Ihlelwe nguPeter Sarnow, Stanford University School of Medicine, Stanford University, California, yavunywa ngoDisemba 25, 2020 (ibuyekezwe ngo-Okthoba 25, 2020)
Sibika ukusebenzisana phakathi kwamayunithi amancane ekuphindaphindweni kwezakhiwo zokulotshwa kwe-coronavirus, ezibalulekile ekuphindaphindeni nasekulondolozeni ukuguquguquka.Sinikeze ubufakazi bokuthi isizinda se-NiRAN esihlotshaniswa ne-nsp12 sinomsebenzi wokudlulisa we-nucleoside monophosphate (NMP) ku-trans, futhi sihlonze i-nsp9 (iphrotheni ebopha i-RNA) njengokuhlosiwe kwayo.I-NiRAN yenza umzamo wokunamathisela okunamathiselwe kwengxenye ye-NMP endaweni egciniwe ye-nsp9 amino terminus ngokusabela okuncike kuma-ion angu-Mn2+ nezinsalela eziseduze ezilondoloziwe ze-Asn.Kutholwe ukuthi umsebenzi we-NiRAN kanye ne-nsp9 NMPylation kubalulekile ekuphindaphindeni kwe-coronavirus.Idatha isivumela ukuthi sixhumanise lo msebenzi womaka we-enzayimu yegciwane esidlekeni ekuqaphelisweni kwangaphambilini kumbono wokuthi ukuqaliswa kokuhlanganiswa kwe-RNA ekilasini lamagciwane e-RNA kusebenza futhi kuyaguquguquka.
I-RNA encike ku-RNA polymerase (RdRps) ye-Nidovirales (Coronaviridae, Arterioviridae, neminye imindeni engu-12) ixhunywe kusizinda se-amino-terminal (N-terminal) kuphrotheni engeyona yesakhiwo (nsp) ekhishwe ku-polyprotein, ebizwa ngokuthi i-NiRAN. I-1ab yakhiwe nge-viral main protease (Mpro).Ngaphambilini, umsebenzi we-GMPylation/UMPylation we-arterial virus we-NiRAN-RdRp nsp wabikwa, futhi kwaphakanyiswa ukuthi kukhiqizwe okwesikhashana ukuze kudluliselwe i-nucleoside monophosphate (NMP) kugciwane (okwamanje elingaziwa) kanye/noma izinto zeseli biopolymerization.Lapha, sibonisa ukuthi i-coronavirus (Human Coronavirus [HCoV]-229E kanye neSevere Acute Respiratory Syndrome 2) nsp12 (NiRAN-RdRp) inomsebenzi we-NMPylation oncike ku-Mn2+, osuselwe ku-nsp9 ngokwakhiwa kwe-Mpro-mediated nsp9 After i-N-terminal flanking nsps ikhishwa nge-proteolytically, i-phosphoramidate iboshelwe ku-amine eyinhloko (N3825) ku-N-terminal ye-nsp9.I-uridine triphosphate iyi-nucleotide ethandwayo kulokhu kusabela, kodwa i-adenosine triphosphate, i-guanosine triphosphate, ne-cytidine triphosphate nazo zingama-co-substrates afanelekayo.Izifundo zoguquko zisebenzisa i-recombinant coronavirus nsp9 kanye ne-nsp12 amaprotheni kanye nezakhi zofuzo eziguqulwe i-HCoV-229E zinqume izinsalela ezidingekayo ku-NiRAN-mediated nsp9 NMPylation kanye nokuphindaphindeka kwegciwane kusiko lamaseli.Idatha iqinisekise ukubikezelwa kwezinsalela zesayithi ezisebenzayo ze-NiRAN futhi yanquma indima ebalulekile yezinsalela ze-nsp9 N3826 ku-nsp9 NMPylation kanye nokuphindaphinda kwegciwane ku-vitro.Lezi zinsalela ziyingxenye yokulandelana kwe-N-terminal NNE tripeptide elondoloziwe futhi kubonakale kuwukuphela kwensalela eguquguqukayo ye-nsp9 nama-homologs ayo emndenini we-coronavirus.Lolu cwaningo lunikeza isisekelo esiqinile socwaningo olusebenzayo lomsebenzi we-NMPylation wamanye amagciwane afakwe esidlekeni futhi luhlongoza okuhlosiwe okungase kube khona ekuthuthukisweni kwemithi elwa namagciwane.
Igciwane le-RNA elinemicu ye-Nidovirales positive lithelela izilwane ezinomgogodla nezilwane ezingenamgogodla (1, 2).Lo myalelo njengamanje uhlanganisa imindeni eyi-14 (3), umndeni wayo weCoronavirus osufundwe kabanzi kule minyaka engama-20 edlule.Ngaleso sikhathi, ama-zoonotic coronaviruses amathathu avela ezilwaneni futhi abangela ukuqubuka okukhulu kwezifo ezinzima zokuphefumula kubantu.Kubandakanya izifo eziwubhadane eziqhubekayo ezibangelwa izifo ezinzima ezithathelwanayo.I-Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (4âââ7).Ama-Nidoviruses ahlanganyela ngenhlangano evamile yofuzo, futhi ingxenye encane ye-membrane-bound replication-transcription complex (RTC) ifakwe ikhodi ku-5-?²-terminal two-thirds kanye nengxenye eyinhloko yesakhiwo yezinhlayiya zegciwane, kanye nezinye izesekeli. .Iphrotheni, efakwe ku-3??² ekugcineni kwe-genome (1).Ngaphandle komndeni owodwa wamagciwane e-planarian (i-Monoviridae) (8), wonke amagciwane anesidleke afaka amayunithi amancane e-RTC kumafreyimu amabili amakhulu okufunda avulekile (ORF) ORF1a kanye ne-ORF1b, ahunyushwe esuka ku-genomic RNA ye.I-ORF1a ihlanganisa i-polyprotein (pp) 1a, kanye ne-ORF1a ne-ORF1b encode ngokuhlanganyela i-pp1ab.Ngokubamba iqhaza okuvamile kwe-main protease (Mpro) ebhalwe ngekhodi yi-ORF1a, kokubili i-pp1a kanye ne-pp1ab kucutshungulwa ngokwephrotheni ibe amaphrotheni angewona wesakhiwo (nsps), eyaziwa nangokuthi i-3CLpro, ngoba ine-homology ne-3Cpro ye-picornavirus ( 9).Lawa ma-nsps kucatshangwa ukuthi aqoqwe abe yi-RTC enkulu eguquguqukayo, enze ukwakheka kwe-genomic RNA (replication) kanye nesethi ye-subgenomic RNA (okulotshiweyo), futhi asetshenziselwa ukuxhumanisa inkulumo ye-ORF etholakala ezansi nomfula we-ORF1b (10? ? ?12).
I-RTC ewumgogodla ihlanganisa i-RNA polymerase (RdRp) encike ku-RNA (RdRp) (13), i-superfamily 1 helicase (HEL1) (14, 15) kanye nama-enzyme amaningana okucubungula i-RNA, afakwe ikhodi ku-ORF1b kanye nomndeni we-coronavirus Iqukethe i-nsp12-nsp16 kanye i-nsp9-nsp12 emndenini we-Arterioviridae (bona isithenjwa 10ââ 12).I-RdRp kanye ne-HEL1 zimelela izizinda ezimbili (ingxenye eyodwa kwesihlanu) ezilondoloziwe zegciwane lesidleke senyoni futhi zine-homology phakathi kwamanye amagciwane e-RNA.I-Core replicase kukholakala ukuthi isizwa amanye ama-subunits, okuhlanganisa nama-nsps ambalwa amancane akhishwe endaweni ye-carboxy-terminal (C-terminal) ye-pp1a, ezansi nomfula we-Mpro (coronavirus nsp5 kanye ne-arterial virus nsp4, ngokulandelana).Banokuvikela okukhethekile komndeni kanye nemisebenzi eyahlukene (kubuyekezwe kunkomba 10ââ12).
Kamuva nje, isizinda esinezici ezihlukile zokulandelana kwe-motif sitholwe ku-amino terminus (N-terminus) eduzane ne-RdRp kuwo wonke amagciwane avalelwe, kodwa awekho amanye amagciwane e-RNA (16).Ngokusekelwe endaweni yayo kanye nomsebenzi we-nucleotide transferase (nucleoside monophosphate [NMP] transferase) umsebenzi, lesi sizinda siqanjwe ngokuthi i-NiRAN (i-Nestvirus RdRp-related nucleotide transferase).Inhlanganisela yezizinda ezimbili ze-NiRAN-RdRp yakha i-nsp12 emndenini we-Coronaviridae kanye ne-nsp9 emndenini we-Arterioviridae, futhi kwenye i-nestoviridae, i-NiRAN-RdRp kulindeleke ukuthi ikhishwe njenge-nsp ezimele evela ku-polyprotein yegciwane.Ku-coronavirus, isizinda se-NiRAN siqukethe ??1/450 izinsalela futhi ixhunywe kusizinda se-C-terminal RdRp ngesifunda esixhumanisa (16?19).Ku-Equine Arteritis Virus (EAV) (Arteriviridae), i-recombinant nsp9 ibonisa imisebenzi ye-Mn2+ ion-dependent (self) UMPylation kanye ne-GMPylation, encike ezisekelweni ezintathu zokulandelana ezilondoloziwe ku-nestovirus, AN, BN kanye ne-CN Izinsalela ngokulandelana.Lapho u-N emele i-NiRAN) (16).I-N-terminal flanking yalezi motifs iyi-motif encane ye-preAN.Ezinye zalezi zinsalela ziphinde zilondolozwe ku-protein kinases ehlobene kude, lapho zikhonjiswe ukuthi zibandakanyeka ekubopheni i-nucleoside triphosphate (NTP) kanye nomsebenzi we-catalytic (20, 21).Ngokuvumelana nalokhu kuqashelwa, izinsalela zezindawo ezimbalwa ezibalulekile ezisebenzayo ku-pseudokinase SelO ezivela ku-Pseudomonas syringae zingahlanganiswa ne-SARS-CoV-2 nsp7/8/12/13 supercomplex esanda kushicilelwa.Izinsalela ze-Conserved Coronavirus NiRAN ezibekwe phezulu ku-electron microstructure.I-recombinant protein (17).Kucatshangwa ukuthi i-U/GMPylation ebhaliwe (self) izokhiqiza isimo sesikhashana sokudlulisa i-NMP ku-substrate (okwamanje engaziwa) (16), kanye nokufana kwesakhiwo phakathi kwe-NiRAN ne-protein kinase (17, 19) ) Ingabe umbono wokuthi I-NiRAN ishintsha amanye amaprotheni.
Izici eziningi, okuhlanganisa ukuhlotshaniswa kwayo okuhleliwe okuyingqayizivele nokuyingqayizivele namagciwane avalelwe kanye nokuhlukaniswa kwezakhi zofuzo ku-RdRp, kwenza i-NiRAN ibe i-enzyme eyinhloko yokulawula yamagciwane avalelwe, okubalulekile ekuveleni kwawo kanye nobunikazi bawo.Ngaphambilini, imisebenzi emithathu engaba khona ehilela i-NiRAN ukuze ilawule ukuhumusha kwe-genome/subgenomic noma ukuphindaphinda/ukuloba ibizwe.Uma kucutshungulwa idatha eyivelakancane nengaphelele etholakala ngaleso sikhathi, umsebenzi ngamunye unezinzuzo kanye nokubi kwawo (16).Kulolu cwaningo, sihlose ukuhlanganisa izifundo zamakhemikhali ezinto eziphilayo kanye nokubuyisela emuva izifundo zofuzo zama-coronavirus amelela lezi zinhlobo ezimbili, futhi sibeke esikutholile kusizinda sokuziphendukela kwemvelo soguquko lwemvelo lomndeni we-coronavirus, ukuze sithole ukuqonda kulo mbuso Oyimfihlakalo.Sibika intuthuko enkulu ekuqondeni kwe-NiRAN ngokuhlonza okuhlosiwe kwemvelo ku-RTC, okuthi (phakathi kwemibono emithathu etholakalayo) enikele endimeni yalesi sizinda ekuqaliseni ukuhlanganiswa kwe-RNA yegciwane esidlekeni.Lolu cwaningo luphinde luvule amathuba kwezinye izindima ze-NiRAN kusixhumi esibonakalayo sokusingatha igciwane.
Ukuze kubonakale izici ze-enzymatic zesizinda se-NiRAN esihlobene ne-corona virus nsp12, sikhiqize uhlobo lwe-human coronavirus 229E (HCoV-229E) nsp12 ku-E. coli, olunophawu lwe-His6 ku-C-terminus, futhi sahlanganisa amaprotheni ane- [α32-P ] Fudumeza kanye ne-NTP ebukhoneni be-MnCl2 njengoba kuchazwe Ezintweni Nezindlela.Ukuhlaziywa komkhiqizo wokusabela kubonise ukuba khona kwephrotheni ene-radiolabeled efuduka ne-nsp12 (106 kDa), okubonisa ukuthi i-coronavirus nsp12 igqugquzela ukwakheka kwe-covalent protein-NMP adducts, eyakhelwe ngokukhethekile nge-uridine monophosphate (UMP) (Figure 1A) Futhi B).Ukuhlaziywa kokulinganisa kubonise ukuthi uma kuqhathaniswa namanye ama-nucleotide, ukushuba kwesignali yokufakwa kwe-UMP kwanda nge-2 izikhathi ze-3 (Umfanekiso we-1C).Le datha ihambisana nomsebenzi obikezelwe we-NMP wokudlulisa wesizinda se-NiRAN se-coronavirus (16), kodwa ibonisa ukuthi izintandokazi ze-nucleotide zesizinda se-NiRAN se-coronavirus kanye negciwane le-arterial kuhlukile.
Umsebenzi we-Self-NMPylation we-HCoV-229E nsp12.(A) I-HCoV-229E nsp12-His6 (106 kDa) ifakwe i-[α-32P] NTP ebekiwe phambi kuka-6 mM MnCl2 imizuzu engu-30 (bona Izinto Ezibalulekile Nezindlela ukuze uthole imininingwane).Imikhiqizo yokusabela yahlukaniswa yi-SDS-PAGE futhi yangcoliswa nge-Coomassie brilliant blue.(B) Iphrotheni ene-radiolabeled ibonakala ngesithombe se-phosphorous.Izimo ze-nsp12-His6 kanye nezimpawu zesisindo samangqamuzana amaprotheni (ngama-kilodaltons) ziboniswa ku-A no-B. (C) Amandla esignali ye-radioactive (mean ± SEM) anqunywa kusukela ekuhloleni okuthathu okuzimele.*P≤0.05.Amandla esignali (iphesenti) ahlobene ne-UTP.
Nakuba imisebenzi ye-enzyme ehlobene ne-NiRAN iye yaboniswa ibalulekile ekuphindaphindeni kwe-EAV ne-SARS-CoV kusiko lamaseli (16), umsebenzi othize we-NiRAN kanye nezinhloso ezingaba khona akukanqunywa.Ukufana kwesakhiwo okusanda kubikwa phakathi kwe-NiRAN nomndeni wamaprotheni anama-protein kinase-like folds (17, 22) kusishukumisele ukuba sihlole i-hypothesis yokuthi i-NiRAN yenza i-NMPylation yamanye amaprotheni.Senze isethi yezinjongo ezingaba yi-homologous, okuhlanganisa amaprotheni angewona wesakhiwo abhalwe ngekhodi i-HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), ngalinye liqukethe ithegi ye-C-terminal His6 (i-appendix ye-SI, Ithebula S1) , Futhi faka lawa maprotheni nge-[α32-P] uridine triphosphate ([α32-P]UTP) lapho kukhona noma ingekho i-nsp12.I-albumin ye-bovine serum kanye ne-MBP-LacZα ye-fusion protein ekhiqizwe ku-E. coli isebenze njengezilawuli (Umfanekiso 2A, imizila 1 kuya ku-7).Iphrotheni ene-radiolabeled yahlaziywa nge-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) kanye ne-autoradiography, futhi kwatholakala ukuthi kukhona isignali eqinile yomsakazo ekuphenduleni okuqukethe i-nsp12 ne-nsp9.Indawo yesignali ihambisana nesisindo samangqamuzana e-nsp9, okubonisa i-Nsp12-mediated UMPylation ye-nsp9 (Umfanekiso 2B, ithrekhi 7).Awekho amanye amaprotheni okuhlola atholakale ene-UMPylated, okuholele ekutheni siphethe ngokuthi i-nsp9 iyingxenye ethile ye-nsp12.Ngokuvumelana nedatha ye-self-NMPylation eboniswe kuMfanekiso 1, i-nsp12 iyakwazi ukudlulisa wonke ama-NMP amane ku-nsp9, nakuba ukusebenza kahle kuhlukile, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Isithombe).3 A kanye B).Ngaphansi kwezimo ezisetshenziswe kulokhu kuhlolwa (nciphisa ukusabela kanye nesikhathi sokuchayeka, nciphisa ukugxila kwe-nsp12; izinto zokwakha nezindlela), i-self-NMPylation ye-nsp12 ayikwazanga ukutholwa (qhathanisa noMfanekiso 2B, umzila 7, kanye noMfanekiso 1B), o kubonakale kusebenza ngempumelelo (Nemizuliswano eminingi) i-UMP isuke ku-nsp12 iye ku-nsp9.Umsebenzi wokudlulisa i-UMP udinga ukuba khona kwama-ion angu-Mn2+, njengoba kukhonjisiwe kuMfanekiso 3C, kuyilapho umsebenzi wokudlulisa we-UMP omncane kuphela obonwe lapho kukhona i-Mg2+, futhi akukho msebenzi phambi kwamanye ama-divalent cations ahloliwe.Idatha efanayo yatholwa ekuhlolweni kwe-NMPylation okuqukethe i-cytidine triphosphate (CTP), i-guanosine triphosphate (GTP), ne-adenosine triphosphate (ATP) (isithasiselo se-SI, Umfanekiso S1).
I-HCoV-229E nsp12-mediated UMPylation ye-nsp9.Uchungechunge lwama-protein substrates (okuhlanganisa i-albumin ye-bovine serum, i-MBP-lacZα, nochungechunge lwe-HCoV-229E nsps olulebulwe nge-C-terminal His6 ebhalwe nge-ORF1a) lusetshenziswe ukuhlola umsebenzi we-UMPylation we-HCoV-229E nsp12-His6⁺-mediated amaprotheni.Faka iphrotheni nge-[α-32P] UTP imizuzu engu-10 uma ungekho (A) noma ubukhona (B) be-nsp12 njengoba kuchazwe ezintweni nasezindleleni.Phezulu kuka-A no-B, ijeli ye-SDS-polyacrylamide egcotshwe nge-Coomassie Brilliant Blue iyaboniswa, futhi ngaphansi kuka-A no-B, kuboniswa ama-autoradiogram ahambisanayo.Indawo ye-protein molecular mass marker (ngama-kilodaltons) inikezwe kwesokunxele.Isikhundla se-nsp12-His6 (B, phezulu) kanye nesignali ye-radioactive ebonwe ngesikhathi sokufakwa kwe-nsp12-His6 nge-nsp9-His6 (B, umzila 7) nakho kubonisiwe, okubonisa ukuthi i- [α-32P]UMP ku-nsp9-His6 (12.9 kDa), engazange ibonwe kwamanye amaprotheni ahlolwe.
I-HCoV-229E NiRAN-mediated biochemical and virological characterization ye-nsp9 NMPylation.(A no-B) Indima ye-nucleotide co-substrate esetshenziswe ekuphenduleni.I-Nsp12-His6 kanye ne-nsp9-His6 zixutshwe futhi zifukanyelwe lapho kukhona ama-NTP [α-32P] ahlukene ekuhlolweni okujwayelekile kwe-NMPylation.(A, phezulu) I-Coomassie-stained nsp9-His6 ihlukaniswe yi-SDS-PAGE.(A, phansi) I-Autoradiograph yendawo efanayo yejeli.(B) Umsebenzi ohlobene (okusho ukuthi ± SEM) phambi kwe-cofactor ye-nucleotide ekhethiwe kunqunywa ekuhloleni okuthathu okuzimele.*P≤0.05.(C) Indima ye-ion yensimbi.Kuboniswa ukuhlolwa okujwayelekile kwe-NMPylation phambi kwe-[α-32P] UTP nama-ion ensimbi ahlukene, ngayinye inokuhlanganiswa okungu-1 mM.Ku-C, phezulu, kuboniswa i-Coomassie stained nsp9-His6, futhi ku-C, ngezansi, kuboniswa i-autoradiography ehambisanayo.Usayizi wephrotheni enelebula (ngama-kilodaltons) uboniswa kwesokunxele sika-A no-C. (D) Ifomu eliguquguqukayo le-HCoV-229E nsp12-His6 eliphethe ukufaka esikhundleni se-amino acid okucacisiwe liku-[α-32P]UTP, njengoba kuchaziwe Ezisetshenziswa kanye Nezindlela.I-nsp9-His6 ene-radiolabele ekhiqizwe ku-NMPylation reaction itholwa nge-phosphorylation imaging (D, phezulu).Umsebenzi ohlobene uma uqhathaniswa nephrotheni yohlobo lwasendle (wt) uboniswa kokuthi D, futhi okuphansi kuthathwa njengesilinganiso (±SEM) kusukela ekuhlolweni okuthathu Okuzimele.Izinkanyezi zibonisa ukushintshwa kwezinsalela ezingalondoloziwe.(E) I-virus titer ku-culture supernatant yamaseli e-p1 atholwe emahoreni angama-24 ngemva kokutholakala kokutheleleka nge-plaque assay.Ukushintshanisa kwe-codon esizindeni se-NiRAN se-mutant enjiniyela ye-HCoV-229E kubonisiwe (izinombolo eziyinsalela zisekelwe endaweni yazo ku-pp1ab).I-RdRp yesayithi esebenzayo ye-mutant nsp12_DD4823/4AA engakwazi ukuphindaphinda isetshenziswe njengesilawuli.
Ukuze sithole ukuqonda okujulile kwesayithi elisebenzayo le-NiRAN futhi sinqume izinsalela ezihlobene nomsebenzi we-nsp9-specific NMP transferase, senze ukuhlaziya ukuguqulwa kwezinguquko, lapho sishintsha khona izinsalela ezilondolozayo ku-NiRAN AN, BN kanye ne-CN motifs ( I-16) I-Ala (isithasiselo se-SI, Umfanekiso S2).Ngaphezu kwalokho, umthelela wokushintshashintsha kwe-Arg-to-Ls noma i-Lys-to-Arg iye yahlolwa ezimweni ezimbili.Njengokulawula (okubi), izinsalela ezingalondoloziwe noma ezingalondoloziwe kancane esizindeni se-NiRAN sama-coronavirus namanye amagciwane afakwe esidlekeni athathelwa indawo i-Ala. Ishintsha i-K4116A (in motif preAN), K4135A (AN), R4178A (BN), D4188A (motif I-BN) kanye ne-D4280A (CN) inciphisa kakhulu noma iqede i-nsp9 NMPylation ngokusebenzisa i-nsp12, kuyilapho amaprotheni ane-conservative substitutes (R4178K) , K4116R) agcina u-60% kanye no-80% womsebenzi wawo, okubonisa ukuthi ukuxegiswa kwemikhawulo ohlangothini lwawo. amaketanga azwela ngokwe-physicochemically (Umfanekiso 3D).Ukushintsha ezinye izinsalela ezimbalwa ezilondoloziwe i-E4145A, D4273A, F4281A kanye ne-D4283A akuyingozi kangako, futhi i-nsp9 UMPylation yehliswa ngokusesilinganisweni kuphela.Imiphumela efanayo yatholwa ku-nsp9 NMPylation reactions ehilela amanye ama-NTP (Figure 3D kanye ne-SI appendix, Figure S3), eqinisekisa ukuthi imiphumela ephawuliwe ekufakweni okukhethekile kwe-amino acid izimele ohlotsheni lwe-nucleotide co-substrate esetshenzisiwe.Okulandelayo, sihlole umthelela ongaba khona walokhu kushintshwa kwe-nsp12 ekuphindaphindeni kwama-coronavirus kusiko lamaseli.Kuze kube manje, sisebenzise izifanekiso ezifanele ze-DNA (cDNA) ezakhiwe ngofuzo ezihlanganiswe egciwaneni lokugoma lokuhlangana kabusha (23, 24) ukuze silobe amaseli angu-5 -7.Ukuhlelwa kwegciwane elithathelwanayo lenzalo ekhiqizwe kulawa maseli kubonise ukuthi eziningi eziguquguqukayo ze-HCoV-229E NiRAN zazingenzeki (Umfanekiso 3E).Iqembu lezakhi eziguquguqukayo zegciwane ezingaphili lihlanganisa ezinye izindlela eziye zaboniswa ukususa noma ukunciphisa kakhulu umsebenzi wokudlulisa we-NMP ku-vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), kodwa kukhona ezinye izindlela ezimbili (K4116R, E4145A) % agodliwe?Umsebenzi wabo we-in vitro NMPylation uphakamisa ukuthi imikhawulo eyengeziwe iyabandakanyeka.Ngokufanayo, ezinye izinguquko ezimbili (R4178K, F4281A) ezibangele ukwehla okumaphakathi komsebenzi we-NiRAN in vitro NMPylation kukhiqize amagciwane aphilayo, nokho, lawa magciwane anciphisa kakhulu ama-titers ngokuphindaphinda.Ngokuhambisana nedatha yomsebenzi we-in vitro eboniswe kuMfanekiso 3D, esikhundleni sezinye izinsalela ezine ezingalondoloziwe ku-coronavirus kanye/noma amanye amagciwane avalelwe (K4113A, D4180A, D4197A, D4273A) (8, 16) akhiqiza amagciwane aphilayo Inzalo, naphezu kokuba nayo i-titer encishisiwe ngokumaphakathi uma iqhathaniswa negciwane lohlobo lwasendle (Umfanekiso 3E).
Ukuze ufunde ukuthi ingabe umsebenzi wokudlulisa we-NMP we-NiRAN-mediated we-NMP uncike esizindeni esisebenzayo se-RdRp, izinsalela ze-Asp ezimbili ezilondoloziwe ezibandakanyeka ekuhlelweni kwama-ion ensimbi e-divalent (11) ku-RdRp motif C zithathelwe indawo i-Ala. Iphrotheni engu-nsp12_DD4823/4AA igcina umsebenzi wayo we-NMPylation we-nsp9, okubonisa ukuthi umsebenzi we-nsp12-mediated in vitro nsp9 NMPylation awudingi umsebenzi we-polymerase (Isithasiselo se-SI, Umfanekiso S4).
Ngemva kokusungula umsebenzi wokudlulisa we-NMP othize we-nsp9 we-nsp12, sizame ukuveza i-NMP-nsp9 adduct nge-mass spectrometry (MS).I-spectrum ye-protein mass ye-recombinant HCoV-229E nsp9 ibonise inani eliphakeme ku-12,045 Da (Umfanekiso 4A).Ukwengezwa kwe-nsp12 akuzange kushintshe ikhwalithi ye-nsp9, okubonisa ukuthi i-nsp12 ne-nsp9 ngeke yakhe inkimbinkimbi ezinzile ngaphansi kwemibandela esetshenzisiwe (i-denaturation) (Umfanekiso 4A).Ebukhoneni be-UTP ne-GTP, ukukalwa kwesisindo sokusabela okuqukethe i-nsp9 kanye ne-nsp12 ngokulandelanayo kwabonisa ukuthi isisindo samaprotheni e-UTP sinyakaze u-306 Da, futhi isisindo samaprotheni e-GTP sahambisa u-345 Da, okubonisa ukuthi i-molecule ngayinye ye-nsp9 ibopha i-UMP noma i-GMP. (Isithombe 4) C kanye no-D).Kucatshangwa ukuthi amandla adingekayo ku-NiRAN-mediated nsp9 NMPylation avela ku-NTP hydrolysis kanye nokukhululwa kwe-pyrophosphate.Nakuba i-molar ephindwe ka-10 ye-nsp9 (okuhlosiwe) kune-nsp12 (i-enzyme) isetshenziswe kulokhu kusabela, i-NMPylation ephelele ye-nsp9 yabonwa, okubonisa ukuthi ukusebenzisana phakathi kwe-nsp12 ne-nsp9 kuhlala isikhathi esifushane, futhi i-nsp12 ingakwazi i-NMPylate ngaphezulu kwe-nsp9 in vitro molecule.
I-NMPylation eyodwa ye-nsp9 phambi kwe-nsp12 ne-UTP noma i-GTP.Kuboniswa i-deconvoluted ephelele ye-protein mass spectrum ye-HCoV-229E nsp9 (i-appendix ye-SI, Ithebula S1) (AD).(A) nsp9 iyodwa, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 phambi kwe-UTP, (D) nsp9 + nsp12-His6 phambi kwe-GTP.
Ukuze kutholakale izinsalela ze-nsp9 UMPylated by nsp12, i-nsp9-UMP yahlukaniswa nge-trypsin.Ama-peptide angumphumela ahlukaniswa yi-nano-high performance liquid chromatography (HPLC) futhi ahlaziywa nge-tandem mass spectrometry (MS/MS) ku-inthanethi.Ukuhlaziywa kwedatha kusetshenziswa iphakheji yesofthiwe ye-Byonic (Amamethrikhi amaprotheni) kubonise i-UMPylation ye-N-terminal amino acid.Lokhu kuqinisekiswa mathupha.I-tandem mass spectrum ye-peptide eyandulelayo [UMP]NNEIMPGK (i-SI appendix, Figure S5A) iveze ucezu kokuthi 421 m/z, okubonisa ukuthi i-UMP ibophezela kokusalela okungu-1 kwe-nsp9.
Ku-N-terminus ye-nsp9, i-Asn ilondolozwe phakathi kwamalungu e-Orthocoronavirinae (isithasiselo se-SI, Umfanekiso S6).Nakuba sikholelwa ukuthi i-N-terminal primary amine nitrogen iyona engase yamukele i-UMP, sinqume ukuthola ubufakazi obengeziwe bokubopha kwe-NMP ku-N-terminal.Ngalesi sizathu, i-peptide ye-N-terminal nsp9 engeyona i-NMPylated ne-NMPylated ehlanjululwe yi-HPLC yatholwa phambi kwe-acetone ne-sodium cyanoborohydride.Ngaphansi kwalezi zimo, ama-amine ayinhloko amahhala kuphela angashintshwa nge-propyl (25).I-peptide etholakala ku-N-terminal nsp9 enochungechunge lwe-NNEIMPGK iqukethe ama-amine amabili ayinhloko, eyodwa ku-N-terminus ye-Asn kanti enye kuchungechunge oluseceleni lwe-Lys ku-C-terminus.Ngakho-ke, amaqembu e-propyl angethulwa kuzo zombili iziphetho.Amakhromatogram e-ion akhishiwe ama-peptide angewona ama-NMPylated akhonjisiwe kusithasiselo se-SI, Umfanekiso S5B.Njengoba kulindelekile, i-N-terminal ne-C-terminal (mono)propylated (i-SI appendix, Figure S5B, upper lane) kanye nama-peptide ane-dipropylated (i-SI appendix, Umfanekiso S5B, umzila ophansi) angabonakala.Le phethini iyashintsha ngokusetshenziswa kwe-NMPylated N-terminal peptide ye-nsp9.Kulesi simo, ama-peptide e-C-terminal propylated kuphela angabonakala, kodwa i-N-terminal propylated peptide kanye nama-peptide e-dipropylated awabonakali (Isithasiselo se-SI, Umfanekiso S5C), okubonisa ukuthi i-UMP idluliselwe ku-N-terminal primary amine Ukuze uvimbele lokhu. iqembu ekwenzeni izinguquko.
Okulandelayo, sishintsha (nge-Ala noma i-Ser) noma sisuse izinsalela ezilondoloziwe ku-N-terminus ye-nsp9 ukuze sichaze imikhawulo eqondene ngqo nethagethi.Ngokusekelwe kudatha yethu ye-MS ebonisa ukuthi i-NiRAN yenza i-nsp9-NMP adduct ene-amine eyinhloko yensalela ye-N-terminal ye-nsp9, sacabanga ukuthi i-nsp9 NMPylation idinga i-viral master protease (Mpro, nsp5) ukuze ikhulule i-nsp9 N-terminal kusuka i-polyprotein precursor yayo.Ukuhlola le nkolelo-mbono, sikhiqize iphrotheni eyandulela i-nsp7-11 equkethe i-nsp9 ku-E. coli futhi senza ukuhlolwa okujwayelekile kwe-NMPylation phambi kwe- [α-32P] UTP (izinto nezindlela).Njengoba kuboniswe kuMfanekiso 5A (umzila 3), isandulela se-nsp7-11 esingasikiwe asinawo umsakazo we-nsp12.Ngokuphambene, uma i-nsp7-11 igoqwe yi-nsp5 ehlangene ukuze ikhulule i-nsp9 (kanye nezinye i-nsps) kusukela kusandulelo, iphrotheni ene-radiolabeled ehamba ne-nsp9 iyatholakala, okuqinisekisa isiphetho sethu sokuthi i-NiRAN kanye ne-N- Selective ukwakheka kwe-covalent nsp9-NMP adducts. .Itheminali eyinhloko ye-amine ye-N-terminal Asn (isikhundla 3825 kokuthi pp1a/pp1ab).Lesi siphetho siphinde sisekelwe ukuhlolwa kusetshenziswa i-nsp9 construct, equkethe izinsalela ezengeziwe eyodwa noma ezimbili ku-N-terminus.Kuzo zombili izimo, i-NiRAN-mediated UMPylation ye-nsp9 yaqedwa (Isithasiselo se-SI, Umfanekiso S7).Okulandelayo, sikhiqize iphrotheni enezinsalela ze-Asn eyodwa noma ezimbili ezisusiwe ekulandeleni kwe-peptide engu-3825-NNEIMPK-3832 ku-N-terminal ye-nsp9.Kuzo zombili izimo, i-nsp9 UMPylation ivinjwe ngokuphelele (Umfanekiso 5B), inikeza ubufakazi obengeziwe bokuthi i-nsp9 N-terminus yangempela isebenza njengesamukeli se-NMP.
Ukucutshungulwa kwe-proteolytic kwe-nsp9 kanye nendima yezinsalela ze-N-terminal ku-NSP12-mediated UMPylation.(A) I-nsp9 UMPylation idinga i-nsp9 N-terminal yamahhala.I-Nsp7-11-His6 ifakwe ngaphambili kokuthi 30 °C kusigcinalwazi sokutholwa kwe-NMPylation esiqukethe i-UTP lapho kukhona noma ingekho i-recombinant Mpro (nsp5-His6).Ngemva kwamahora angu-3, qala i-NMPylation assay ngokungeza i-nsp12-His6 njengoba kuchazwe kokuthi Izinto Ezibalulekile Nezindlela.Ukusabela okuqukethe i-nsp5-His6 (umzila 1) kanye ne-nsp9-His6 (umzila 2) kusetshenziswe njengesilawuli.Ngemuva kwemizuzu eyi-10, ukusabela kuye kwanqanyulwa futhi ingxube yokusabela yahlukaniswa yi-SDS-PAGE.Iphrotheni yayigcotshwe nge-Coomassie Brilliant Blue (A, phezulu).Isandulela se-Nsp7-11-His6 kanye nomkhiqizo ocutshunguliwe ophuma ku-nsp5-His6 mediated cleavage kukhonjiswa kwesokudla.Sicela uqaphele (ngenxa yobukhulu bazo obuncane) ukuthi i-nsp7 ne-nsp11-His6 azibonakali kule jeli, futhi ukusabela kuhambisana ne-nsp5-His6 (imizila 1 no-4; indawo ye-nsp5-His6 iboniswa yindilinga eqinile) noma i-nsp9-His6 (Umzila 2) iqukethe inani elincane le-MBP (ekhonjiswe yimibuthano evulekile) njengokungcola okuyinsalela ngoba ivezwa njengamaprotheni e-MBP ahlanganisayo (isithasiselo se-SI, Ithebula S1).(B) Okuhlukile kwe-Nsp9-His6 akunazo izinsalela ze-N-terminal ye-An eyodwa noma ezimbili (inombolo eyinsalela ngokuya ngendawo ethi pp1a/pp1ab) futhi iyahlanzwa futhi ifakwe i-nsp12-His6 kanye ne-[α-32P] UTP.B, SDS-PAGE egcotshwe nge-Coomassie ikhonjiswe phezulu, B, i-autoradiograph ehambisanayo ikhonjiswe ngezansi.Indawo yomaka wesisindo se-molecular (ngama-kilodaltons) iboniswa kwesokunxele.(C) Izinsalela ze-HCoV-229E nsp9-His6 N-terminal ezilondoloziwe zathathelwa indawo i-Ala noma i-Ser, futhi inani elifanayo lephrotheni lasetshenziswa ku-nsp12-His6 mediated UMPylation reaction.Imikhiqizo yokusabela yahlukaniswa yi-SDS-PAGE futhi yagcotshwa nge-Coomassie Brilliant Blue (C, phezulu), futhi i-radiolabeled nsp9-His6 itholwe nge-phosphorescence imaging (C, middle).Kusetshenziswa amaprotheni ohlobo lwasendle (wt) njengereferensi (isethelwe ku-100%), umsebenzi we-NMPylation ohlobene (okusho ukuthi ± SEM) ubalwa kusukela ekuhloleni okuthathu okuzimele.(D) Ama-virus titer ku-p1 cell culture supernatant yamaseli e-Huh-7 atheleleke ngamaseli e-HCoV-229E wild-type Huh-7, kanye nezinguquko eziphethe okushintshiwe okumisiwe kwe-amino acid ku-nsp9 kwanqunywa ngokuhlolwa kwe-plaque.I-RdRp motif C eguquguqukayo ephindwe kabili engu-DD4823/4AA engakwazi ukuphindaphinda isetshenziswe njengokulawula okungekuhle.
I-N-terminus ye-nsp9 (ikakhulukazi izikhundla 1, 2, 3, kanye ne-6) ilondolozwe kakhulu phakathi kwamalungu omndeni omncane we-Orthocoronavirinae (i-SI appendix, Figure S6).Ukuze kufundwe indima engaba khona yalezi zinsalela ku-nsp12-mediated nsp9 NMPylation, izinsalela ze-Asn ezimbili ezilandelanayo ku-N-terminus ye-nsp9 zathathelwa indawo i-Ala noma i-Ser (zodwa noma zihlangene).Uma kuqhathaniswa nohlobo lwasendle i-nsp9, esikhundleni se-N3825 nge-Ala noma i-Ser kuholele ekunciphiseni okuphindwe kabili kwe-Nsp12-mediated UMPylation (Umfanekiso 5C).Ngokuvumelana nesiphetho sethu sokuthi i-NMPylation yenzeka ku-N-terminal primary amine esikhundleni sochungechunge oluseceleni lwensalela ye-N-theminali, sibone i-NMPylation eyinsalela ebalulekile ngokushintshwa kwe-N3825A kanye ne-N3825S.Kuyathakazelisa ukuthi uma i-Asn yesibili ithathelwa indawo yi-Ala noma i-Ser, i-nsp9 UMPylation incishiswa ngokuqinile (ngaphezu kwezikhathi ze-10), kuyilapho ukufakwa esikhundleni kuka-Ala ezikhundleni 3, 4, no-6 kunomphumela olinganiselayo kuphela ku-nsp9 UMPylation (Umfanekiso 2). ).5C).Imiphumela efanayo yatholwa kusetshenziswa i-ATP, CTP noma i-GTP (isithasiselo se-SI, Umfanekiso S8).Ngokuhlangene, le datha ibonisa indima ebalulekile ye-N2826 (isikhundla 2 ku-nsp9) ku-nsp9 NMPylation.
Ukuze kutholwe ubufakazi obengeziwe bokuhlobana okusebenzayo phakathi kwe-N-terminus ye-nsp9 ne-NMPylation, senze ukuqondanisa okulandelanayo okuningi (MSA) kokulandelana kwe-nsp9 komndeni weCoronavirus (okuhluka phakathi kwezinsalela eziyi-104 neziyi-113) (Isithasiselo se-SI, Umfanekiso S6).Sezizonke, ezinhlotsheni ezingu-47 (ezaziwayo nezifakayo) zezinhlobo ezi-5 zomndeni omncane we-Orthocoronavirinae ezithelela izilwane ezincelisayo, izinyoni, nezilwane ezihuquzelayo, ziyi-8 kuphela izinsalela ezitholakale zingaguquguquki.Izinguquko ezibanzi kakhulu, ezihlanganisa ukususwa nokufakwa, zibonwe emijikelezweni phakathi kwezakhi zesakhiwo sesibili ze-nsp9, njengoba kunqunywe izifundo zesakhiwo zangaphambilini (26 ??28).Kutholwe izinsalela ezinhlanu ezingaguquki ku-β strand kanye ne-α helix yengxenye ye-C-terminal ye-nsp9.Izinsalela ezintathu ezingaguquki zakha motif ye-NNE yetheminali engu-N ye-nsp9.Kuvezwe ukuthi i-Asn yesibili yalesi motif iyona kuphela insalela engaguquki, ebuye yabelwa i-nsp9 yokucabanga ye-frog coronavirus ehlobene, futhi imele uhlobo lwe-Microhyla letovirus 1 emndenini omncane i-Letovirinae ye-Alphaletovirus.Ukongiwa kwezinsalela kuma-elementi esakhiwo sesibili se-nsp9 kungalungiswa ngokucatshangelwa kwesakhiwo ukuze kugcinwe ukugoqa noma izakhiwo zokubopha ezaziwayo ze-RNA.Kodwa-ke, lokhu kucabanga akubonakali kusebenza ekongiweni kwe-NNE, futhi ngaphambi kwalolu cwaningo, imvelo yezingqinamba ezikhawulela ukuhluka kokulandelana kwe-tripeptide yayifihliwe ngokuphelele.
Ukuze kunqunywe ukubaluleka kwe-nsp9-NMPylation nokongiwa kwe-NNE ekuphindaphindeni kwe-coronavirus, sikhiqize izinguquko eziguquguqukayo ze-HCoV-229E, ezithwala ukushintshwa okukodwa noma okukabili kwezinsalela ze-nsp9 N-terminal, okubonisa ukuthi i-nsp9 NMPylation iyingozi ku-vitro.Ngaphambi kokuthi siqale, sizama ukuphendula umbuzo wokuthi ingabe lokhu kushintsha (eduze kwesayithi le-nsp8|9 lokuhlukanisa) kuthinta ukucubungula kwe-proteolytic yesifunda se-C-terminal pp1a.Isethi ye-nsp7-11 polyprotein constructs equkethe ukushintshwa okuhambisanayo ku-N-terminus ye-nsp9 yakhiqizwa ku-E. coli futhi yasikwa nge-recombinant Mpro.Ukuqhekeka kwe-proteolytic kweziza ezine (okuhlanganisa isiza se-nsp9 flanking) akuthintwa kakhulu yinoma yikuphi okunye okwethulwe esikhundleni (i-appendix ye-SI, Umfanekiso S9), ngaphandle kwezinguquko zesakhiwo kulawa maprotheni aphazamisana ne-Mpro-mediated nsp8|9 cleavage ( Noma okunye) iwebhusayithi.
Amaseli e-Huh-7 adluliselwe ngobude be-genome HCoV-229E RNA, efaka esikhundleni se-Ala noma se-Ser kuma-tripeptides e-NNE alondoloziwe (N3825, N3826, kanye ne-E3827) ku-nsp9 N terminus, okubonisa ukuthi ukuguqulwa okuningi kuyabulala.Sikwazile ukuhlenga leli gciwane ngokushintsha i-Ser noma i-Ala ye-N-terminal Asn (N2835A noma i-N2835S), kodwa sehlulekile ukubuyisela leli gciwane ngokunye ukuguqulwa okukodwa nokukabili ngokulandelana kwe-NNE (N3826A, N3826S, NN3825/6AA, NN3825/6SS) , E3827A) (Umfanekiso 5D).
Le miphumela ikhombisa ukuthi ukuphindaphindeka kwama-coronavirus kusiko lezicubu kukhawulelwe (okufanayo noma okufanayo), kukhawulela ukuguqulwa kwemvelo kwezindawo ze-nsp9 NMPylation emzimbeni, futhi kusekela indima ebalulekile yale mpendulo emjikelezweni wokuphila wama-coronavirus.
Kusethi yokugcina yokuhlolwa, sikhiqize i-C-terminal His6 ebhalwe ukuthi SARS-CoV-2 nsp12 kanye ne-nsp9, kanye nezinhlobo ezimbili eziguqukayo ze-nsp12 ku-E. coli.Izinsalela zesayithi ezisebenzayo ezizindeni ze-NiRAN ne-RdRp bezikade zilandelana Sebenzisa i-Ala esikhundleni (Umfanekiso 6A kanye nesithasiselo se-SI, Ithebula S2).I-K4465 ku-SARS-CoV-2 nsp12 ihambisana ne-K4135 ku-HCoV-229E (Isithasiselo se-SI, Umdwebo S2), okubonakale kudingekile kumsebenzi we-NiRAN kanye nokuphindaphinda kwe-HCoV-229E (Umfanekiso 3D no-E).Lezi zinsalela futhi zihambisana nensalela ye-arterial virus EAV nsp9 K94, eboniswe ngaphambilini ukuthi iyadingeka ku-NiRAN self-UMPylation/self-GMPylation (16).Njengoba kuboniswe kuMfanekiso 6B, i-SARS-CoV-2 nsp12 inomsebenzi wokudlulisa we-UMP isebenzisa i-nsp9 njengengxenye engaphansi, kuyilapho i-nsp12_K4465A i-mutant yesayithi esebenzayo ingasebenzi.Ukushintshanisa okukabili ekulandeleni kwesici se-SDD se-RdRp motif C akuthinti umsebenzi wokudlulisa we-UMP (Umfanekiso 6B), okubonisa ukuthi umsebenzi we-RdRp awunawo umthelela oqondile ku-nsp9 UMPylation.Idatha efanayo yatholwa kusetshenziswa i-CTP, i-GTP ne-ATP (isithasiselo se-SI, Umfanekiso S10).Kafushane, le datha ibonisa ukuthi i-NiRAN-mediated nsp9 NMPylation inomsebenzi wokonga kuma-coronavirus omele isizukulwane esihlukile somndeni ongaphansi we-orthocoronavirus.
I-NMPylation ye-SARS-CoV-2 nsp12-mediated ye-nsp9.(A) Ijeli ye-Coomassie ene-SDS-polyacrylamide ebonisa iphrotheni ehlanganisiwe esetshenziswe kuhlolo lwe-NMPylation.Njengokulawula, iphrotheni eguquguqukayo enendawo esebenzayo esikhundleni sesizinda se-NiRAN (K4465A) kanye nesizinda se-RdRp (DD5152/3AA) ye-SARS-CoV-2 nsp12 yasetshenziswa.Izinombolo zezinsalela zisekelwe endaweni ku-pp1ab.(B) I-Autoradiograph yokutholwa kwe-UMPylation isebenzisa i-nsp9-His6 kanye ne-[α-32P]UTP njenge-substrate ye-nsp12-His6 (uhlobo lwasendle [wt] ne-mutant).Isisindo samangqamuzana (ngama-kilodalton) sephrotheni enelebula iboniswa kwesokunxele.
Izizinda ze-NiRAN zivame ukulondolozwa ku-Nidovirales (16), okubonisa ukuthi zenza ukusabela kwe-enzymatic kubalulekile ekuphindaphindeni kwe-Nidovirus.Kulolu cwaningo, sikwazile ukufakazela ukuthi isizinda se-NiRAN se-coronavirus sidlulisa i-NMP (ekhiqizwe ku-NTP) iye ku-nsp9, iphrotheni ebopha i-RNA engaqondakali ehilelekile ekuphindaphindekeni kwegciwane (26 ?? 29 ), ukuyicacisa njengento ehlosiwe yemvelo futhi umlingani we-coronavirus RTC.
Isizinda se-NiRAN sabelana ngezisusa ezintathu zokulandelana (AN, BN, kanye ne-CN), eziqukethe inani elincane kakhulu lezinsalela ezilondolozwe kuyo yonke imindeni ku-oda le-Nidovirales le-monophyletic kodwa elihluke kakhulu (8, 16).Ucwaningo lwakamuva lubonise ukuthi ahlobene ngokuhlelekile nomndeni ongenasici wamaprotheni anjenge-protein kinase, ekuqaleni ayebizwa ngokuthi umndeni wakwaSelO (17, 19, 22, 30, 31).Amaprotheni ahlobene ne-SelO ane-kinase folds, kodwa awanazo izinsalela zesayithi ezisebenzayo ezimbalwa kuma-kinases akudala (22, 32).Ngokusekelwe ekuhlehleni kwama-molecule e-ATP aboshwe endaweni esebenzayo futhi azinziswe ukusebenzisana okuthile, i-SelO yacatshangelwa futhi yaqinisekiswa kamuva ukuthi idlulisela i-AMP (esikhundleni se-phosphate) ku-protein substrate (22), kuyilapho enye iphrotheni efana ne-SelO efana ne-YdiU yebhaktheriya ine-. muva nje kuboniswe ukukhuthaza ukunamathiselwe kwe-UMP e-Tyr kanye nezinsalela Zakhe zamaphrotheni ahlukahlukene angaphansi (33).
Ukuze kuqinisekiswe futhi kwandiswe ukubikezela kwezinsalela zesayithi ezisebenzayo zesizinda se-coronavirus ye-NiRAN, sisebenzise izindlela ze-biochemical ne-reverse genetics ukwenza ukuhlaziya ukuguqulwa kwe-coronavirus nsp12 (Umfanekiso 3D no-E kanye nesithasiselo se-SI, Umfanekiso S3 kanye nethebula) S1â S4).Idatha ibonisa ukuthi ukushintshwa kwe-HCoV-229E K4135, R4178 kanye ne-D4280 nge-Ala kuqeda umsebenzi wokudlulisa we-NMP ku-vitro kanye nokuphindaphinda kwegciwane kusiko lweseli (Umfanekiso 3D kanye no-E kanye nezithasiselo ze-SI, Umfanekiso S3), zisekela ubukhona bazo ku-NTP γ-phosphate ( K4135, R4178) kanye nokuxhumana kwama-ion ensimbi esayithi asebenzayo (D4280).Ukushintshwa kwe-E4145A kweGlu elondoloziwe ebangeni legciwane lesidleke senyoni okwabikezelwa ukuthi kuzozinzisa indawo ye-K4135 (17) kwaboniswa ukuqeda ukuphindaphindeka kwegciwane, kodwa okumangazayo, umsebenzi wagcinwa ku-in vitro NMPylation assay (Umfanekiso 3D no-E kanye Isithasiselo se-SI, Umfanekiso S3 Kanye namathebula S1–S4).Ukubhekwa okufanayo kwenziwa lapho ukufaka esikhundleni okuhambisanayo kwethulwa ku-YdiU homolog ye-Salmonella typhimurium (E130A) (33).Sekuhlangene, le datha isekela umsebenzi wokulawula wale zinsalela ezilondoloziwe esikhundleni somsebenzi we-catalytic.
Ukushintsha izinsalela ze-Phe ezilondoloziwe (F4281A) phakathi kohlu lwe-nestovirus kusizinda se-HCoV-229E NiRAN (8) kuholele ekwehleni komsebenzi we-NMPylation ku-vitro kanye nokwehla okuphawulekayo kokuphindaphindeka kwegciwane kusiko lamaseli (Umfanekiso 3D, E kanye ne-SI) isithasiselo, Umfanekiso S3).Idatha ihambisana nomsebenzi obalulekile wokulawula wale nsalela, njengensalela ye-DFG ye-Homologous Phe eboniswe ngaphambilini.Ku-classical protein kinases, iyingxenye ye-loop ebophayo ye-Mg2+ futhi isiza ukuhlanganisa nokulawula umgogodla???Iyadingeka emsebenzini osebenzayo we-catalytic (32, 34).Ukufaka esikhundleni i-Ala ne-Arg kwezinsalela ze-K4116 (ku-preAN motif), ngokulandelana, kwaqeda ukuphindaphindeka kwegciwane futhi, njengoba bekulindelekile, kwaba nemiphumela ehlukile kumsebenzi wokudlulisa we-NMP ku-vitro, kuya ngeketango le-amino acid eseceleni elethulwe (Umfanekiso 3D Kanye no-E kanye nezithasiselo ze-SI. , Umfanekiso S3).Idatha yokusebenza ihambisana nolwazi lwesakhiwo, okubonisa ukuthi le nsalela isungule ukusebenzisana ne-ATP phosphate (17).Esizindeni se-NiRAN seminye imindeni yamagciwane akhiwe esidlekeni, isikhundla se-HCoV-229E pp1a/pp1ab K4116 sihlala u-Lys, Arg noma u-His (8), okubonisa ukuthi umkhawulo wokusebenza wale nsalela ethile uxegisiwe.Ukufakwa esikhundleni kwe-D4188A ne-D4283A kuqeda noma kunciphisa kakhulu umsebenzi we-enzyme futhi kuqede ukuphindaphinda kwegciwane (Umfanekiso 3).Lezi zinsalela ezimbili zilondolozwa kumagciwane amaningi (kodwa hhayi wonke) avalelwe (8), okubonisa umsebenzi obalulekile oqondene nomndeni othile kodwa okungenzeka ukuthi ongewona othuthukisayo.Ukushintshwa kwe-Ala kwezinye izinsalela ze-Lys ne-Asp (K4113A, D4180A, D4197A kanye ne-D4273A) ezingalondoloziwe ku-Coronaviridae noma eminye imindeni ye-Nestioviridae (8) isetshenziswe njengezilawuli.Njengoba kulindelekile, lezi zinguquko zibekezelelwa kakhulu, ngokuncipha kancane komsebenzi we-enzyme kanye nokuphindaphinda kwegciwane kwezinye izimo (Umfanekiso 3 kanye nesithasiselo se-SI, Umfanekiso S3).Sekukonke, idatha ye-coronavirus mutagenesis ihambisana kakhulu ne-self-GMP kanye nedatha yofuzo ehlehlayo ye-EAV NiRAN-RdRp (16), lapho i-EAV nsp9 (coronavirus nsp12 ortholog) insalela ye-K94 (ihambisana ne-HCoV- 229E K4135) imisebenzi ebalulekile), i-R124 (ihambisana ne-R4178), i-D132 (ihambisana ne-D4188), i-D165 (ihambisana ne-D4280), i-F166 (ihambisana ne-F4281).Ngaphezu kwalokho, idatha ye-HCoV-229E mutagenesis iyahambisana futhi yandiswa kusukela kudatha yofuzo ehlanekezelwe ye-SARS-CoV (16), efana kakhulu naleyo ephawulwe ku-CN motif ehambisanayo Phe-to-Ala mutant SARS-CoV_nsp12 i-phenotype echazwe -F219A ne-HCoV-229E_F4281A (Umfanekiso 3 D no-E kanye nesithasiselo se-SI, Umfanekiso S3 kanye neThebula S1-S4).
Uma kuqhathaniswa ne-EAV orthologs (16), ethanda ngokucacile i-UTP ne-GTP (ekuphenduleni kwe-self-NMPylation), ucwaningo lwethu lubonisa ukuthi isizinda se-coronavirus NiRAN (emelwe i-HCoV-229E kanye ne-SARS-CoV-2) singasebenza ngempumelelo. idlulisele Omane ama-NMP, nakuba kukhona okuncanyelwayo okuncane kwe-UMP (Izibalo 1 kanye no-3).Ukucaciswa okuphansi okuphansi kwe-co-substrate ethile ye-NTP kuhambisana nesakhiwo esisanda kubikwa se-SARS-CoV-2 nsp7/8/12/13, lapho i-ADP-Mg2+ ibophezela endaweni esebenzayo ye-NiRAN, kodwa hhayi ne-adenine Ingxenye. yokwakhiwa kokusebenzelana okuthile (17).Esifundweni sethu, uhlobo lwe-nucleotide olusetshenziswa ekuphenduleni kwe-NMPylation alunawo umthelela ohlukile emsebenzini we-protein eguquguqukayo (Isithasiselo se-SI, Umfanekiso S3), okubonisa ukuthi ayikho kulezi zinsalela ehlobene eduze nokubopha kwe-nucleobase ethile.Isisekelo sesakhiwo kanye nokubaluleka kwebhayoloji okungaba khona kokuthandwayo okuhlukile kwe-NTP co-substrate okuphawulwe ezizindeni ze-NiRAN zama-coronavirus kanye namagciwane e-arterial kusazofundwa;zingase zibe yiqiniso noma zibe ngenxa yemikhawulo yezifundo zazo.Njengamanje, ngeke kukhishwe ukuthi umsebenzi we-NMPylator ongase ube khona wesizinda se-arterial virus NiRAN (uma kuqhathaniswa nomsebenzi owake wabonakala ngaphambili we-self-NMPylation) unokuthandwa okuhlukile kwe-co-substrate, kucatshangelwa ukuthi ukufana phakathi kwe-arterial kanye ne-coronavirus. Isizinda se-NiRAN sisemkhawulweni waso.Qhathanisa Ngokulandelana (16).Uma kuqhathaniswa ne-pseudokinase SelO, esebenzisa i-Mg2+ njenge-cofactor, umsebenzi we-coronavirus kanye ne-arterial virus i-NiRAN incike ku-Mn2+ (16) (Figure 3C kanye ne-SI appendix, Figure S1).Ukuncika kwe-Mn2+ nokuthandwa okusobala kwe-UTP kuyisici esingajwayelekile samaphrotheni e-NMPylator, futhi kusanda kuqinisekiswa kuphrotheni ye-YdiU ye-Salmonella typhimurium, eyenza i-UMPylation encike ku-Mn2+ encike ku-Mn2+-protein chaperone ukuvikela amaseli echibini lokungeniswa kwe-stress Cell ATP ( 33).
Ukufana okusanda kuchazwa kwesakhiwo phakathi kwesizinda se-coronavirus NiRAN kanye ne-cell protein kinases (17, 19) kunikeza ukwesekwa okwengeziwe kwekhono le-NiRAN lokuxhumanisa i-NMP namanye amaprotheni esiwabikile kulolu cwaningo.Sigxile ekusesheni kwethu ekuqondeni okungaba khona kwe-NiRAN kumaphrotheni afakwe ikhodi yi-HCoV-229E ORF1a, aziwa ngokusiza ngokuqondile noma ngokungaqondile ikhophi efakwe ikhodi ye-RTC ye-ORF1b (12, 35).Ukuhlola kwethu kunikeza ubufakazi obuqand' ikhanda be-NMPylation esebenzayo neqondile ye-nsp9 (Umfanekiso 2).Uma iphrotheni eqondiwe isetshenziswa ngokweqisa kwe-molar ephindwe izikhathi eziyisi-8 kuye kweziyi-10 kunaleyo ye-enzyme (nsp12), kuyaqinisekiswa ukuthi i-nsp9 iphelele (mono)NMPized (Umfanekiso 4).Siphethe ngokuthi ukusebenzisana phakathi kwe-nsp12 ne-nsp9 okwesikhashana futhi ngeke kwakhiwe inkimbinkimbi ezinzile nge-nsp9 (uma engekho amanye amayunithi e-RTC).Lesi siphetho sisekelwa izifundo zokusebenzisana kwamaprotheni ku-SARS-CoV proteome (35).Ukuhlaziywa kwe-MS kuhlonze i-amine eyinhloko yensalela ye-N-terminal ye-nsp9 njengendawo ye-NMPylation (isithasiselo se-SI, Umfanekiso S5).Ukwakhiwa kwebhondi ye-phosphoramidate kanye neqembu le-amino le-N-terminal lihlukanisa umsebenzi we-NMPylation we-NiRAN-mediated kusukela ku-Pseudomonas syringae SelO-mediated AMPylation reaction, eyenza ukwakheka kwe-O-linked AMP e-Ser, Thr, noma Tyr residues Peptide adduct ( 22), kanye ne-S. typhimurium YdiU yakha i-O-linked (ne-Tyr) kanye ne-N-linked (with His) peptide-UMP adducts.Ulwazi olulinganiselwe olutholakala emndenini we-SelO wamaphrotheni lubonisa ukuthi amalungu alo mndeni omkhulu wamaprotheni ahluke kakhulu ekwakhekeni kwe-peptide-NMP adducts.Lokhu ukuqaphela okuthakazelisayo okudinga ukufundwa okwengeziwe.
Idatha etholwe kulolu cwaningo isiholele ekucabangeni ukuthi i-NMPylation ye-nsp9 idinga i-N-terminus yamahhala.Kumongo wokuphindaphinda kwegciwane, lokhu kuzohlinzekwa ngokuqhekeka kwe-proteolytic kwesayithi lokucubungula le-nsp8|nsp9 ku-replicase polyprotein pp1a elamula i-Mpro kanye ne-pp1ab.Kuma-coronavirus amaningi, umehluko phakathi kwalesi siza esithile (i-VKLQ|NNEI ku-HCoV-229E) kanye nazo zonke ezinye izingosi ze-Mpro cleavage ze-coronavirus yi-Asn (kunezinye izinsalela ezincane, njenge-Ala, i-Ser Noma Gly) ithatha i-P1â???Indawo (36).Idatha ye-peptide cleavage etholwe ocwaningweni lwangaphambili ibonise ukuthi ukusebenza kahle kwe-cleavage kwesayithi le-nsp8|nsp9 kwakungaphansi kunakwamanye amasayithi, okubonisa ukuthi 1) lesi sizindalwazi singase sibe nendima yokulawula ekucubunguleni okuhambisana ngesikhathi kwe-C-terminal. pp1a isifunda, noma 2) a Indima ye-nsp9 N-terminus ekhethekile elondoloziwe ekuphindaphindeni kwegciwane (37).Idatha yethu (Umfanekiso 5A) ibonise ukuthi uhlobo lokuphinda lwenziwe lwe-nsp9 oluphethe ukulandelana kwe-N-terminal lwangempela lwenziwe nge-NMP ngempumelelo yi-nsp12.Ukulandelana kwe-N-terminal flanking kususwe yi-factor Xa (nsp9-His6; i-appendix ye-SI, Ithebula S1) noma i-Mpro-mediated cleavage (nsp7-11-His6; Umfanekiso 5A kanye ne-SI appendix, Ithebula S1).Okubalulekile, isandulela esine-nsp9 esine-nsp9 esingasikiwe i-nsp7-11-His6 sibonise ukumelana ne-NMPylation ye-nsp12, ehambisana nedatha yethu, okubonisa ukuthi i-adduct ye-nsp9-NMP yakhiwe nge-N-terminal primary amine ( isithasiselo se-SI, Umfanekiso S5) .Ukuze sithole ukuqonda okujulile kokucaciswa kwe-substrate ye-NiRAN, sibe sesigxila kuzinsalela eziseduze ze-N-terminal ze-nsp9.Uma engekho amanye amaprotheni, ayakwazi ukuguquguquka ngokwesakhiwo, avimbele ukuthi angabonwa ngendlela engabhaliwe ye-nsp9 (26 28, 38), ebonisa ukuhlukahluka kwemvelo okulinganiselwe Lokhu kungenxa yokulandelana okubalulekile okuqondile (hhayi okuhlobene nesakhiwo sesibili) umsebenzi we-nsp9 N-terminal fragment.Ukushintshaniswa kwe-Ala kwezinsalela ezilondoloziwe kulesi sifunda (Izibalo 5C kanye ne-D kanye nesithasiselo se-SI, Umfanekiso S8) kuveza ukuthi i-N3826 ibalulekile ku-nsp9 NMPylation in vitro, kuyilapho esikhundleni se-N3825A kanye ne-E3827A kuholela ekwehleni kwe-NMPylation, kuyilapho i-M3829A ne-P38 ishintshashintsha .Ngokusobala kuthinta i-nsp9 NMPylation.Nakuba ukufakwa esikhundleni kwe-N-terminal Asn (N3825A, N3825S) kunomphumela omaphakathi kuphela ku-nsp9 NMPylation kanye nokuphindaphindeka kwegciwane kusiko leseli (Umfanekiso 5C no-D), ukususwa kokulandelana kwezinsalela ze-Asn ku-N-terminal 3825-NN dipeptide. kufakazelwe ukuthi Kuyingozi kumagciwane, okubonisa ukuthi insalela ye-Asn eyodwa iyadingeka ngaphambi kwenye insalela ku-N-terminus, okungcono kakhulu ku-Asn, nakuba kubonakala sengathi ukushintshwa kwezinsalela ezifanayo kungabekezelelwa ngokwengxenye (Umfanekiso 5B, C, kanye no-D).Siphetha ngokuthi i-dipeptide ye-3825-NN, ikakhulukazi izinsalela ze-N3826 ezilondoloziwe nezibalulekile ngaphakathi kwebanga le-coronavirus (i-appendix ye-SI, Umfanekiso S6), iqinisekisa ukubophezela okulungile nokuma kwe-nsp9 N-terminus kusayithi elisebenzayo le-NiRAN.
Ukufaka esikhundleni i-Ala (E3827A) ye-Glu egciniwe yayo yonke imindeni emincane igcina i-nsp9 NMPylation in vitro kodwa ibulala amagciwane kusiko lweseli (Umfanekiso 5C no-D), okubonisa umsebenzi owengeziwe wale nsalela, ngokwesibonelo, ekusebenzelaneni okubalulekile (i-NMPylated noma engaguquliwe. ) nsp9 I-N-terminus nezinye izici ezihilelekile ekuphindaphindeni kwegciwane.Ukuguqulwa kwe-Nsp9 akuzange kuthinte inqubo ye-proteolytic ye-nsp9 nanoma iyiphi i-nsps eseduze (39) (I-SI Appendix, Figure S9), okubonisa ukuthi ama-phenotypes abulalayo wezinguquko ezimbalwa ze-nsp9 eziboniwe awazange abalwe ukungasebenzi kahle kwenqubo ye-C proteolytic-terminal pp1a .
Idatha engenhla ihlinzeka ngobufakazi bokuthi ngemva kokuphathwa kwe-Mpro-mediated ye-nsp8|9 cleavage site ku-pp1a/pp1ab, i-N-terminus ye-nsp9 ingaba UMPylated (noma ilungiswe kancane ngenye i-NMP).Ukwengeza, ukongiwa okuhle kakhulu kwe-N-terminus ye-nsp9 (kufaka phakathi ubunye kanye nezinsalela ze-Asn ezingaguquki emndenini we-coronavirus) kanye nedatha yofuzo ehlanekezelwe etholwe kulolu cwaningo (Izibalo 3E kanye no-5D) kusenze saphetha ngokuthi i-nsp9 NMPylation echaziwe. ihlobene nebhayoloji futhi ibalulekile ekuphindaphindeni kwe-coronavirus.Imiphumela yokusebenza yalokhu kuguqulwa kusazocutshungulwa, ngokwesibonelo, mayelana nomsebenzi ochazwe ngaphambilini (ongacacisiwe) we-nsp9 (ifomu elingashintshiwe) umsebenzi wokubopha we-RNA (2628).I-N-terminal NMPylation ingase futhi ithinte ukusebenzisana kwe-nsp9 namaphrotheni noma ama-substrates e-RNA noma ukwakheka kwemihlangano ehlukene yamazinga amane.Lokhu kubonwe ezifundweni zesakhiwo futhi kuqinisekisiwe ukuthi kuhlobene ngokusebenza nokuphindaphinda kwe-coronavirus, nakuba ikakhulukazi uma kungekho Esimeni salokhu kuguqulwa (26- âââ29, 40).
Yize ukucaciswa okuqondisiwe kwesizinda se-coronavirus ye-NiRAN kusadinga ukucaciswa kabanzi, idatha yethu ibonisa ukuthi ukucaciswa kwephrotheni okuqondiwe kwesizinda se-coronavirus ye-NiRAN kuncane kakhulu.Nakuba ukulondolozwa kwezinsalela zesizinda esisebenzayo (8, 16) esizindeni se-NiRAN sayo yonke imindeni ye-nidovirus kusekela ngokuqinile umsebenzi we-NMPylator egciniwe lawa maprotheni, ubunikazi bezinsalela zephaketheni ezibophayo ze-substrate zalesi sizinda Ukongiwa kanye nokongiwa kusazobonakala. , futhi ingase yehluke phakathi kwemindeni eyahlukene yezinhloso ze-Nidovirales.Ngokufanayo, okuhlosiwe okufanelekile kwamanye amagciwane avalelwe kusazonqunywa.Angase abe ama-orthologs akude e-nsp9 noma amanye amaprotheni, ngoba ukulandelana okungaphandle kwezizinda eziyisihlanu eziphindaphindayo ezivame ukulondolozwa kumagciwane afakwe esidlekeni azilondolozwa kancane (8), okuhlanganisa uhlu lwe-genome phakathi kwe-Mpro ne-NiRAN, Phakathi kwazo, i-nsp9 itholakala endaweni i-coronavirus.
Ukwengeza, okwamanje asikwazi ukukhipha ukuthi kungenzeka ukuthi isizinda se-NiRAN sinezinjongo ezengeziwe (kuhlanganise namaselula).Kulokhu, kufanelekile ukusho ukuthi ama-homologues amagciwane kule phrotheni evelayo i-NMPylators (NMPylators) (30, 31) ibonakala "inezilawuli eziyinhloko"?I-NMP ilungisa amaprotheni amaselula ahlukahlukene ukuze alawule noma asuse imisebenzi yawo ephansi, ngaleyo ndlela abambe iqhaza ezinqubweni ezihlukahlukene zebhayoloji, njengokuphendula kwengcindezi yeselula kanye ne-redox homeostasis (22, 33).
Kulolu cwaningo (Izibalo 2 no-4 kanye neSithasiselo se-SI, Izibalo S3 kanye ne-S5), sikwazile ukufakazela ukuthi i-nsp12 idlulisele ingxenye ye-UMP (NMP) endaweni eyodwa (elondoloziwe) ku-nsp9, kuyilapho amanye amaprotheni engazange ashintshwe esetshenziswa Ngaphansi kwezimo, ukucaciswa kwe-substrate okuchazwe kahle (kunokuba kuxege) kuyasekelwa.Ngokuvumelana nalokhu, uma kuqhathaniswa ne-N-terminal nsp9 NMPylation, umsebenzi we-NMPylation we-nsp12 uphansi kakhulu, ukutholwa kwawo kudinga isikhathi eside sokuchayeka kwe-autoradiography, futhi kusetshenziswa ukwanda okuphindwe ka-10 kokugxilisa ingqondo kwe-nsp12.Ngaphezu kwalokho, ukuhlaziya kwethu kwe-MS kuhlulekile ukunikeza ubufakazi be-NMPylation ye-nsp12, okuphakamisa ukuthi isizinda se-NiRAN self-NMPylation (okungcono kakhulu) kuwumsebenzi wesibili.Kodwa-ke, kufanele kuqashelwe ukuthi ezinye izifundo zinikeze ubufakazi bokuqala bokuthi isimo se-self-AMPylation se-NMPylator yebhaktheriya singalawula umsebenzi wabo we-NMPylation kwamanye ama-protein substrates (22, 33).Ngakho-ke, ucwaningo olwengeziwe luyadingeka ukuze kuphenywe imiphumela engaba khona yokusebenza kwemisebenzi ye-self-NMPylation ebikwe ku-EAV nsp9 (16) kanye ne-coronavirus nsp12 (lolu cwaningo), okuhlanganisa nomthelela ohlongozwayo ofana ne-chaperone ekugoqweni kwesizinda se-C-terminal RdRp ( 16) .
Ngaphambilini, imibono eminingana ephathelene nemisebenzi engenzeka engezansi yomfula wesizinda se-nidoviral se-NiRAN iye yacatshangelwa, okuhlanganisa i-RNA ligase, i-RNA -capped guanylate transferase kanye nomsebenzi wokuqala wamaprotheni (16), kodwa ayikho neyodwa yazo ehambisana nemisebenzi etholakalayo eya phansi.Ulwazi olutholwe ezikhundleni ezilandelayo luyisikhathi esifanayo ncamashi ngaphandle kokwenza ukuqagela okwengeziwe.Idatha etholwe kulolu cwaningo ihambisana kakhulu (kodwa ayikwazi ukufakazela) ukuthi isizinda se-NiRAN sihilelekile ekuqalisweni kwe-RNA synthesis eyenziwe ngamaprotheni.Ngaphambilini bekukholelwa ukuthi umsebenzi wesizinda se-NiRAN ngo-5 ??²-I-RNA capping noma ukusabela kwe-RNA ligation akuthintwa yilokhu kanye Nosekelo lwenye idatha.Ngakho-ke, isibonelo, indawo esebenzayo ye-NiRAN ibhekwa njengebandakanya i-Asp elondoloziwe njengesisekelo esijwayelekile (D252 ku-Pseudomonas syringae SelO; D4271 ku-HCoV-229E pp1ab; D208 ku-SARS-CoV-2 nsp12) (Isithasiselo se-SI, umfanekiso 2 ).S2) (17, 22, 33), kuyilapho i-catalysis ku-ATP-encike ku-RNA ligase kanye ne-RNA capping enzyme yenziwa yi-covalent enzyme-(lysyl-N)-NMP intermediate, ehilela insalela ye-Lys engaguquki. 41).Ngaphezu kwalokho, ukucaciswa okuphawulekayo okusekelwe ekulandelaneni okuphawulekayo kwe-coronavirus NiRAN kokuhlosiwe kwamaprotheni alondoloziwe kanye nokucaciswa okukhululekile kwama-co-substrates e-NTP (ikhetha i-UTP) kuphikisa i-enzyme ye-capping ye-NiRAN-mediated noma imisebenzi efana ne-RNA ligase.
Ngokusobala, mningi umsebenzi owengeziwe odingekayo ukuze kuqinisekiswe futhi, uma kufakazelwe, kucaciswe ngeqhaza elingase libe khona le-nsp9-UMP (nsp9-NMP) ku-RNA synthesis eyenziwe ngamaprotheni, ezoxhumanisa imibiko eminingana ethokozisayo kodwa (kuze kube manje) ebikwe ngaphambilini. .Okuphawuliwe okukodwa.Isibonelo, kunqunywe ukuthi isiphetho se-RNA-negative-strand ye-coronavirus siqala nge-oligo(U) strand (42, 43).Lokhu kuqaphela kuhambisana nombono wokuthi ukuhlanganiswa kwe-RNA yomucu ongemuhle kuqaliswa ngokubophezela uhlobo lwe-UMPylated lwe-nsp9 ku-poly(A) umsila (izibangeli), ezingathuthukiswa ngokubopha kwayo i-RNA Umsebenzi kanye/noma ukusebenzisana enye iphrotheni ye-RTC.Ingxenye ye-UMP ehlinzekwe yi-nsp9 ingase isetshenziswe “njengesiqalo” se-oligouridylation engu-nsp7/8/nsp12, kusetshenziswa umsila ongu-3??²-poly(A) ku-genomic RNA noma Okunye ukulandelana okuqukethe i-oligo (A) isebenza njengesifanekiso, esifana nendlela esungulwe iphrotheni ye-picornavirus VPg (44).Kuthiwani uma isiphakamiso “singesona esivamile”????Ukuqaliswa kokuhlanganiswa kwe-RNA (i-protein-induced) negative-strand RNA kunikeza isixhumanisi kokuphawuliwe, okubonisa ukuthi i-coronavirus negative-strand RNA ine-UMP (esikhundleni se-UTP) ekupheleni kwayo (42), okucatshangwa ukuthi ikhombisa ukuthi i-nucleic acid Dicer ihlukanisa isiphetho se-phosphorylated yi-endonuclease eqondene ne-uridine engaziwa.Uma kuqinisekisiwe, lo msebenzi we-nucleic acid hydrolytic ungasiza ukukhulula uhlobo lwe-oligomeric UMPylated lwe-nsp9 kusukela ekugcineni okungu-5 ² komucu ongenayo ongenayo.Indima engaba khona ye-nsp9 ekuqalisweni kwamaprotheni iphinde ihambisane nezifundo zofuzo ezihlanekezelwe ezedlule, ezibonise ukuthi i-nsp9 (kanye ne-nsp8) isebenzisana ngokujulile futhi ikakhulukazi nesici esilondoloziwe se-RNA esisebenza eduze nesiphetho esi-3 se-coronavirus genome.45).Ngokwalo mbiko, lokhu okubhekwe ngaphambili manje kungabuyekezwa futhi kwandiswe ngocwaningo olwengeziwe.
Kafushane, idatha yethu inqume umsebenzi othile wethegi ye-enzyme yegciwane exhunywe ku-RdRp ku-N-terminus.Ku-coronavirus, lo msebenzi osanda kutholwa we-NiRAN-mediated UMPylator/NMPylator usetshenziselwa ukuthembela ku-Mn2+ nezinsalela eziseduze zase-Asn futhi ubangele ukwakheka kwamabhondi (amandla aphansi) we-phosphoramidate ne-N-terminal primary amine.Ngokuqhekeka kwe-Mpro-mediated endaweni ye-nsp8|9 yokuhlukanisa, okuqondiwe kwe-nsp9 kungasetshenziswa ku-NMPylation, okubonisa ukuhlangana okusebenzayo phakathi kwe-protease nesizinda se-NiRAN, esidlulela ku-RdRp.Ukongiwa kwezinsalela ezibalulekile endaweni esebenzayo ye-Nsp12 NiRAN kanye nethagethi ye-nsp9, kuhlanganiswe nedatha etholwe kuma-coronavirus amabili okuhlanganisa ne-SARS-CoV-2, kunikeza ubufakazi obuqinile bokuthi i-nsp9 NMPylation iyi-coronavirus izici ze-Conservative nazo ziyisinyathelo esibalulekile ekuphindaphindeni kwegciwane.Idatha etholakalayo isiholela ekuphetheni ngokuthi indima ethile ye-NMPylated ifomu le-nsp9 ekuhlanganiseni kwe-RNA eyenziwe ngamaprotheni iyisimo esinengqondo se-coronavirus namanye amagciwane avalelwe, futhi i-NiRAN ingase futhi iqondise amanye amaprotheni angaziwa.Lawula igciwane.Ukusebenzisana kosokhaya.Uma kuqinisekisiwe, ukubandakanyeka kwama-protein primers ekuhlanganiseni kwe-RNA yegciwane kuzokhulisa ukulandelana kwesizinda se-Mpro/3CLpro ne-RdRp phakathi kwe-coronavirus etholwe ngaphambilini kanye ne-picornavirus-like supergroup (9), manje esehlanganiswe ku-Pisonivirites esanda kusungulwa ( 46) esigabeni.
Idatha yethu iphinda ibonise ukuthi imisebenzi eyisisekelo, ekhethayo futhi elondolozayo ye-enzyme ekhonjwe kulolu cwaningo ingasetshenziswa njengezinhloso zemithi elwa namagciwane.Amakhompiyutha aphazamisa ukubophezela (kanye nokuguqulwa okulandelayo) kwe-nsp9 N-terminus egciniwe endaweni esebenzayo ye-NiRAN ingathuthukiswa ibe yimithi elwa namagciwane esebenzayo futhi eguquguqukayo, elungele ukwelapha ama-coronavirus ezilwane kanye nabantu asuka ku-(sub)infections ehlukene. , okuhlanganisa i-SARS-CoV-2 kanye ne-Middle East Respiratory Syndrome Coronavirus.
Ukulandelana kwekhodi yephrotheni ye-coronavirus ekhiqizwe kulolu cwaningo kwandiswa yi-RT-PCR isebenzisa i-RNA ehlukanisiwe ku-Huh-7 etheleleke nge-HCoV-229E noma i-Vero E6 etheleleke nge-SARS-CoV-2, futhi yafakwa kusetshenziswa izinqubo ezijwayelekile zokuhlanganisa.pMAL-c2 (New England Biological Laboratory) noma pASK3-Ub-CHis6 (47) i-vector expression (SI Appendix, Tables S1 and S2).Ukushintshwa kwe-codon eyodwa kwethulwe yi-PCR-based site-directed mutagenesis (48).Ukuze kukhiqizwe iphrotheni ehlanganisiwe ye-MBP, amaseli e-E. coli TB1 ashintshiwe ngokwakhiwa kwe-plasmid ye-pMAL-c2 efanele (isithasiselo se-SI, Ithebula S1).Iphrotheni ehlanganisiwe yahlanzwa nge-amylose affinity chromatography futhi yahlanganiswa ne-factor Xa.Kamuva, iphrotheni ethegi ye-C-terminal His6-tagged yahlanzwa yi-Ni-immobilized metal affinity chromatography (Ni-IMAC) njengoba kuchazwe ngaphambilini (49).Ukuze kukhiqizwe amaprotheni e-ubiquitin fusion, amaseli e-E. coli TB1 asebenzise ukwakhiwa kwe-plasmid ye-pASK3-Ub-CHIs6 efanele (Isithasiselo se-SI, Amathebula S1 kanye ne-S2) kanye ne-pCGI plasmid DNA efaka ikhodi ye-ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Uguquko (47).I-C-terminal His6-tagged coronavirus protein yahlanzwa njengoba kuchazwe ngaphambilini (50).
Ukuhlolwa kwe-self-NMPylation kwe-HCoV-229E nsp12-His6 kwenziwe njengoba kuchazwe ku-EAV nsp9 (16).Ngamafuphi, i-nsp12-His6 (0.5 µM) iqukethe u-50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH, pH 8.0, 5 mM dithiothreitol ( DTT), 6 mM MnCl2, 25, µM buffer i-NTP eshiwo no-0.17 µM kufane ne-[α32-P]NTP (3,000 Ci/mmol; Hartmann Analytic) ku-30 °C imizuzu engu-30.Kuzo zonke ezinye izivivinyo (ezijwayelekile) ze-NMPylation ze-nsp12-mediated nsp9 NMPylation, izimo zokusabela zilungiswa ngale ndlela elandelayo: nsp12-His6 (0.05 µM) kanye ne-nsp9-His6 (4 µM) ebukhoneni buka-50 mM HEPES-KOH (pH 8.0). ), 5 mM DTT, 1 mM MnCl2, 25 µM ekhonjisiwe ye-NTP, kanye ne-0.17 µM ifane ne-[α32-P]NTP.Ngemva kokufukamela imizuzu engu-10 ku-30°C, isampula yokusabela yaxutshwa nesampula ye-SDS-PAGE buffer: 62.5 mM tris(hydroxymethyl)aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol kanye no-0.005% bromophenol eluhlaza okwesibhakabhaka.Iphrotheni yenziwe yashintshwa ngokushisisa ku-90 °C imizuzu emi-5 futhi yahlukaniswa ngo-12% SDS-PAGE.Ijeli igxilile futhi ingcoliswe ngesisombululo se-Coomassie Brilliant Blue (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), ikhishwe umbala, futhi ivezwe esikrinini sesithombe se-phosphorescent amahora angu-20 (ukuthola i-nsp12 ku-NMPylation) noma (ubuningi) Amahora angu-2 (ukuhlola i-nsp9 NMPylation).Isithombe se-Typhoon 9200 (GE Healthcare) sisetshenziswe ukuskena isikrini futhi i-ImageJ yasetshenziselwa ukuhlaziya ukuqina kwesignali.
Ekuhlaziyweni kwe-MS, i-1 µM nsp12-His6 kanye ne-10 µM nsp9 (ngaphandle kwethegi ye-hexahistidine) zisetshenziswe ekuhlaziyweni kwe-NMPylation (isithasiselo se-SI, Ithebula S1) kanye nokugxila okukhuphukile okungu-500 µM UTP ne-GTP kwasetshenziswa.Kuye ngokugxilisa kwawo kanye nekhwalithi yamaprotheni elindelekile, isistimu ye-Waters ACQUITY H-Class HPLC efakwe ikholomu ye-MassPrep (Amanzi) yasetshenziselwa ukukhipha usawoti ongu-1 kuya ku-10 µL wezisombululo zamaprotheni ezivinjiwe ku-inthanethi.Iphrotheni encibilikisiwe ikhishwa emthonjeni we-ion ye-electrospray ye-Synapt G2Si mass spectrometer (Amanzi) ngokusebenzisa igradient elandelayo ye-buffer A (amanzi/0.05% formic acid) kanye ne-buffer B (acetonitrile/0.045% formic acid), kanye nezinga lokushisa lekholomu 60 ° C kanye nezinga lokugeleza elingu-0.1 mL/min: i-elution isocratically ngo-5% A imizuzu emi-2, bese kuba i-gradient yomugqa iye ku-95% B phakathi nemizuzu engu-8, bese igcina u-95% B eminye imizuzu emi-4.
Ama-ion amahle anobubanzi obungu-500 kuya ku-5000 m/z ayatholakala.I-Glu-fibrinopeptide B ikalwa njalo ngemizuzwana engama-45 ukuze ilungiswe ngokuzenzakalelayo.Sebenzisa isofthiwe yensimbi ye-MassLynx enesandiso se-MaxEnt1 ukuze uhlukanise i-spectrum emaphakathi ngemva kokudonsa isisekelo nokushelela.
I-UMPylated HCoV-229E nsp9 yagaywa ngokungeza i-trypsin eguquliwe yezinga elilandelanayo (Serva) futhi yafakwa ngobusuku obungu-37 °C.Ikholomu ye-Chromabond C18WP spin (ingxenye inombolo 730522; Macherey-Nagel) yasetshenziselwa ukukhipha usawoti kanye nokugxilisa ama-peptide.Ekugcineni, i-peptide yancibilika ku-25 µL wamanzi, aqukethe i-acetonitrile engu-5% kanye ne-0.1% ye-formic acid.
Amasampuli ahlaziywa yi-MS kusetshenziswa i-Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Uhlelo lokugcina lwe-nanoâ HPLC (i-Dionex), ifakwe isiphetho esinqunyiwe esifakwe ku-50 cm??75 μm C18 RP ikholomu epakishwe ngobuhlalu kazibuthe obungu-2.4 μm (uDkt. Albin Maisch High Performance LC GmbH) Xhuma ku-mass spectrometer ku-inthanethi ngomthombo we-Proxeon nanospray;jova u-6 µL wekhambi lokugaya i-trypsin ku-300 µm odayamitha wangaphakathi ×??1 cm C18 Ikholomu ye-PepMap yokugxila ngaphambili (Thermo Scientific).Kusetshenziswa amanzi/0.05% ye-formic acid njengesincibilikisi, isampula livaleleke ngokuzenzakalelayo futhi lakhishwa usawoti ngenani lokugeleza elingu-6 µL/min.
Ama-gradient alandelayo amanzi/0.05% formic acid (solvent A) kanye ne-80% acetonitrile/0.045% formic acid (solvent B) asetshenziswa ukuze kuzuzwe ukuhlukaniswa kwama-tryptic peptides ngesilinganiso sokugeleza esingu-300 nL/min: 4% B for Imizuzu emi-5, bese kuba ngu-30 I-gradient yomugqa iye ku-45% B phakathi nemizuzu, kanye nokwenyuka komugqa kube ngu-95% we-solvent B phakathi nemizuzu emi-5.Xhuma ikholomu ye-chromatographic ku-nano-emitter yensimbi engagqwali (Proxeon), bese ufafaza i-event ngokuqondile ku-capillary eshisayo ye-mass spectrometer usebenzisa amandla angu-2,300 V. Ukuskena kwenhlolovo okunokulungiswa okungu-60,000 ku-Orbitrap mass analyzer kuhlotshaniswa okungenani okuthathu kwedatha ye-MS/MS scan, ekhishwe ngaphandle ngamandla imizuzwana engu-30, kusetshenziswa ukungqubuzana kwe-ion trap okudalwe ukushayisana noma ukuhlukaniswa kwamandla aphezulu okushayisana kuhlanganiswe nokutholwa kwe-orbitrap, Ukulungiswa kungu-7,500.
Isikhathi sokuthumela: Aug-03-2021