1a/pp1ab, hiki ke hoʻololi ʻia ka N-terminus o nsp9 (a i ʻole hoʻololi ʻia me kekahi NMP).Eia kekahi, ʻo ka mālama maikaʻi loa o ka N-terminus o nsp9 (me nā koena Asn hoʻokahi a me ka invariant i ka ʻohana coronavirus) a me nā ʻikepili genetics i loaʻa i kēia noiʻi (Figures 3E a me 5D) alakaʻi iā mākou e hoʻoholo i ka nsp9 NMPylation i wehewehe ʻia. pili olaola a pono no ka replication coronavirus.E aʻo ʻia nā hopena hana o kēia hoʻololi, no ka laʻana, e pili ana i ka hana hoʻopaʻa RNA (2628) i wehewehe mua ʻia (non-specific) nsp9 (ʻano hoʻololi ʻole).Hiki i ka N-terminal NMPylation ke hoʻopili i ka pilina o nsp9 me ka protein a i ʻole RNA substrates a i ʻole ka hoʻokumu ʻana o nā hui ʻehā ʻano like ʻole.Ua ʻike ʻia kēia mau mea i loko o nā haʻawina structural a ua hōʻoia ʻia e pili ana i ka hoʻopiʻi coronavirus, ʻoiai inā ʻaʻole i loaʻa i ka hihia o kēia hoʻololi (26- ââ29, 40).
ʻOiai ʻo ka kikoʻī kikoʻī o ka coronavirus NiRAN domain e pono e hōʻike ʻia i nā kikoʻī hou aku, hōʻike kā mākou ʻikepili i ka kikoʻī o ka protein target specificity o ka coronavirus NiRAN domain.ʻOiai ke kākoʻo ikaika nei ka mālama ʻana i nā koena pūnaewele ikaika (8, 16) ma ka ʻaoʻao NiRAN o nā ʻohana nidovirus a pau i ka hana a ka NMPylator i mālama ʻia i kēia mau protein, ʻo ke ʻano o ka substrate hoʻopaʻa ʻana i nā koena ʻeke o kēia ʻāina. , a ʻokoʻa paha ma waena o nā ʻohana like ʻole o nā kumu Nidovirales.Pēlā nō, ʻaʻole i hoʻoholo ʻia nā pahuhopu kūpono o nā maʻi maʻi pūnana ʻē aʻe.He mau orthologs mamao paha lākou o nsp9 a i ʻole nā protein ʻē aʻe, no ka mea, ʻaʻole mālama ʻia nā kaʻina ma waho o nā kikowaena replicase ʻelima i mālama mau ʻia i nā maʻi maʻi pūnana (8), me ka pūʻulu genome ma waena o Mpro a me NiRAN, Aia ma waena o lākou, aia ka nsp9 i ka coronavirus.
Eia hou, ʻaʻole hiki iā mākou ke hoʻokuʻu i kēia manawa i ka hiki ke loaʻa i ka domain NiRAN nā pahuhopu hou (me ka cellular).I kēia hihia, pono e haʻi ʻia ʻo nā homologues bacteria i loko o kēia pūmua e puka mai nei NMPylators (NMPylators) (30, 31) me he mea lā he "master regulators"?Hoʻololi ʻo NMP i nā ʻano protein cellular e hoʻoponopono a hoʻopau paha i kā lākou mau hana i lalo, a laila e pāʻani ana i kahi hana i nā ʻano hana olaola like ʻole, e like me ka pane ʻana i ke kaumaha cellular a me ka homeostasis redox (22, 33).
Ma kēia haʻawina (Nā Kiʻi 2 a me 4 a me SI Appendix, Nā Kiʻi S3 a me S5), ua hiki iā mākou ke hōʻoia i ka neʻe ʻana o nsp12 i ka ʻāpana UMP (NMP) i kahi kūlana hoʻokahi (mālama ʻia) ma nsp9, ʻoiai ʻaʻole i hoʻololi ʻia nā protein ʻē aʻe i ka hoʻohana ʻia Ma lalo o nā kūlana, kākoʻo ʻia ke ʻano kikoʻī i wehewehe maikaʻi ʻia (ma mua o ka wehe).Kūlike me kēia, ke hoʻohālikelike ʻia me N-terminal nsp9 NMPylation, haʻahaʻa loa ka hana NMPylation ponoʻī o nsp12, pono ka ʻike ʻana i ka manawa hōʻike autoradiography lōʻihi, a hoʻohana ʻia ka piʻi ʻana he 10-fold o ka nsp12.Eia kekahi, ʻaʻole i hāʻawi ʻia kā mākou loiloi MS i nā hōʻike no NMPylation o nsp12, e hōʻike ana ʻo NiRAN domain self-NMPylation (ma ka maikaʻi) kahi hana lua.Eia naʻe, pono e hoʻomaopopo ʻia ua hāʻawi nā haʻawina ʻē aʻe i nā hōʻike mua e hiki ai i ke kūlana self-AMPylation o NMPylator bacteria ke hoʻomalu i kā lākou hana NMPylation ma nā substrates protein ʻē aʻe (22, 33).No laila, pono ka noiʻi hou aʻe e noiʻi i nā hopena hana hiki ke hana ʻia o nā hana NMPylation ponoʻī i hōʻike ʻia no EAV nsp9 (16) a me ka coronavirus nsp12 (kēia haʻawina), me ka hopena like chaperone i manaʻo ʻia ma ka pelu ʻana o ka C-terminal RdRp domain ( 16) ).
Ma mua, ua noʻonoʻo ʻia kekahi mau kuhiakau e pili ana i nā hana i lalo o ka domain nidoviral NiRAN, me RNA ligase, RNA -capped guanylate transferase a me ka protein priming activity (16), akā ʻaʻohe o lākou i kūpono me nā hana i lalo.ʻO ka ʻike i loaʻa ma nā kūlana ma lalo nei ka manawa like me ka hana ʻole ʻana i nā manaʻo hou.ʻO ka ʻikepili i loaʻa ma kēia haʻawina ʻoi aku ka kūlike me (akā ʻaʻole hiki ke hōʻoia) ua komo ka domain NiRAN i ka hoʻomaka ʻana o ka synthesis RNA i hoʻokomo ʻia i ka protein.Ua manaʻo mua ʻia ka hana o ka domain NiRAN ma 5??ʻAʻole pili ʻia ka ²-RNA capping a i ʻole RNA ligation reactions e kēia a me ke kākoʻo o nā ʻikepili ʻē aʻe.No laila, no ka laʻana, ua manaʻo ʻia ka pūnaewele ikaika o NiRAN e komo i ka Asp i mālama ʻia ma ke ʻano he kumu maʻamau (D252 i Pseudomonas syringae SelO; D4271 i HCoV-229E pp1ab; D208 i SARS-CoV-2 nsp12) (SI Appendix, helu 2 ).S2) (17, 22, 33), aʻo ka catalysis i ka ATP-dependent RNA ligase a me ka RNA capping enzyme e lawe ʻia e ka covalent enzyme-(lysyl-N) -NMP intermediate, e pili ana i kahi koena Lys non- Changed ( 41).Eia kekahi, ʻo ka mea kikoʻī e pili ana i ke kaʻina o ka coronavirus NiRAN no nā pahuhopu protein mālama ʻia a me ka kikoʻī hoʻomaha no nā co-substrates NTP (makemake UTP) e kūʻē i ka NiRAN-mediated capping enzyme a i ʻole RNA ligase-like hana.
ʻIke loa, he nui nā hana hou e pono ai e hōʻoia a, inā i hōʻoia ʻia, e wehewehe i ka hiki ke hana o nsp9-UMP (nsp9-NMP) i loko o ka protein-induced RNA synthesis, kahi e hoʻopili ai i kekahi mau mea hoihoi akā (i kēia manawa) i hōʻike mua ʻia. .Nānā kaʻawale.No ka laʻana, ua hoʻoholo ʻia e hoʻomaka ka hopena o ka RNA-ʻole o ka coronavirus me kahi kaula oligo(U) (42, 43).Ua kūlike kēia nānā ʻana me ka manaʻo e hoʻomaka ʻia ka synthesis o ka negative-strand RNA ma o ka hoʻopaʻa ʻana i ke ʻano UMPylated o nsp9 i ka huelo poly(A) ( triggers), hiki ke hoʻoikaika ʻia e kāna paʻa RNA ʻO ka hana a me ka launa pū ʻana me. kekahi protein RTC.Hiki ke hoʻohana ʻia ka ʻāpana UMP i hāʻawi ʻia e nsp9 ma ke ʻano he "primer" no ka nsp7/8/nsp12-mediated oligouridylation, me ka hoʻohana ʻana i ka huelo 3??²-poly(A) i loko o ka genomic RNA a i ʻole kekahi oligo (A) -containing sequence. lawelawe ʻo ia ma ke ʻano he laʻana, e like me ka mīkini i hoʻokumu ʻia no ka picornavirus VPg protein (44).He aha inā he "non-normative" ka manaʻo????ʻO ka hoʻomaka ʻana o ka (protein-induced) negative-strand RNA synthesis e hāʻawi i kahi loulou i nā ʻike, e hōʻike ana he UMP (ma kahi o UTP) i ka hopena (42) i ka coronavirus negative-strand RNA. nucleic acid Hoʻokaʻawale ʻo Dicer i ka hopena phosphorylated e kahi endonuclease kikoʻī uridine i ʻike ʻole ʻia.Inā hoʻokūpaʻa ʻia, hiki i kēia hana nucleic acid hydrolytic ke kōkua i ka hoʻokuʻu ʻana i ke ʻano oligomeric UMPylated o nsp9 mai ka hopena 5 ² o ka strand maikaʻi ʻole.ʻO ka hiki ke hana o nsp9 i loko o ka protein priming ua kūlike pū me nā haʻawina genetics mua, i hōʻike ʻia e pili ana ka nsp9 (a me nsp8) me ke koʻikoʻi a kikoʻī me ka mea cis-acting RNA i mālama ʻia ma kahi kokoke i ka hopena 3 o ka genome coronavirus.45).Wahi a kēia hōʻike, hiki ke nānā hou ʻia kēia mau ʻike mua a hoʻonui ʻia ma o ka noiʻi hou ʻana.
I ka hōʻuluʻulu manaʻo, ua hoʻoholo kā mākou ʻikepili i ka hana kikoʻī o kahi huaʻōlelo enzyme virus nested proprietary i hoʻopili ʻia me RdRp ma ka N-terminus.I ka coronavirus, ua hoʻohana ʻia kēia hana hou i ʻike ʻia e NiRAN-mediated UMPylator/NMPylator e hilinaʻi i ka Mn2+ a me nā koena Asn e pili ana a hoʻokumu i ka hoʻokumu ʻana o nā paʻa phosphoramidate (haʻahaʻa haʻahaʻa) me ka N-terminal primary amine.Ma o Mpro-mediated cleavage ma ka nsp8|9 cleavage site, hiki ke hoʻohana ʻia ka nsp9 target no NMPylation, e hōʻike ana i ka hoʻopili hana ma waena o ka protease a me ka domain NiRAN, e hoʻonui i RdRp.ʻO ka mālama ʻana i nā koena koʻikoʻi ma ka nsp12 NiRAN kahua ikaika a me ka nsp9 target, i hui pū ʻia me ka ʻikepili i loaʻa mai nā coronaviruses ʻelua me SARS-CoV-2, hāʻawi i nā hōʻike ikaika ʻo nsp9 NMPylation he coronavirus ʻO nā hiʻohiʻona Conservative kekahi hana koʻikoʻi i ka hoʻopiʻi virus.ʻO ka ʻikepili i loaʻa e alakaʻi iā mākou e hoʻoholo i ka hana kikoʻī o ke ʻano NMPylated o nsp9 i ka protein-induced RNA synthesis he hiʻohiʻona kūpono no ka coronavirus a me nā maʻi maʻi nested ʻē aʻe, a hiki i ka NiRAN ke kuhikuhi i nā protein ʻike ʻole ʻia.Hoʻoponopono i ka maʻi maʻi.Hoʻopili hoʻokipa.Inā hoʻokūpaʻa ʻia, ʻo ke komo ʻana o nā protein primers i ka viral RNA synthesis e hoʻonui i ke kaʻina affinity o nā kikowaena Mpro/3CLpro a me RdRp ma waena o ka hui coronavirus i ʻike mua ʻia a me ka picornavirus-like supergroup (9), i hoʻohui ʻia i kēia manawa i nā Pisonivirites i hoʻokumu hou ʻia. 46) ma ka māhele.
Hōʻike pū kā mākou ʻikepili e hiki ke hoʻohana ʻia nā hana enzyme maʻamau, koho a conservative i ʻike ʻia i loko o kēia haʻawina i mau pahuhopu no nā lāʻau antiviral.Hiki ke hoʻomohala ʻia nā mea hoʻohui i ka hoʻopaʻa ʻana (a me ka hoʻololi hou ʻana) o ka nsp9 N-terminus i mālama ʻia i ka pūnaewele ikaika o NiRAN i mau lāʻau antiviral maikaʻi a versatile, kūpono no ka mālama ʻana i nā coronaviruses o nā holoholona a me nā kānaka mai nā ʻano maʻi like ʻole. , me SARS-CoV-2 a me Middle East Respiratory Syndrome Coronavirus.
Ua hoʻonui ʻia ke kaʻina coding o ka protein coronavirus i hana ʻia i loko o kēia noiʻi e RT-PCR me ka hoʻohana ʻana i ka RNA i hoʻokaʻawale ʻia mai ka Huh-7 i loaʻa i ka HCoV-229E a i ʻole Vero E6 i loaʻa i ka SARS-CoV-2, a hoʻokomo ʻia me ka hoʻohana ʻana i nā kaʻina hana cloning maʻamau.pMAL-c2 (New England Biological Laboratory) a i ʻole pASK3-Ub-CHis6 (47) hōʻike vector (SI Appendix, Papa S1 a me S2).Ua hoʻokomo ʻia nā hoʻololi codon hoʻokahi e PCR-based site-directed mutagenesis (48).No ka hanaʻana i ka protein fusion MBP, ua hoʻololiʻia nā pūnaewele E. coli TB1 me ka pMAL-c2 plasmid construct (SI appendix, Table S1).Hoʻomaʻemaʻe ʻia ka protein fusion e ka amylose affinity chromatography a ʻoki ʻia me ka factor Xa.Ma hope mai, ua hoʻomaʻemaʻe ʻia ka protein C-terminal His6-taged e Ni-immobilized metal affinity chromatography (Ni-IMAC) e like me ka mea i wehewehe mua ʻia (49).No ka hanaʻana i ka protein fusion ubiquitin, ua hoʻohana nā pūnaewele E. coli TB1 i ka pASK3-Ub-CHis6 plasmid construct (SI Appendix, Tables S1 a me S2) a me ka pCGI plasmid DNA e hoʻopili ana i ka ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Hoʻololi (47).Ua hoʻomaʻemaʻe ʻia ka C-terminal His6-tagged coronavirus protein e like me ka mea i wehewehe mua ʻia (50).
Ua hana ʻia ka hoʻāʻo self-NMPylation o HCoV-229E nsp12-His6 e like me ka wehewehe ʻana ma EAV nsp9 (16).I ka pōkole, aia ka nsp12-His6 (0.5 µM) 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 µM buffer. ua like ka NTP a me ka 0.17 µM i ka [α32-P]NTP (3,000 Ci/mmol; Hartmann Analytic) ma 30 °C no 30 mau minuke.Ma nā mea ʻē aʻe (maʻamau) NMPylation assays o nsp12-mediated nsp9 NMPylation, ua hoʻoponopono ʻia nā ʻano hopena penei: nsp12-His6 (0.05 µM) a me nsp9-His6 (4 µM) i mua o 50 mM HEPES-KOH (pH 8.0). ), 5 mM DTT, 1 mM MnCl2, 25 µM i hōʻike ʻia i ka NTP, a me 0.17 µM i like me [α32-P]NTP.Ma hope o ka incubating no 10 mau minuke ma 30 ° C, ua hui pū ʻia ka hāpana hopena me ka SDS-PAGE sample buffer: 62.5 mM tris(hydroxymethyl)aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol a me 0.005% bromophenol polū.Ua denatured ka protein e ka hoʻomehana ʻana ma 90 ° C no 5 mau minuke a hoʻokaʻawale ʻia e 12% SDS-PAGE.Hoʻopaʻa ʻia ka gel a hoʻopaʻa ʻia me ka Coomassie Brilliant Blue solution (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), decolorized, a ʻike ʻia i kahi pale kiʻi phosphorescent no 20 mau hola (e ʻike i ka nsp12 mai NMPylation) a i ʻole (kiʻekiʻe) 2 mau hola (e loiloi i ka nsp9 NMPylation).Ua hoʻohana ʻia kahi kiʻi Typhoon 9200 (GE Healthcare) e nānā i ka pale a ua hoʻohana ʻia ʻo ImageJ e nānā i ka ikaika o ka hōʻailona.
No ka loiloi MS, ua hoʻohana ʻia ka 1 µM nsp12-His6 a me 10 µM nsp9 (me ka ʻole hexahistidine tag) i ka nānā ʻana o NMPylation (SI appendix, Table S1) a ua hoʻohana ʻia ka piʻi ʻana o ka 500 µM UTP a me GTP.Ma muli o ko lākou noʻonoʻo ʻana a me ka maikaʻi o ka protein i manaʻo ʻia, ua hoʻohana ʻia kahi ʻōnaehana Waters ACQUITY H-Class HPLC me kahi kolamu MassPrep (Waters) no ka hoʻopau ʻana i ka 1 a 10 µL o nā hoʻonā protein buffered online.Hoʻokuʻu ʻia ka protein desalted i loko o ke kumu electrospray ion o Synapt G2Si mass spectrometer (Waters) ma o ka gradient aʻe o ka buffer A (wai/0.05% formic acid) a me ka buffer B (acetonitrile/0.045% formic acid), a ʻo ka mahana o ke kolamu. 60 ° C a me kahi kahe kahe o 0.1 mL / min: elution isocratically me 5% A no 2 mau minuke, a laila he laina laina i 95% B i loko o 8 mau minuke, a mālama i 95% B no kekahi 4 mau minuke.
ʻIke ʻia nā ion maikaʻi me ka laulā nui o 500 a 5000 m/z.Ana ʻia ʻo Glu-fibrinopeptide B i kēlā me kēia 45 kekona no ka hoʻoponopono ʻana i ka hoʻoheheʻe nui.E hoʻohana i ka lako polokalamu mea kani MassLynx me ka hoʻonui MaxEnt1 e hoʻokaʻawale i ke kiko awelika ma hope o ka unuhi ʻana i ka baseline a me ka hoʻomaʻamaʻa.
Ua ʻeli ʻia ʻo UMPylated HCoV-229E nsp9 e ka hoʻohui ʻana i ka trypsin hoʻololi ʻia (Serva) a hoʻoulu ʻia i ka pō ma 37 °C.Ua hoʻohana ʻia kahi kolamu wili Chromabond C18WP (ʻāpana helu 730522; Macherey-Nagel) e hoʻopau i ka paʻakai a hoʻopaʻa i nā peptides.ʻO ka hope, ua hoʻoheheʻe ʻia ka peptide i 25 µL o ka wai, aia ka 5% acetonitrile a me 0.1% formic acid.
Ua kālailai ʻia nā laʻana e MS me ka Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Ka hope loa nanoâ HPLC nenoai (Dionex), lako me ka maʻamau hopena-kau 50 knm ??75 μm C18 RP kolamu i hoʻopaʻa ʻia me 2.4 μm magnetic beads (Dr. Albin Maisch High Performance LC GmbH) Hoʻohui i ka spectrometer mass online ma o ke kumu Proxeon nanospray;e hoʻokomo i 6 µL o ka hoʻoheheʻe trypsin i loko o 300 µm anawaena o loko ×??1 knm C18 PepMap kolamu pre-concentration (Thermo Scientific).Me ka hoʻohana ʻana i ka wai/0.05% formic acid ma ke ʻano he mea hoʻoheheʻe, ua paʻa ʻokoʻa ka hāpana a hoʻokaʻawale ʻia ma kahi kahe kahe o 6 µL/min.
Ua hoʻohana ʻia nā kaha kaha o ka wai/0.05% formic acid (solvent A) a me 80% acetonitrile/0.045% formic acid (solvent B) no ka hoʻokō ʻana i ka hoʻokaʻawale ʻana o nā peptides tryptic ma kahi kahe kahe o 300 nL/min: 4% B no 5 mau minuke, a laila 30 A linear gradient i 45% B i loko o nā minuke, a me ka hoʻonui laina i 95% solvent B i loko o 5 mau minuke.E hoʻohui i ke kolamu chromatographic i kahi nano-emitter (Proxeon), a kāpīpī pololei i ka eluent i ka capillary wela o ka spectrometer me ka hoʻohana ʻana i ka hiki o 2,300 V. ʻO ka nānā ʻana me ka hoʻonā ʻana o 60,000 i ka Orbitrap mass analyzer pili. me ka liʻiliʻi loa i ʻekolu ʻikepili MS/MS scans, dynamically kāpae ʻia no 30 kekona, me ka hoʻohana ʻana i ka linear ion trap collision induced dissociation a i ʻole ka ʻoi aku o ka ikehu collision dissociation i hui pū ʻia me ka orbitrap detection , ʻO ka hoʻonā ʻana he 7,500.
Ka manawa hoʻouna: ʻAukake-03-2021