Yosinthidwa ndi Peter Sarnow, Stanford University School of Medicine, Stanford University, California, yovomerezeka pa Disembala 25, 2020 (yawunikiridwa pa Okutobala 25, 2020)
Timapereka lipoti la kuyanjana pakati pa magawo ang'onoang'ono pakubwereza kwa ma coronaviruses-transcription complexes, omwe ndi ofunikira pakubwereza komanso kusungitsa chisinthiko.Tidapereka umboni kuti dera la NiRAN lolumikizidwa ndi nsp12 lili ndi ntchito ya nucleoside monophosphate (NMP) transase mu trans, ndipo tidazindikira nsp9 (protein yomanga ya RNA) ngati chandamale chake.NiRAN imathandizira kuphatikizika kwa gulu la NMP ku nsp9 amino terminus yosungidwa momwe imadalira ma ayoni a Mn2+ ndi zotsalira zosungidwa za Asn.Zinapezeka kuti zochitika za NiRAN ndi nsp9 NMPylation ndizofunikira pakubwereza kwa coronavirus.Detayo imatilola kulumikiza izi za chizindikiro cha enzyme ya nested virus ku zomwe zawonedwa kale mumalingaliro akuti kuyambika kwa kaphatikizidwe ka RNA m'gulu la ma virus a RNA kumagwira ntchito komanso kusinthasintha.
RNA-yodalira RNA polymerase (RdRps) ya Nidovirales (Coronaviridae, Arterioviridae, ndi mabanja ena 12) imalumikizidwa ndi dera la amino-terminal (N-terminal) mu mapuloteni osakhazikika (nsp) otulutsidwa kuchokera ku polyprotein, yotchedwa NiRAN. 1ab imapangidwa ndi viral main protease (Mpro).M'mbuyomu, kachilombo koyambitsa matenda a NiRAN-RdRp nsp mwiniwake wa GMPylation/UMPylation adanenedwa, ndipo adanenedwa kuti apangitse kanthawi kochepa kusamutsidwa kwa nucleoside monophosphate (NMP) kupita ku kachilombo (kosadziwika) kachiromboka ndi/kapena cell biopolymerization Zinthu.Apa, tikuwonetsa kuti coronavirus (Human Coronavirus [HCoV]-229E and Severe Acute Respiratory Syndrome Coronavirus 2) nsp12 (NiRAN-RdRp) ili ndi ntchito ya NMPylation yodalira Mn2+, yomwe imachokera ku nsp9 kudzera mukupanga kwa Mpro-mediated nsp9 After N-terminal flanking nsps imatulutsidwa proteolytically, phosphoramidate imamangirizidwa ku primary amine (N3825) pa N-terminal ya nsp9.Uridine triphosphate ndiye nucleotide yomwe amakonda kwambiri pochita izi, koma adenosine triphosphate, guanosine triphosphate, ndi cytidine triphosphate nawonso ndi othandizana nawo.Maphunziro a masinthidwe pogwiritsa ntchito ma recombinant coronavirus nsp9 ndi nsp12 mapuloteni komanso osinthika a HCoV-229E opangidwa ndi majini adatsimikiza zotsalira zofunika pa NiRAN-mediated nsp9 NMPylation ndi kubwereza kwa ma virus mu chikhalidwe cha ma cell.Detayo idatsimikizira kuneneratu kwa zotsalira za NiRAN zomwe zikugwira ntchito ndikutsimikiza gawo lofunikira la zotsalira za nsp9 N3826 mu nsp9 NMPylation ndi kubwereza kachilombo mu vitro.Chotsalira ichi ndi gawo la njira yosungidwa ya N-terminal NNE tripeptide ndipo idakhala yokhayo yotsalira ya nsp9 ndi ma homologs ake m'banja la coronavirus.Kafukufukuyu akupereka maziko olimba a kafukufuku wogwira ntchito wa ntchito ya NMPylation ya ma virus ena okhala m'chisa ndipo akupereka mipherezero yomwe ingatheke pakupanga mankhwala oletsa ma virus.
Kachilombo ka RNA ka Nidovirales positive-stranded RNA imayambitsa mitundu yosiyanasiyana ya vertebrates ndi invertebrates (1, 2).Lamuloli pano likuphatikiza mabanja 14 (3), omwe banja la Coronavirus laphunziridwa kwambiri zaka 20 zapitazi.Panthawiyo, ma zoonotic coronaviruses atatu adatuluka kuchokera ku nyama zomwe zidayambitsa matenda akulu akulu kupuma mwa anthu.Kuphatikizapo kulimbikira miliri chifukwa kwambiri pachimake matenda opatsirana.Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (4âââ7).Ma Nidoviruses amagawana gulu lodziwika bwino la ma genome, ndipo gawo laling'ono la membrane-bound replication-transcript complex (RTC) limasungidwa mu 5-?²-terminal two-third ndi gawo lalikulu la tinthu ta kachilomboka, komanso zida zina. .Mapuloteni, osungidwa mu 3??² kumapeto kwachitatu kwa genome (1).Kupatula banja limodzi la mavairasi a planarian (Monoviridae) (8), mavairasi onse okhala ndi zisa amaika magulu a RTC m'mafelemu awiri akuluakulu otseguka (ORF) ORF1a ndi ORF1b, omwe amamasuliridwa kuchokera ku genomic RNA ya.ORF1a imayika ma polyprotein (pp) 1a, ndi ORF1a ndi ORF1b pamodzi amatsekera pp1ab.Ndi kutenga nawo gawo kwa protease yayikulu (Mpro) yolembedwa ndi ORF1a, onse pp1a ndi pp1ab amasinthidwa kukhala mapuloteni osiyanasiyana osamapangidwe (nsps), omwe amadziwikanso kuti 3CLpro, chifukwa ali ndi homology ndi 3Cpro ya picornavirus ( 9).Ma nspswa amaganiziridwa kuti amasonkhanitsidwa kukhala RTC yayikulu yosunthika, imathandizira kaphatikizidwe ka genomic RNA (replication) ndi seti ya subgenomic RNA (zolemba), ndipo amagwiritsidwa ntchito kugwirizanitsa mawu a ORF yomwe ili kumunsi kwa ORF1b (10? ? ?12).
Pakatikati RTC imaphatikizapo RNA-yodalira RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) ndi ma enzyme angapo opangira RNA, omwe makamaka amasungidwa mu ORF1b komanso m'banja la coronavirus Ili ndi nsp12-nsp16 ndi nsp9-nsp12 m'banja la Arterioviridae (onani maumboni 10ââ 12).RdRp ndi HEL1 zimayimira madera awiri (gawo limodzi mwachisanu) otetezedwa a kachilombo ka mbalame ndipo ali ndi homology pakati pa ma virus ena a RNA.Kufanizira kwapakati kumakhulupirira kuti kumathandizidwa ndi ma subunits ena, kuphatikiza ma nsps angapo ang'onoang'ono otulutsidwa kudera la carboxy-terminal (C-terminal) la pp1a, kumunsi kwa Mpro (coronavirus nsp5 ndi arterial virus nsp4, motsatana).Ali ndi chitetezo chochepa cha mabanja ndi zochitika zosiyanasiyana (zowunikiridwa muzofotokozera 10ââ12).
Posachedwapa, dera lomwe lili ndi mawonekedwe apadera otsatizana linapezeka pa amino terminus (N-terminus) moyandikana ndi RdRp mu ma virus onse okhala ndi zisa, koma palibe ma virus ena a RNA (16).Kutengera malo ake ndi nucleotide transferase (nucleoside monophosphate [NMP] transferase), domain iyi imatchedwa NiRAN (Nestvirus RdRp-related nucleotide transferase).Kuphatikiza kwa zigawo ziwiri za NiRAN-RdRp kumapanga nsp12 mubanja la Coronaviridae ndi nsp9 mubanja la Arterioviridae, ndipo mu nestoviridae ina, NiRAN-RdRp ikuyembekezeka kutulutsidwa ngati nsp yodziyimira payokha kuchokera ku viral polyprotein.Mu coronavirus, dera la NiRAN lili ndi ??1/450 zotsalira ndipo zimalumikizidwa ndi dera la C-terminal RdRp kudzera m'chigawo cholumikizira (16?19).Mu Equine Arteritis Virus (EAV) (Arteriviridae), recombinant nsp9 ikuwonetsa zochitika za Mn2+ ion-dependent (self) UMPylation ndi GMPylation, zomwe zimadalira magawo atatu otetezedwa a nestovirus, AN, BN ndi CN Zotsalira motsatizana.Pomwe N imayimira NiRAN) (16).Kuzungulira kwa N-terminal kwazithunzizi ndizosakhazikika kwambiri za preAN.Zina mwazotsalirazi zimasungidwanso mu protein kinases, pomwe zawonetsedwa kuti zimagwira ntchito yomanga ya nucleoside triphosphate (NTP) ndi catalytic (20, 21).Mogwirizana ndi izi, zotsalira zingapo zazikulu zomwe zimagwira ntchito mu pseudokinase SelO kuchokera ku Pseudomonas syringae zitha kusonkhanitsidwa ndi SARS-CoV-2 nsp7/8/12/13 supercomplex yomwe yasindikizidwa posachedwa.Zotsalira za Conserved Coronavirus NiRAN zomwe zili pamwamba pa ma electron microstructure.Mapuloteni ophatikizanso (17).Zikuganiziridwa kuti zolembedwa (self) U/GMPylation zidzatulutsa nyengo yosakhalitsa kuti isamutsire NMP ku gawo lapansi (losadziwika) (16), ndi kufanana kwapangidwe pakati pa NiRAN ndi protein kinase (17, 19) ) Kodi lingaliro lakuti NiRAN imasintha mapuloteni ena.
Zinthu zambiri, kuphatikiza mwapadera komanso mwapadera kuyanjana kwake ndi ma virus okhala ndi zisa komanso kupatukana kwa majini ku RdRp, zimapangitsa NiRAN kukhala puloteni yoyenera yowongolera ma virus okhala ndi zisa, zomwe ndizofunikira kwambiri kuti ziwonekere komanso kudziwika kwawo.M'mbuyomu, ntchito zitatu zomwe zingakhalepo zokhudzana ndi NiRAN zowongolera kumasulira kwa ma genome/subgenomic kapena kubwereza/kulemba zidatchedwa.Poganizira zakusowa komanso zosakwanira zomwe zinalipo panthawiyo, ntchito iliyonse imakhala ndi zabwino ndi zoyipa zake (16).Mu kafukufukuyu, tikufuna kuphatikiza maphunziro a biochemical ndikusintha ma genetic a ma coronavirus omwe akuyimira mibadwo iwiriyi, ndikuyika zomwe tapeza pakusintha kwachilengedwe kwa banja la coronavirus, kuti tidziwe bwino za malo Odabwitsawa.Timafotokoza kupita patsogolo kwakukulu pakumvetsetsa kwa NiRAN kudzera pakuzindikiritsa zolinga zachilengedwe mu RTC, zomwe (pakati paziganizo zitatu zomwe zilipo) zimathandizira gawo la domain iyi poyambitsa kaphatikizidwe ka kachilombo ka RNA.Kafukufukuyu amatsegulanso mwayi wa maudindo ena a NiRAN pa mawonekedwe a virus host.
Kuti tiwonetsere momwe kachilombo ka corona kachilomboka kamayenderana ndi nsp12 yokhudzana ndi dera la NiRAN, tidapanga mawonekedwe ophatikizananso a human coronavirus 229E (HCoV-229E) nsp12 mu E. coli, yokhala ndi tag ya His6 pa C-terminus, ndikuphatikiza mapuloteni okhala ndi [α32-P] Ikani pamodzi ndi NTP pamaso pa MnCl2 monga momwe tafotokozera mu Zida ndi Njira.Kuwunika kwa zomwe zidachitika kukuwonetsa kukhalapo kwa mapuloteni opangidwa ndi radiolabed omwe amasamuka ndi nsp12 (106 kDa), kuwonetsa kuti coronavirus nsp12 imathandizira mapangidwe a protein-NMP adducts, opangidwa makamaka ndi uridine monophosphate (UMP) (Chithunzi 1A) Ndipo B).Kusanthula kwachulukidwe kunawonetsa kuti poyerekeza ndi ma nucleotide ena, mphamvu ya chizindikiro cha kuphatikiza kwa UMP idakula ndi 2 mpaka 3 nthawi (Chithunzi 1C).Izi zikugwirizana ndi zomwe zanenedweratu za NMP transferase domain ya NiRAN domain of the coronavirus (16), koma zikuwonetsa kuti zokonda za nucleotide za NiRAN domain ya coronavirus ndi kachilombo ka corona ndizosiyana.
Ntchito yodzipangira nokha ya HCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) idalumikizidwa ndi [α-32P] NTP pamaso pa 6 mM MnCl2 kwa mphindi 30 (onani Zida ndi Njira kuti mumve zambiri).Zomwe zidachitika zidalekanitsidwa ndi SDS-PAGE ndikudetsedwa ndi Coomassie buluu wonyezimira.(B) Mapuloteni opangidwa ndi radiolabele amawonetsedwa ndi kujambula kwa phosphorous.Malo a nsp12-His6 ndi mapuloteni a molecular mass markers (mu kilodaltons) akuwonetsedwa mu A ndi B. (C) Kuchuluka kwa chizindikiro cha radioactive (kutanthauza ± SEM) kunatsimikiziridwa kuchokera ku mayesero atatu odziimira okha.*P≤0.05.Mphamvu ya siginecha (peresenti) imagwirizana ndi UTP.
Ngakhale zochita za ma enzyme zokhudzana ndi NiRAN zawonetsedwa kuti ndizofunikira pakubwereza kwa EAV ndi SARS-CoV mu cell culture (16), ntchito yeniyeni ya NiRAN ndi zomwe zikuyembekezeka sizinadziwikebe.Kufanana kwaposachedwa kwapangidwe pakati pa NiRAN ndi banja la mapuloteni okhala ndi mapuloteni kinase-ngati makutu (17, 22) zidatipangitsa kuyesa lingaliro lakuti NiRAN imathandizira NMPylation ya mapuloteni ena.Tidapanga zokonda zomwe zitha kukhala zofanana, kuphatikiza mapuloteni osakhazikika osungidwa ndi HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), iliyonse ili ndi C-terminal His6 tag (SI appendix, Table S1) , Ndipo amaika mapuloteniwa ndi [α32-P] uridine triphosphate ([α32-P]UTP) pamaso kapena palibe nsp12.Bovine seramu albumin ndi MBP-LacZα maphatikizidwe mapuloteni opangidwa mu E. coli ankatumikira monga amazilamulira (Chithunzi 2A, misewu 1 mpaka 7).Mapuloteni opangidwa ndi radiolabele adawunikidwa ndi sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ndi autoradiography, ndipo zinapezeka kuti panali chizindikiro champhamvu cha radioactive chomwe chinali ndi nsp12 ndi nsp9.Malo a chizindikirocho amagwirizana ndi kuchuluka kwa maselo a nsp9, kusonyeza nsp12-mediated UMPylation ya nsp9 (Chithunzi 2B, track 7).Palibe mapuloteni ena oyesera omwe adapezeka kuti ndi UMPylated, zomwe zidatipangitsa kunena kuti nsp9 ndi gawo linalake la nsp12.Mogwirizana ndi chidziwitso cha self-NMPylation chomwe chikuwonetsedwa mu Chithunzi 1, nsp12 imatha kusamutsa ma NMP onse anayi kupita ku nsp9, ngakhale kuti ntchitoyo ndi yosiyana, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Chithunzi).3 A ndi B).Pansi pamikhalidwe yomwe imagwiritsidwa ntchito poyesa izi (kufupikitsa zomwe zimachitika ndi nthawi yowonekera, kuchepetsa kuchuluka kwa nsp12; zida ndi njira), kudziyimira pawokha kwa NMPylation ya nsp12 sikunadziwike (yerekezerani ndi Chithunzi 2B, kanjira 7, ndi Chithunzi 1B), yakhala yothandiza (Ndi maulendo angapo) UMP idasuntha kuchoka ku nsp12 kupita ku nsp9.Ntchito yosinthira ya UMP imafuna kukhalapo kwa ma ion a Mn2 +, monga momwe tawonetsera pa Chithunzi 3C, pomwe ntchito yocheperako ya UMP yokha idawonedwa pamaso pa Mg2 +, ndipo palibe chochita pamaso pa ma cation ena awiri omwe adayesedwa.Zomwezo zinapezedwa muzoyesa za NMPylation zomwe zili ndi cytidine triphosphate (CTP), guanosine triphosphate (GTP), ndi adenosine triphosphate (ATP) (SI appendix, Chithunzi S1).
HCoV-229E nsp12-mediated UMPylation ya nsp9.Magawo angapo a mapuloteni (kuphatikizapo bovine serum albumin, MBP-lacZα, ndi mndandanda wa HCoV-229E nsps wolembedwa ndi C-terminal His6 yolembedwa ndi ORF1a) adagwiritsidwa ntchito kuwunika ntchito ya UMPylation ya HCoV-229E nsp12-His6⁺-mediated mapuloteni.Ikani mapuloteni ndi [α-32P] UTP kwa mphindi 10 popanda (A) kapena kukhalapo (B) ya nsp12 monga momwe tafotokozera mu zipangizo ndi njira.Pamwamba pa A ndi B, gel wa SDS-polyacrylamide wodetsedwa ndi Coomassie Brilliant Blue akuwonetsedwa, ndipo pansi pa A ndi B, ma autoradiogram ofanana akuwonetsedwa.Malo a protein molecular mass marker (mu kilodaltons) amaperekedwa kumanzere.Malo a nsp12-His6 (B, pamwamba) ndi chizindikiro cha radioactive chomwe chimawonedwa panthawi ya incubation ya nsp12-His6 ndi nsp9-His6 (B, lane 7) imasonyezedwanso, zomwe zimasonyeza kuti [α-32P] UMP ku nsp9-His6 (12.9 kDa), yomwe siinawonedwe kwa mapuloteni ena oyesedwa.
HCoV-229E NiRAN-mediated biochemical and virological characterization ya nsp9 NMPylation.(A ndi B) Ntchito ya nucleotide co-substrate yomwe imagwiritsidwa ntchito pochita.Nsp12-His6 ndi nsp9-His6 zimasakanizidwa ndikuyikidwa pamaso pa [α-32P] NTPs mu muyezo wa NMPylation assay.(A, pamwamba) Coomassie-stained nsp9-His6 yolekanitsidwa ndi SDS-PAGE.(A, pansi) Autoradiograph ya dera lomwelo la gel osakaniza.(B) Ntchito yachibale (kutanthauza ± SEM) pamaso pa nucleotide cofactor yosankhidwa imatsimikiziridwa kuchokera ku mayesero atatu odziimira okha.*P≤0.05.(C) Udindo wa ayoni zitsulo.Zowonetsedwa ndizoyezetsa wamba ya NMPylation pamaso pa [α-32P] UTP ndi ayoni achitsulo osiyanasiyana, iliyonse imakhala ndi 1 mM.Mu C, pamwamba, Coomassie wodetsedwa nsp9-His6 akuwonetsedwa, ndipo mu C, pansi, autoradiography yofananira ikuwonetsedwa.Kukula kwa mapuloteni olembedwa (mu ma kilodaltons) akuwonetsedwa kumanzere kwa A ndi C. (D) Mtundu wosinthika wa HCoV-229E nsp12-His6 wonyamula m'malo mwa amino acid womwe watchulidwa uli mu [α-32P]UTP, monga tafotokozera. mu Zipangizo ndi Njira.Ma radiolabeled nsp9-His6 opangidwa mu NMPylation reaction amapezeka ndi phosphorylation imaging (D, pamwamba).Zochita zofananira poyerekeza ndi mapuloteni amtundu wakuthengo (wt) akuwonetsedwa mu D, ndipo pansi amatengedwa ngati avareji (± SEM) kuchokera ku kuyesa katatu kwa Independent.Nyenyezi zimasonyeza kulowetsa m'malo mwa zotsalira zosasungidwa.(E) Titer ya virus mu chikhalidwe chapamwamba kwambiri cha maselo a p1 omwe adapeza patatha maola 24 matenda atatsimikiziridwa ndi kuyesa kwa plaque.Ma codon omwe ali mu domeni ya NiRAN ya osinthika a HCoV-229E akuwonetsedwa (manambala otsalira amatengera malo awo mu pp1ab).Malo osasinthika a RdRp osasinthika a nsp12_DD4823/4AA adagwiritsidwa ntchito ngati chiwongolero.
Kuti timvetse mozama za malo ogwira ntchito a NiRAN ndikuwona zotsalira zokhudzana ndi ntchito ya nsp9-specific NMP transferase, tinapanga masinthidwe osinthika, momwe tinasinthira zotsalira zotsalira mu NiRAN AN, BN ndi CN motifs ( 16) Ndi Ala (SI appendix, Chithunzi S2).Kuphatikiza apo, kukhudzika kwa ma Conservative Arg-to-Ls kapena Lys-to-Arg m'malo adawunikidwa pawiri.Monga chowongolera (choyipa), zotsalira zomwe sizinasungidwe kapena kusungidwa kudera la NiRAN la ma coronaviruses ndi ma virus ena okhala m'chisa amasinthidwa ndi Ala. Kusintha K4116A (in motif preAN), K4135A (AN), R4178A (BN), D4188A (motif BN) ndi D4280A (CN) amachepetsa kwambiri kapena amachotsa nsp9 NMPylation kupyolera mu nsp12, pamene mapuloteni okhala ndi conservative m'malo (R4178K), K4116R) amasunga 60% ndi 80% ya ntchito zawo, zomwe zimasonyeza kuti kupumula kwa zoletsedwa kumbali yawo. unyolo ndi physicochemically sensitive (Chithunzi 3D).Kusintha zotsalira zina zingapo zosungidwa E4145A, D4273A, F4281A ndi D4283A ndizochepa kwambiri, ndipo nsp9 UMPylation imachepetsedwa pang'ono.Zotsatira zofananazo zinapezedwa mu nsp9 NMPylation reactions yokhudzana ndi NTPs zina (Figure 3D ndi SI appendix, Figure S3), kutsimikizira kuti zotsatira zomwe zimawonedwa pazitsulo zinazake za amino acid ndizosiyana ndi mtundu wa nucleotide co-substrate yomwe imagwiritsidwa ntchito.Kenako, tidayesa zomwe zingachitike m'malo mwa nsp12 pakubwerezabwereza kwa ma coronavirus mu chikhalidwe cha ma cell.Kuti izi zitheke, tidagwiritsa ntchito ma tempulo oyenerera a DNA (cDNA) opangidwa ndi kachilombo ka katemera wophatikizanso (23, 24) kuti alembe ma cell 5 -7.Katchulidwe ka ma virus omwe amapangidwa m'maselowa adawonetsa kuti masinthidwe ambiri a HCoV-229E NiRAN sanali zotheka (Chithunzi 3E).Gulu la osinthika ma virus omwe sali otheka akuphatikizapo njira zina zomwe zasonyezedwa kuthetsa kapena kuchepetsa kwambiri ntchito ya NMP transferase in vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), koma pali njira zina ziwiri (K4116R, E4145A) % yasungidwa?Ntchito yawo ya in vitro NMPylation ikuwonetsa kuti zoletsa zina zikukhudzidwa.Momwemonso, masinthidwe ena awiri (R4178K, F4281A) omwe adachepetsa pang'onopang'ono mu ntchito ya NiRAN mu vitro NMPylation adapanga ma virus amoyo, komabe, ma viruswa adachepetsa kwambiri titers kudzera kubwereza.Mogwirizana ndi zomwe zikuwonetsedwa mu chithunzi 3D, kuchotsa zotsalira zina zinayi zomwe sizinasungidwe mu coronavirus ndi/kapena ma virus ena okhala ndi zisa (K4113A, D4180A, D4197A, D4273A) (8, 16) adatulutsa ma virus otheka Ana, ngakhale anali nawo. a amtengo yafupika titer poyerekeza ndi zakuthengo-mtundu kachilombo (Chithunzi 3E).
Kuti muphunzire ngati ntchito yosinthira ya NiRAN-mediated NMP imatengera dera lomwe likugwira ntchito la RdRp, zotsalira ziwiri za Asp zosungidwa zomwe zidaphatikizidwa pakulumikizana kwa ma ion achitsulo (11) mu RdRp motif C zidasinthidwa ndi Ala. ntchito yake ya nsp9 NMPylation, kusonyeza kuti nsp12-mediated in vitro nsp9 NMPylation ntchito safuna polymerase ntchito (SI Zowonjezera, Chithunzi S4).
Titakhazikitsa nsp9-specific NMP transferase activity ya nsp12, tinayesa kuwonetsa kutsatsa kwa NMP-nsp9 ndi mass spectrometry (MS).Mapuloteni athunthu amtundu wa recombinant HCoV-229E nsp9 adawonetsa pachimake pa 12,045 Da (Chithunzi 4A).Kuwonjezera kwa nsp12 sikunasinthe khalidwe la nsp9, kusonyeza kuti nsp12 ndi nsp9 sizipanga zovuta zokhazikika pansi pa zomwe zimagwiritsidwa ntchito (denaturation) (Chithunzi 4A).Pamaso pa UTP ndi GTP, kuyeza kwa kuchuluka kwa zomwe zili ndi nsp9 ndi nsp12 motsatana zidawonetsa kuti mapuloteni a UTP adasuntha 306 Da, ndipo kuchuluka kwa mapuloteni a GTP kudasuntha 345 Da, kuwonetsa kuti molekyulu iliyonse ya nsp9 imamanga UMP kapena GMP. (Chithunzi 4) C ndi D).Akuti mphamvu yofunikira pa NiRAN-mediated nsp9 NMPylation imachokera ku NTP hydrolysis ndi pyrophosphate release.Ngakhale kuchuluka kwa nsp9 (chandamale) ka 10 kuposa nsp12 (enzyme) kudagwiritsidwa ntchito pochita izi, pafupifupi NMPylation ya nsp9 yathunthu idawonedwa, kuwonetsa kuti kuyanjana pakati pa nsp12 ndi nsp9 kumakhala kwakanthawi, ndipo nsp12 imatha NMPylate kwambiri nsp9 mu vitro molekyulu.
NMPylation Imodzi ya nsp9 pamaso pa nsp12 ndi UTP kapena GTP.Kuwonetseredwa ndi kuchuluka kwa protein mass spectrum ya HCoV-229E nsp9 (SI appendix, Table S1) (AD).(A) nsp9 yokha, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 pamaso pa UTP, (D) nsp9 + nsp12-His6 pamaso pa GTP.
Kuti mudziwe zotsalira za nsp9 UMPylated ndi nsp12, nsp9-UMP idang'ambika ndi trypsin.Ma peptides omwe adatsatirawo adasiyanitsidwa ndi nano-high performance liquid chromatography (HPLC) ndikuwunikidwa ndi tandem mass spectrometry (MS/MS) pa intaneti.Kusanthula deta pogwiritsa ntchito phukusi la mapulogalamu a Byonic (Protein Metrics) kunawonetsa UMPylation ya N-terminal amino acid.Izi zimatsimikiziridwa pamanja.Tandem mass spectrum ya precursor peptide [UMP]NNEIMPGK (SI appendix, Figure S5A) inavumbulutsa chidutswa cha 421 m/z, kusonyeza kuti UMP imamangiriza ku 1 yotsalira ya nsp9.
Pa N-terminus ya nsp9, Asn amasungidwa pakati pa mamembala a Orthocoronavirinae (SI appendix, Chithunzi S6).Ngakhale timakhulupirira kuti N-terminal primary amine nitrogen ndiyemwe angavomereze UMP, tinaganiza zopeza umboni wowonjezera wa NMP kumangirira pa N-terminal.Pachifukwa ichi, non-NMPylated ndi NMPylated N-terminal peptide nsp9 yoyeretsedwa ndi HPLC inatengedwa pamaso pa acetone ndi sodium cyanoborohydride.Pansi pazimenezi, ma amine oyambirira aulere okha ndi omwe angasinthidwe ndi propyl (25).Peptide yochokera ku N-terminal nsp9 yokhala ndi mndandanda wa NNEIMPGK ili ndi ma amine awiri oyambira, imodzi pa N-terminus ya Asn ndi ina kumbali ya Lys pa C-terminus.Choncho, magulu a propyl akhoza kuyambitsidwa pamapeto onse awiri.Ma chromatogram a ion a ma peptide omwe si a NMPylated akuwonetsedwa muzowonjezera za SI, Chithunzi S5B.Monga zikuyembekezeredwa, N-terminal ndi C-terminal (mono) propylated (SI appendix, Chithunzi S5B, chapamwamba lane) ndi dipropylated peptides (SI appendix, Chithunzi S5B, njira yapansi) ikhoza kudziwika.Njirayi imasintha pogwiritsa ntchito NMPylated N-terminal peptide ya nsp9.Pachifukwa ichi, ma peptide a C-terminal propylated okha amatha kudziwika, koma N-terminal propylated peptides ndi dipropylated peptide sizidziwika (SI Zowonjezera, Chithunzi S5C), kusonyeza kuti UMP yasamutsidwa ku N-terminal primary amine Kupewa izi. gulu kusintha.
Kenako, timasintha (ndi Ala kapena Ser) kapena kuchotsa zotsalira zomwe zasungidwa pa N-terminus ya nsp9 kuti tifotokoze zopinga zomwe mukufuna.Kutengera ndi data yathu ya MS yowonetsa kuti NiRAN imapanga nsp9-NMP adduct yokhala ndi amine woyambira wa N-terminal residue ya nsp9, tidalingalira kuti nsp9 NMPylation imafuna viral master protease (Mpro, nsp5) kuti amasule nsp9 N-terminal kuchokera. kalambulabwalo wake wa polyprotein.Kuti tiyese kulingalira kumeneku, tinapanga puloteni ya precursor nsp7-11 yomwe ili ndi nsp9 mu E. coli ndipo tinayesa mayeso a NMPylation pamaso pa [α-32P] UTP (zida ndi njira).Monga momwe tawonetsera pa Chithunzi 5A (njira 3), cholozera cha nsp7-11 chosadulidwa sichinalembedwe ndi nsp12.Mosiyana ndi izi, ngati nsp7-11 imang'ambika ndi recombinant nsp5 kuti itulutse nsp9 (ndi ma nsps ena) kuchokera ku kalambulabwalo, mapuloteni ojambulidwa ndi radiolabele omwe amasamuka ndi nsp9 amapezeka, kutsimikizira mfundo yathu kuti NiRAN ndi N-Selective mapangidwe a covalent nsp9-NMP adducts. .Terminal primary amine ya N-terminal Asn (malo 3825 mu pp1a/pp1ab).Izi zimathandizidwanso ndi kuyesa pogwiritsa ntchito nsp9 construct, yomwe ili ndi chotsalira chimodzi kapena ziwiri zowonjezera pa N-terminus.Muzochitika zonsezi, NiRAN-mediated UMPylation ya nsp9 inathetsedwa (SI Zowonjezera, Chithunzi S7).Kenako, tidapanga puloteni yokhala ndi zotsalira za Asn imodzi kapena ziwiri zomwe zachotsedwa pamayendedwe a 3825-NNEIMPK-3832 peptide pa N-terminal ya nsp9.Pazochitika zonsezi, nsp9 UMPylation inatsekedwa kwathunthu (Chithunzi 5B), kupereka umboni wowonjezera kuti nsp9 N-terminus yeniyeni imakhala ngati cholandirira cha NMP.
Proteolytic processing ya nsp9 ndi gawo la zotsalira za N-terminal mu nsp12-mediated UMPylation.(A) nsp9 UMPylation imafuna nsp9 N-terminal yaulere.Nsp7-11-His6 imayikidwa kale pa 30 °C mu NMPylation detector buffer yomwe ili ndi UTP pamaso kapena kusakhalapo kwa recombinant Mpro (nsp5-His6).Pambuyo pa maola atatu, yambani kuyesa kwa NMPylation powonjezera nsp12-His6 monga tafotokozera mu Zida ndi Njira.Zomwe zili ndi nsp5-His6 (njira 1) ndi nsp9-His6 (njira 2) zidagwiritsidwa ntchito ngati kuwongolera.Pambuyo pa mphindi 10, zomwe zidachitikazo zidathetsedwa ndipo kusakanikirana kwake kudalekanitsidwa ndi SDS-PAGE.Puloteniyo idadetsedwa ndi Coomassie Brilliant Blue (A, pamwamba).Kalambulabwalo wa Nsp7-11-His6 ndi zomwe zidakonzedwa kuchokera ku nsp5-His6 mediated cleavage zikuwonetsedwa kumanja.Chonde dziwani (chifukwa cha kukula kwawo kochepa) kuti nsp7 ndi nsp11-His6 siziwoneka mu gel osakaniza, ndipo zomwe zimachitika zimaphatikizidwa ndi nsp5-His6 (mizere 1 ndi 4; malo a nsp5-His6 akuwonetsedwa ndi bwalo lolimba) kapena nsp9-His6 (Lane 2) ili ndi zochepa za MBP (zosonyezedwa ndi mabwalo otseguka) monga zonyansa zotsalira chifukwa zimawonetsedwa ngati mapuloteni a MBP fusion (SI appendix, Table S1).(B) Nsp9-His6 yosiyana ilibe zotsalira za N-terminal Asn imodzi kapena ziwiri (zotsalira zotsalira malinga ndi malo a pp1a/pp1ab) ndipo zimayeretsedwa ndi kuikidwa ndi nsp12-His6 ndi [α-32P] UTP.B, SDS-PAGE yodetsedwa ndi Coomassie ikuwonetsedwa pamwamba, B, autoradiograph yofananira ikuwonetsedwa pansi.Malo a cholembera cholemetsa cha maselo (mu ma kilodaltons) akuwonetsedwa kumanzere.(C) HCoV-229E nsp9-His6 N-terminal yosungirako zotsalira zinasinthidwa ndi Ala kapena Ser, ndipo kuchuluka kwa mapuloteni komweko kunagwiritsidwa ntchito mu nsp12-His6 mediated UMPylation reaction.Zomwe zidachitikazo zidalekanitsidwa ndi SDS-PAGE ndikudetsedwa ndi Coomassie Brilliant Blue (C, pamwamba), ndipo nsp9-His6 yolembedwa ndi radio idazindikirika ndi kujambula kwa phosphorescence (C, pakati).Pogwiritsa ntchito mapuloteni amtundu wakutchire (wt) monga chofotokozera (chokhazikitsidwa ku 100%), ntchito ya NMPylation (kutanthauza ± SEM) inawerengedwa kuchokera ku mayesero atatu odziimira okha.(D) Titers a virus mu p1 cell culture supernatant ya ma cell a Huh-7 omwe ali ndi ma cell a HCoV-229E wild-type Huh-7, ndi masinthidwe onyamula ma amino acid osankhidwa mu nsp9 adatsimikiziridwa ndi plaque assay.RdRp motif C double mutant DD4823/4AA yolephera kubwereza idagwiritsidwa ntchito ngati kuwongolera koyipa.
N-terminus ya nsp9 (makamaka malo 1, 2, 3, ndi 6) imasungidwa kwambiri pakati pa mamembala a Orthocoronavirinae subfamily (SI appendix, Chithunzi S6).Kuti muphunzire za kuthekera kwa zotsalirazi mu nsp12-mediated nsp9 NMPylation, zotsalira ziwiri zotsatizana za Asn pa N-terminus ya nsp9 zidasinthidwa ndi Ala kapena Ser (pazokha kapena kuphatikiza).Poyerekeza ndi nyama zakutchire nsp9, kuchotsa N3825 ndi Ala kapena Ser kunapangitsa kuti kuchepetsedwa kawiri mu nsp12-mediated UMPylation (Chithunzi 5C).Mogwirizana ndi kunena kwathu kuti NMPylation imapezeka pa N-terminal primary amine m'malo mwa unyolo wam'mbali wa zotsalira za N-terminal, tinawona NMPylation yotsalira yotsalira ndi kusinthidwa kwa N3825A ndi N3825S.Chochititsa chidwi, ngati Asn wachiwiri wasinthidwa ndi Ala kapena Ser, nsp9 UMPylation imachepetsedwa kwambiri (kuposa nthawi za 10), pamene m'malo mwa Ala pa maudindo 3, 4, ndi 6 ali ndi zotsatira zochepa pa nsp9 UMPylation (Chithunzi 2). ).5C).Zotsatira zofananazo zinapezedwa pogwiritsa ntchito ATP, CTP kapena GTP (SI appendix, Figure S8).Pamodzi, izi zikuwonetsa gawo lalikulu la N2826 (malo 2 mu nsp9) mu nsp9 NMPylation.
Kuti tipeze umboni wowonjezera wa kulumikizana kwa magwiridwe antchito pakati pa N-terminus ya nsp9 ndi NMPylation, tidapanga ma sequence alignment (MSA) a nsp9 sequence of the Coronavirus family (kusiyana pakati pa 104 ndi 113 zotsalira) (SI Zowonjezera, Chithunzi S6 ndi).Pazonse, mu mitundu 47 (yodziwika ndi yoyika) yamitundu isanu yamagulu ang'onoang'ono a Orthocoronavirinae omwe amapatsira nyama zoyamwitsa, mbalame, ndi zokwawa zosiyanasiyana, zotsalira 8 zokha zonse zidapezeka kuti sizisintha.Zosintha zochulukirapo, kuphatikiza kufufutidwa ndi kuyikapo, zidawonedwa m'mizere pakati pazinthu zachiwiri za nsp9, monga momwe zidakhazikitsira maphunziro am'mbuyomu (26 ??28).Zotsalira zisanu zosasinthika zinapezeka mu β strand ndi α helix ya C-terminal part ya nsp9.Zotsalira zitatu zosasinthika zimapanga NNE motif ya N terminus ya nsp9.Zawululidwa kuti Asn wachiwiri wa motif iyi ndiye chotsalira chokhacho chosasinthika, chomwe chimagawidwanso ndi malingaliro ongoyerekeza a nsp9 a frog coronavirus, ndikuyimira mitundu ya Microhyla letovirus 1 mu subfamily Letovirinae ya Alphaletovirus.Kusungidwa kwa zotsalira muzinthu zachiwiri za nsp9 zitha kutsatiridwa ndi malingaliro apangidwe kuti asunge zopindika kapena zodziwika za RNA zomangira.Komabe, lingaliro ili silikuwoneka kuti likugwira ntchito pa kusungidwa kwa NNE, ndipo phunziroli lisanachitike, chikhalidwe cha zopinga zomwe zimalepheretsa kusintha kwa maulendo a tripeptide zinali zobisika kwathunthu.
Kuti tidziwe kufunikira kwa kuteteza kwa nsp9-NMPylation ndi NNE pakubwerezabwereza kwa coronavirus, tidapanga masinthidwe a HCoV-229E, omwe amanyamula zotsalira za nsp9 N-terminal, kusonyeza kuti nsp9 NMPylation ndi yovulaza mu vitro.Tisanayambe, timayesa kuyankha funso ngati zoloŵa m'malozi (pafupi ndi nsp8|9 cleavage site) zikukhudza ma proteolytic processing a dera la C-terminal pp1a.Magulu a nsp7-11 polyprotein constructs okhala ndi zolowa m'malo zofananira pa N-terminus ya nsp9 adapangidwa mu E. coli ndikudulidwa ndi recombinant Mpro.The proteolytic cleavage of the four sites (kuphatikiza nsp9 flanking site) sikukhudzidwa kwambiri ndi zolowa m'malo (SI appendix, Chithunzi S9), kupatula kusintha kwamapangidwe a mapuloteniwa omwe amasokoneza Mpro-mediated nsp8|9 cleavage ( Kapena zina) webusayiti.
Ma cell a Huh-7 adasinthidwa ndi HCoV-229E RNA yautali wa genome, ndikuyika ma Ala kapena Ser m'malo mwa NNE tripeptides (N3825, N3826, ndi E3827) pa nsp9 N terminus, kuwonetsa kuti masinthidwe ambiri amapha.Tinatha kupulumutsa kachilomboka posintha Ser kapena Ala ya N-terminal Asn (N2835A kapena N2835S), koma tinalephera kubwezeretsa kachilomboka ndi masinthidwe ena amodzi ndi awiri mu ndondomeko ya NNE (N3826A, N3826S, NN3825 / 6AA, NN3825/6SS) , E3827A) (Chithunzi 5D).
Zotsatira izi zikuwonetsa kuti kubwereza kwa ma coronavirus mu chikhalidwe cha minofu ndikoletsedwa (zofanana kapena zofananira), kuchepetsa kusintha kwachilengedwe kwa malo a nsp9 NMPylation m'thupi, ndikuthandizira gawo lalikulu la kuyankha kumeneku pamayendedwe amoyo wa ma coronavirus.
Muzoyeserera zomaliza, tinapanga C-terminal His6 yolembedwa kuti SARS-CoV-2 nsp12 ndi nsp9, ndi mitundu iwiri yosinthika ya nsp12 mu E. coli.Zotsalira zamasamba zomwe zimagwira ntchito mu madera a NiRAN ndi RdRp zinali motsatana Gwiritsani ntchito Ala m'malo mwake (Chithunzi 6A ndi SI appendix, Table S2).K4465 mu SARS-CoV-2 nsp12 imagwirizana ndi K4135 mu HCoV-229E (SI Zowonjezera, Chithunzi S2), zomwe zidawoneka kuti ndizofunikira pazochitika za NiRAN ndi kubwereza kwa HCoV-229E (Chithunzi 3D ndi E).Zotsalirazi zimagwirizananso ndi kachilombo ka EAV nsp9 K94 zotsalira, zomwe poyamba zinawonetsedwa kuti ndizofunikira kwa NiRAN self-UMPylation/self-GMPylation (16).Monga momwe chithunzi 6B chikuwonetsedwera, SARS-CoV-2 nsp12 ili ndi ntchito yosinthira ya UMP pogwiritsa ntchito nsp9 ngati gawo laling'ono, pomwe nsp12_K4465A yosinthira tsamba sikugwira ntchito.Kulowetsedwa kawiri muzotsatira za SDD za RdRp motif C sizikhudza ntchito ya UMP transferase (Chithunzi 6B), kusonyeza kuti ntchito ya RdRp ilibe mphamvu yeniyeni mu nsp9 UMPylation.Zomwezo zinapezedwa pogwiritsa ntchito CTP, GTP ndi ATP (SI appendix, Chithunzi S10).Mwachidule, izi zikuwonetsa kuti NiRAN-mediated nsp9 NMPylation ili ndi ntchito yosamala mu ma coronavirus omwe amayimira magulu osiyanasiyana a orthocoronavirus subfamily.
SARS-CoV-2 nsp12-mediated NMPylation ya nsp9.(A) Coomassie wodetsedwa ndi SDS-polyacrylamide gel owonetsa mapuloteni ophatikizana omwe amagwiritsidwa ntchito mu mayeso a NMPylation.Monga kuwongolera, puloteni yosinthika yokhala ndi malo olowa m'malo mwa NiRAN domain (K4465A) ndi RdRp domain (DD5152/3AA) ya SARS-CoV-2 nsp12 idagwiritsidwa ntchito.Manambala otsalira amatengera malo mu pp1ab.(B) Autoradiograph ya kuzindikira kwa UMPylation pogwiritsa ntchito nsp9-His6 ndi [α-32P] UTP monga gawo lapansi la nsp12-His6 (mtundu wakutchire [wt] ndi mutant).Mamolekyu (mu kilodaltons) a mapuloteni olembedwa akuwonetsedwa kumanzere.
Madera a NiRAN nthawi zambiri amasungidwa ku Nidovirales (16), kuwonetsa kuti amathandizira kuti ma enzymatic ayambe kubwerezabwereza Nidovirus.Mu kafukufukuyu, tidatha kutsimikizira kuti dera la NiRAN la coronavirus limasamutsa NMP (yopangidwa kuchokera ku NTP) kupita ku nsp9, puloteni yodabwitsa ya RNA yomwe imamangirira ma virus (26 ?? 29), kuti idziwe ngati chandamale chachilengedwe komanso mnzake wa coronavirus RTC.
Dera la NiRAN limagawana zinthu zitatu zotsatizana (AN, BN, ndi CN), zomwe zili ndi zotsalira zochepa kwambiri zomwe zimasungidwa m'mabanja onse mu dongosolo la monophyletic koma losiyana kwambiri ndi Nidovirales (8, 16).Kafukufuku waposachedwa wawonetsa kuti amalumikizana bwino ndi banja losadziwika bwino la mapuloteni ngati ma protein kinase, omwe poyamba amatchedwa banja la SelO (17, 19, 22, 30, 31).Mapuloteni okhudzana ndi SelO ali ndi zopindika za kinase, koma alibe zotsalira zingapo zosungidwa mu classical kinases (22, 32).Kutengera kusinthika kwa mamolekyu a ATP omangika pamalo omwe akugwira ntchito ndikukhazikika ndi kuyanjana kwapadera, SelO idangoganiziridwa ndipo kenako idatsimikiziridwa kuti imasamutsa AMP (m'malo mwa phosphate) kupita ku gawo la mapuloteni (22), pomwe puloteni ina ya bakiteriya ya SelO yonga YdiU ili nayo. posachedwapa zasonyezedwa kuti zikuthandizira mgwirizano wa UMP ku Tyr ndi zotsalira Zake zamapuloteni osiyanasiyana (33).
Pofuna kutsimikizira ndi kukulitsa kuneneratu kwa malo otsalira a malo otsalira a coronavirus NiRAN, tidagwiritsa ntchito njira za biochemical ndi reverse genetics kusanthula masinthidwe a coronavirus nsp12 (Chithunzi 3D ndi E ndi SI appendix, Chithunzi S3 ndi tebulo) S1â S4).Deta ikuwonetsa kuti m'malo mwa HCoV-229E K4135, R4178 ndi D4280 ndi Ala kumathetsa ntchito yosinthira ya NMP mu m'galasi ya NMP ndi kubwereza kachilombo mu chikhalidwe cha cell (Chithunzi 3D ndi E ndi SI zowonjezera, Chithunzi S3), kuchirikiza kupezeka kwawo mu NTP γ-phosphate. (K4135, R4178) ndi kugwirizana kwa yogwira malo zitsulo ayoni (D4280).Kulowetsedwa kwa E4145A kwa Glu wotetezedwa m'malo osiyanasiyana a kachilombo ka mbalame komwe kunanenedweratu kuti kukhazikitse malo a K4135 (17) adawonetsedwa kuti athetse kubwereza kwa ma virus, koma chodabwitsa, ntchitoyi idasungidwa mu in vitro NMPylation assay (Chithunzi 3D ndi E ndi SI appendix, Chithunzi S3 Ndi Matebulo S1–S4).Kuwona kofananako kunapangidwa pamene kulowetsedwa kofanana kunayambika mu YdiU homolog ya Salmonella typhimurium (E130A) (33).Kuphatikizidwa pamodzi, izi zimathandizira ntchito yoyang'anira zotsalira zosungidwazi m'malo mothandizira ntchito.
Kusintha zotsalira za Phe (F4281A) mkati mwa nestovirus mu HCoV-229E NiRAN domain (8) zinapangitsa kuchepa kwa ntchito ya NMPylation mu vitro komanso kuchepa kwakukulu kwa kufalikira kwa kachilombo mu chikhalidwe cha maselo (Chithunzi 3D, E ndi SI) zowonjezera, Chithunzi S3).Detayo ikugwirizana ndi ntchito yofunika yoyendetsera zotsalira izi, monga zotsalira za DFG zotsalira zomwe zawonetsedwa kale.Mu classical protein kinases, ndi gawo la kuzungulira kwa Mg2 + ndipo imathandizira kusonkhanitsa ndikuwongolera msana???Zofunikira pakuchita zolimbitsa thupi (32, 34).Kulowetsa Ala ndi Arg kwa zotsalira za K4116 (mu preAN motif), motsatana, kunathetsa kubwerezabwereza kwa ma virus ndipo, monga zimayembekezeredwa, zinali ndi zotsatira zosiyana pa ntchito ya NMP transferase in vitro, kutengera unyolo wam'mbali wa amino acid womwe unayambitsidwa (Chithunzi 3D Ndi E ndi zowonjezera za SI. , Chithunzi S3).Deta yogwira ntchito ikugwirizana ndi zomwe zimapangidwira, zomwe zimasonyeza kuti zotsalirazi zakhazikitsa mgwirizano ndi ATP phosphate (17).Mudera la NiRAN la mabanja ena omwe ali ndi kachilomboka, malo a HCoV-229E pp1a/pp1ab K4116 amakhala ndi Lys, Arg kapena His (8), kusonyeza kuti kuletsa kwa zotsalira izi kwachepetsedwa.Kulowetsedwa kwa D4188A ndi D4283A kumathetsa kapena kumachepetsa kwambiri ntchito ya enzyme ndikuchotsa kubwereza kwa kachilombo (Chithunzi 3).Zotsalira ziwirizi zimasungidwa m'ma virus ambiri (koma osati onse) omwe amakhala m'zisa (8), kuwonetsa ntchito yofunika kwambiri yabanja koma mwina yosathandiza.Kusintha kwa Ala m'malo ena angapo a Lys ndi Asp (K4113A, D4180A, D4197A ndi D4273A) omwe sanasungidwe mu Coronaviridae kapena mabanja ena a Nestioviridae (8) adagwiritsidwa ntchito ngati zowongolera.Monga zikuyembekezeredwa, zoloŵa m'malozi zimakhala zolekerera, ndi kuchepa pang'ono kwa ntchito ya enzyme ndi kubwereza kwa tizilombo nthawi zina (Chithunzi 3 ndi SI appendix, Chithunzi S3).Ponseponse, data ya coronavirus mutagenesis imagwirizana kwambiri ndi self-GMP ndi reverse genetics data ya EAV NiRAN-RdRp (16), momwe EAV nsp9 (coronavirus nsp12 ortholog) zotsalira K94 (zogwirizana ndi HCoV- 229E K4135) zofunikira), R124 (yogwirizana ndi R4178), D132 (yogwirizana ndi D4188), D165 (yogwirizana ndi D4280), F166 (yogwirizana ndi F4281).Kuphatikiza apo, data ya HCoV-229E mutagenesis imagwirizana ndikukulitsidwa kuchokera ku data yomwe idanenedwapo kale ya SARS-CoV reverse genetics (16), yofanana kwambiri ndi yomwe imawonedwa ndi CN motif Phe-to-Ala mutant SARS-CoV_nsp12 The phenotype yofotokozedwa -F219A ndi HCoV-229E_F4281A (Chithunzi 3 D ndi E ndi SI zowonjezera, Chithunzi S3 ndi Table S1-S4).
Poyerekeza ndi ma orthologs a EAV (16), omwe amakonda kwambiri UTP ndi GTP (mu self-NMPylation reaction), kafukufuku wathu akuwonetsa kuti dera la coronavirus la NiRAN (loyimiridwa ndi HCoV-229E ndi SARS-CoV-2) litha kukhala bwino. anasamutsa Ma NMP onse anayi, ngakhale pali zokonda pang'ono za UMP (Zithunzi 1 ndi 3).Kutsika kwapang'onopang'ono kwa gawo laling'ono la NTP likugwirizana ndi zomwe zanenedwa posachedwa za SARS-CoV-2 nsp7/8/12/13 mawonekedwe apamwamba, momwe ADP-Mg2+ imamangirira pamalo omwe akugwira ntchito ku NiRAN, koma osati ndi gawo la adenine. za kupanga kwa kuyanjana kwapadera (17).Mu phunziro lathu, mtundu wa nucleotide womwe umagwiritsidwa ntchito mu NMPylation reaction ulibe kusiyana kosiyana pa ntchito ya mapuloteni osinthika (SI Zowonjezera, Chithunzi S3), kusonyeza kuti palibe zotsalirazi zomwe zimagwirizana kwambiri ndi kumangirira kwa nucleobase inayake.Maziko okhazikika komanso kufunikira kwachilengedwe kwamitundu yosiyanasiyana ya NTP co-substrate zowonedwa m'magawo a NiRAN a coronaviruses ndi ma virus a arterial zikuyenera kuphunziridwa;zikhoza kukhala zoona kapena chifukwa cha kuchepa kwa maphunziro awo.Pakalipano, sizinganenedwe kuti ntchito ya NMPylator ya kachilombo koyambitsa matenda a NiRAN (poyerekeza ndi ntchito yodzipangira yokha ya NMPylation) ili ndi zokonda zosiyana, poganizira kuti kufanana pakati pa arterial ndi coronavirus. NiRAN domain yatsala pang'ono kutha.Kutengera motsatana (16).Poyerekeza ndi pseudokinase SelO, yomwe imagwiritsa ntchito Mg2 + ngati cofactor, ntchito ya coronavirus ndi arterial virus NiRAN imadalira Mn2 + (16) (Chithunzi 3C ndi SI appendix, Chithunzi S1).Kudalira kwa Mn2 + ndi zodziwika bwino za UTP ndi chinthu chachilendo cha mapuloteni a NMPylators, ndipo zatsimikiziridwa posachedwapa mu mapuloteni a YdiU a Salmonella typhimurium, omwe amachititsa kuti Mn2 + -yomwe amadalira mapuloteni a UMPylation kuti ateteze maselo ku dziwe la ATP la kupsinjika maganizo. 33).
Kufanana komwe kwafotokozedwa posachedwa pakati pa dera la coronavirus la NiRAN ndi ma cell protein kinases (17, 19) kumapereka chithandizo chowonjezera pakutha kwa NiRAN kulumikiza bwino NMP ndi mapuloteni ena omwe tanena mu kafukufukuyu.Tidayang'ana pakusaka zomwe tingakwaniritse za NiRAN pamapuloteni osungidwa ndi HCoV-229E ORF1a, omwe amadziwika kuti amathandiza mwachindunji kapena mwanjira ina ya RTC's ORF1b-encoded replicase (12, 35).Kuyesera kwathu kumapereka umboni wotsimikizirika wa NMPylation yogwira mtima komanso yeniyeni ya nsp9 (Chithunzi 2).Ngati puloteni yomwe ikukhudzidwa ikugwiritsidwa ntchito mopitirira muyeso wa molar womwe ndi 8 mpaka 10 kuposa wa enzyme (nsp12), zimatsimikiziridwa kuti nsp9 kwathunthu (mono) NMPized (Chithunzi 4).Tidatsimikiza kuti kuyanjana pakati pa nsp12 ndi nsp9 kumakhala kwakanthawi ndipo sikungapange zovuta zokhazikika ndi nsp9 (popanda ma subunits ena a RTC).Kutsiliza uku kumathandizidwa ndi maphunziro okhudzana ndi mapuloteni pa SARS-CoV proteome (35).Kusanthula kwa MS kunazindikiritsa amine woyambirira wa N-terminal zotsalira za nsp9 ngati malo a NMPylation (SI appendix, Chithunzi S5).Kupanga mgwirizano wa phosphoramidate ndi gulu la amino la N-terminal limasiyanitsa ntchito ya NiRAN-mediated NMPylation kuchokera ku Pseudomonas syringae SelO-mediated AMPylation reaction, yomwe imayambitsa mapangidwe a O-linked AMP ku Ser, Thr, kapena Tyr zotsalira za Peptide ( 22), ndipo S. typhimurium YdiU imapanga O-olumikizidwa (ndi Tyr) ndi N-yolumikizidwa (ndi Ake) peptide-UMP adducts.Zomwe zilipo pagulu la mapuloteni a SelO zimasonyeza kuti mamembala a banja lalikulu la mapuloteni amasiyana kwambiri pakupanga ma peptide-NMP adducts.Ichi ndi lingaliro lochititsa chidwi lomwe liyenera kuphunzira mowonjezereka.
Zomwe tapeza mu phunziroli zidatipangitsa kulingalira kuti NMPylation ya nsp9 imafuna N-terminus yaulere.Pankhani ya kubwereza kwa ma virus, izi zidzaperekedwa ndi proteolytic cleavage ya nsp8|nsp9 pokonza malo mu replicase polyprotein pp1a mediated ndi Mpro ndi pp1ab.M'ma coronavirus ambiri, kusiyana pakati pa tsamba ili (VKLQ|NNEI mu HCoV-229E) ndi malo ena onse a coronavirus Mpro cleavage ndi Asn (m'malo mwa zotsalira zina zazing'ono, monga Ala, Ser Kapena Gly) zimakhala P1â???Malo (36).Deta ya peptide cleavage yomwe inapezedwa m'mafukufuku oyambirira inasonyeza kuti cleavage ya malo a nsp8|nsp9 inali yochepa kusiyana ndi malo ena, kusonyeza kuti 1) tsamba ili likhoza kukhala ndi gawo loyang'anira pakukonzekera nthawi yake ya C-terminal. pp1a dera, kapena 2) a Ntchito ya nsp9 N-terminus yapadera yotetezedwa pakubwereza kachilombo ka HIV (37).Deta yathu (Chithunzi 5A) idawonetsa kuti mawonekedwe ophatikizanso a nsp9 onyamula mndandanda weniweni wa N-terminal anali bwino NMPized ndi nsp12.Mndandanda wa N-terminal flanking unachotsedwa ndi factor Xa (nsp9-His6; SI appendix, Table S1) kapena Mpro-mediated cleavage (nsp7-11-His6; Chithunzi 5A ndi SI appendix, Table S1).Chofunika kwambiri, nsp9-containing precursor nsp7-11-His6 yosagwirizana ndi NMPylation ya nsp12, yomwe ikugwirizana ndi deta yathu, kusonyeza kuti nsp9-NMP adduct imapangidwa kudzera mu N-terminal primary amine ( SI appendix, Chithunzi S5) .Kuti timvetsetse mozama za gawo la gawo la NiRAN, tidayang'ananso zotsalira za N-terminal za nsp9.Mapuloteni ena akapanda, amakhala osinthika mwamapangidwe, kuwalepheretsa kuti adziwike mu mawonekedwe osalembedwa a nsp9 (26 28, 38), kuwonetsa kusiyanasiyana kwawo kochepa. ntchito ya nsp9 N-terminal fragment.Ala m'malo mwa zotsalira zosungidwa m'derali (Zizindikiro 5C ndi D ndi zowonjezera za SI, Chithunzi S8) zimasonyeza kuti N3826 ndiyofunikira pa nsp9 NMPylation in vitro, pamene N3825A ndi E3827A m'malo mwake zimayambitsa kuchepa kwa NMPylation, pamene M3829A ndi P38 do38 m'malo .Mwachiwonekere zimakhudza nsp9 NMPylation.Ngakhale kulowetsedwa kwa N-terminal Asn (N3825A, N3825S) kumangokhudza pang'onopang'ono pa nsp9 NMPylation ndi kubwereza kachilomboka mu selo chikhalidwe (Chithunzi 5C ndi D), kuchotsedwa kwa zotsalira za Asn kuchokera ku N-terminal 3825-NN dipeptide. zatsimikiziridwa kuti Ndi zakupha kwa mavairasi, kusonyeza kuti zotsalira za Asn zimafunika pasanafike zotsalira zina pa N-terminus, makamaka Asn, ngakhale zikuwoneka kuti m'malo mwa zotsalira zofanana zingathe kulekerera pang'ono (Chithunzi 5B, C, ndi D).Tikuwona kuti dipeptide ya 3825-NN, makamaka zotsalira zosungidwa ndi zofunika za N3826 mkati mwa gulu la coronavirus (SI appendix, Chithunzi S6), imatsimikizira kumangirira kolondola kwa nsp9 N-terminus pamalo omwe akugwira ntchito ku NiRAN.
Kulowetsa Ala (E3827A) kwa Glu yosungidwa yamagulu onse ang'onoang'ono kumasunga nsp9 NMPylation mu vitro koma ndi yowopsa ku ma virus mu cell cell (Chithunzi 5C ndi D), kuwonetsa ntchito yowonjezera ya zotsalirazi, mwachitsanzo, pakulumikizana kwakukulu (NMPylated kapena yosasinthidwa. ) nsp9 N-terminus ndi zinthu zina zomwe zimakhudzidwa ndi kuchulukitsa kwa ma virus.Kusintha kwa Nsp9 sikunakhudze njira ya proteolytic ya nsp9 kapena nsps iliyonse yoyandikana (39) (SI Zowonjezera, Chithunzi S9), kusonyeza kuti ma phenotypes akupha a masinthidwe angapo a nsp9 omwe adawonedwa sanayambitsidwe ndi kusokonekera kwa C proteolytic process-terminal pp1a .
Zomwe zili pamwambazi zimapereka umboni kuti pambuyo pa chithandizo cha Mpro-mediated cha nsp8|9 cleavage site in pp1a/pp1ab, N-terminus ya nsp9 ikhoza kukhala UMPylated (kapena kusinthidwa pang'ono ndi NMP ina).Kuphatikiza apo, kusungidwa kwabwino kwa N-terminus ya nsp9 (kuphatikiza zotsalira za Asn m'banja la coronavirus) komanso zotsalira zamtundu zomwe zapezeka mu kafukufukuyu (Zithunzi 3E ndi 5D) zidatipangitsa kunena kuti nsp9 NMPylation yofotokozedwayo. ndizogwirizana ndi biologically ndipo ndizofunikira pakubwerezabwereza kwa coronavirus.Zotsatira zakusinthaku zikuyenera kuphunziridwabe, mwachitsanzo, ponena za zomwe zafotokozedwa kale (zosadziwika) nsp9 (mawonekedwe osasinthidwa) ntchito yomanga ya RNA (2628).N-terminal NMPylation ingakhudzenso kuyanjana kwa nsp9 ndi mapuloteni kapena ma RNA substrates kapena mapangidwe a magulu anayi osiyana.Izi zawonedwa m'maphunziro amapangidwe ndipo zatsimikiziridwa kuti zimagwirizana ndi kubwerezabwereza kwa coronavirus, ngakhale makamaka pakalibe Pankhani yosinthidwa (26- ââ29, 40).
Ngakhale chandamale chandalama ya coronavirus ya NiRAN ikufunikabe kufotokozedwa mwatsatanetsatane, zambiri zathu zikuwonetsa kuti puloteni yomwe imayang'anira dera la coronavirus la NiRAN ndiyopapatiza kwambiri.Ngakhale kusungidwa kwa zotsalira zazikulu zomwe zimagwira ntchito (8, 16) mu dera la NiRAN la mabanja onse a nidovirus zimathandizira kwambiri ntchito ya NMPylator yosungidwa ya mapuloteni awa, zidziwitso za gawo lapansi zotsalira za m'thumba za derali Kusamalira ndi kusungirako zikuyenera kudziwika , ndipo akhoza kusiyana pakati pa mabanja osiyanasiyana a zolinga za Nidovirales.Momwemonso, zolinga zoyenera za ma virus ena okhala m'chisa sizinatsimikizidwebe.Atha kukhala ma orthologs akutali a nsp9 kapena mapuloteni ena, chifukwa kutsatizana kunja kwa zigawo zisanu zofananira zomwe nthawi zambiri zimasungidwa mu ma virus okhala ndi zisa ndizotetezedwa (8), kuphatikiza ma genome pakati pa Mpro ndi NiRAN, Pakati pawo, nsp9 ili mu kachilombo ka corona.
Kuonjezera apo, sitingathe kutsutsa kuti n'zotheka kuti dera la NiRAN lili ndi zowonjezera (kuphatikizapo ma cellular) zolinga.Pankhaniyi, ndiyenera kunena kuti ma homologues a bakiteriya omwe ali mu mapuloteni omwe akubwerawa a NMPylators (NMPylators) (30, 31) akuwoneka kuti ali ndi "olamulira akuluakulu"?NMP imayang'anira mapuloteni osiyanasiyana am'manja kuti azitha kuyendetsa kapena kuthetsa ntchito zawo zotsika, potero amatenga gawo munjira zosiyanasiyana zachilengedwe, monga kuyankha kwapang'onopang'ono kwa ma cell ndi redox homeostasis (22, 33).
Mu phunziro ili (Zithunzi 2 ndi 4 ndi SI Zowonjezera, Zithunzi S3 ndi S5), tinatha kutsimikizira kuti nsp12 inasamutsa gawo la UMP (NMP) kumalo amodzi (otetezedwa) mu nsp9, pamene mapuloteni ena sanasinthidwe mu kugwiritsidwa ntchito Pansi pazimenezi, kufotokozedwa bwino (m'malo momasuka) gawo lapansi limathandizidwa.Mogwirizana ndi izi, poyerekeza ndi N-terminal nsp9 NMPylation, nsp12's mwiniwake wa NMPylation ntchito ndi yotsika kwambiri, kuzindikira kwake kumafuna nthawi yotalikirapo ya autoradiography, ndipo kuwonjezeka kwa 10 kwa nsp12 kumagwiritsidwa ntchito.Kuphatikiza apo, kusanthula kwathu kwa MS kunalephera kupereka umboni wa NMPylation wa nsp12, zomwe zikuwonetsa kuti NiRAN domain self-NMPylation ndi (bwino kwambiri) ntchito yachiwiri.Komabe, ziyenera kudziwidwa kuti maphunziro ena apereka umboni woyambirira kuti kudziyimira pawokha kwa AMPylation kwa bakiteriya NMPylator kumatha kuwongolera zochita zawo za NMPylation pama protein ena (22, 33).Chifukwa chake, kafukufuku wochulukirapo akufunika kuti afufuze zomwe zingachitike chifukwa cha ntchito zodzipangira okha NMPylation zomwe zanenedwa za EAV nsp9 (16) ndi coronavirus nsp12 (kafukufukuyu), kuphatikiza zomwe akufuna kukhala ngati chaperone pakupinda kwa C-terminal RdRp domain ( 16))
M'mbuyomu, malingaliro angapo okhudzana ndi zomwe zingachitike kumunsi kwa dera la nidoviral NiRAN adaganiziridwa, kuphatikiza RNA ligase, RNA -capped guanylate transferase ndi protein priming activity (16), koma palibe imodzi yomwe imagwirizana ndi ntchito zomwe zimapezeka kumunsi.Zomwe zapezedwa m'malo otsatirawa ndi nthawi yomweyo popanda kupanga malingaliro owonjezera.Zomwe zapezedwa mu phunziroli zimagwirizana kwambiri (koma sizingatsimikizire) kuti dera la NiRAN likukhudzidwa ndi kuyambitsa kaphatikizidwe ka RNA kopangidwa ndi mapuloteni.Poyamba ankakhulupirira kuti ntchito ya NiRAN domain mu 5 ??²-RNA capping kapena RNA ligation reactions sizimakhudzidwa ndi izi komanso Kuthandizira zina.Chifukwa chake, mwachitsanzo, tsamba logwira ntchito la NiRAN limaonedwa kuti likuphatikiza Asp yosungidwa ngati maziko (D252 mu Pseudomonas syringae SelO; D4271 mu HCoV-229E pp1ab; D208 mu SARS-CoV-2 nsp12) (SI Zowonjezera, chithunzi 2 ).S2) (17, 22, 33), pamene catalysis mu ATP-yodalira RNA ligase ndi RNA capping enzyme imachitika ndi covalent enzyme-(lysyl-N)-NMP yapakatikati, yomwe imaphatikizapo zotsalira za Lys zosasinthika. 41).Kuphatikiza apo, kutsatizana kodziwika bwino kwa coronavirus NiRAN pazolinga zosungidwa zamapuloteni komanso kumasuka kwa ma co-substrates a NTP (imakonda UTP) kumatsutsana ndi enzyme ya NiRAN-mediated capping kapena RNA ligase-like function.
Mwachiwonekere, ntchito yowonjezereka yowonjezereka ikufunika kuti zitsimikizidwe, ndipo, ngati zatsimikiziridwa, kufotokozera za ntchito yomwe nsp9-UMP (nsp9-NMP) ingagwiritsidwe ntchito ndi mapuloteni opangidwa ndi RNA synthesis, yomwe idzagwirizanitsa zingapo zosangalatsa koma (mpaka pano) malipoti omwe adanenedwa kale. .Kuwona kwapayekha.Mwachitsanzo, zatsimikiziridwa kuti kutha kwa RNA yoyipa ya coronavirus imayamba ndi chingwe cha oligo(U) (42, 43).Kuwonetsetsa uku kumagwirizana ndi lingaliro lakuti kaphatikizidwe ka strand-strand RNA imayambika ndikumanga mawonekedwe a UMPylated a nsp9 ku poly(A) mchira (zoyambitsa), zomwe zingathe kulimbikitsidwa ndi RNA kumanga Ntchito ndi / kapena kuyanjana ndi puloteni wina wa RTC.Gawo la UMP loperekedwa ndi nsp9 litha kugwiritsidwa ntchito ngati "primer" ya nsp7/8/nsp12-mediated oligouridylation, pogwiritsa ntchito 3??²-poly(A) mchira mu genomic RNA kapena Wina oligo (A) -otsatira mndandanda. imakhala ngati template, yofanana ndi njira yokhazikitsidwa ndi picornavirus VPg protein (44).Bwanji ngati lingalirolo liri "losakhazikika"????Kukhazikitsidwa kwa (protein-induced) negative-strand RNA synthesis kumapereka ulalo pazowonera, kuwonetsa kuti coronavirus negative-strand RNA ili ndi UMP (m'malo mwa UTP) kumapeto kwake (42), zomwe zimadziwika kuti zikuwonetsa kuti nucleic acid Dicer imadula mapeto a phosphorylated ndi endonuclease yosadziwika ya uridine.Ngati zitsimikizidwa, ntchito ya nucleic acid hydrolytic iyi ingathandize kumasula mawonekedwe a oligomeric UMPylated a nsp9 kuchokera kumapeto kwa 5 ² kwa chingwe chopanda pake.Kuthekera kwa nsp9 pakupanga mapuloteni kumagwirizananso ndi kafukufuku wam'mbuyomu wa genetics, omwe awonetsa kuti nsp9 (ndi nsp8) imalumikizana mozama komanso makamaka ndi chinthu chosungidwa cha RNA chomwe chili pafupi ndi 3 kumapeto kwa genome ya coronavirus.45).Malinga ndi lipotili, zowonera zakalezi tsopano zitha kuwunikidwanso ndikukulitsidwa kudzera mu kafukufuku wina.
Mwachidule, deta yathu idatsimikiza zomwe zidachitika ndi tag ya enzyme yomwe idalumikizidwa ndi RdRp pa N-terminus.Mu coronavirus, ntchito yomwe yangopezeka kumene ya NiRAN-mediated UMPylator/NMPylator imagwiritsidwa ntchito kudalira Mn2+ ndi zotsalira zapafupi za Asn ndikuyambitsa kupanga (zochepa mphamvu) zomangira za phosphoramidate ndi N-terminal primary amine.Kupyolera mu Mpro-mediated cleavage pa nsp8|9 cleavage site, nsp9 target ingagwiritsidwe ntchito pa NMPylation, kusonyeza kugwirizanitsa ntchito pakati pa protease ndi NiRAN domain, yomwe imafikira ku RdRp.Kusungidwa kwa zotsalira zazikulu pa tsamba logwira ntchito la nsp12 NiRAN ndi chandamale cha nsp9, kuphatikiza ndi zomwe zapezedwa kuchokera ku ma coronaviruses awiri kuphatikiza SARS-CoV-2, zimapereka umboni wamphamvu wakuti nsp9 NMPylation ndi mawonekedwe a coronavirus Conservative nawonso ndi gawo lofunikira pakubwereza kwa kachilomboka.Zomwe zilipo zimatipangitsa kunena kuti gawo lenileni la NMPylated mawonekedwe a nsp9 mu kaphatikizidwe ka RNA yopangidwa ndi mapuloteni ndizochitika zomveka za ma virus ndi ma virus ena omwe ali ndi zisa, ndipo NiRAN imathanso kulunjika mapuloteni ena osadziwika.Sinthani kachilomboka.Host kucheza.Ngati zitsimikizidwa, kutengapo gawo kwa mapuloteni oyambira mu kaphatikizidwe ka RNA kudzakulitsa kugwirizana kwa madera a Mpro/3CLpro ndi RdRp pakati pa gulu lodziwika bwino la coronavirus ndi picornavirus-like supergroup (9), zomwe tsopano zalumikizidwa mu Pisonivirites yomwe yangokhazikitsidwa kumene. 46) mu gulu.
Zomwe tapeza zikuwonetsanso kuti ntchito zoyambira, zosankha komanso zosunga ma enzyme zomwe zadziwika mu kafukufukuyu zitha kugwiritsidwa ntchito ngati chandamale chamankhwala oletsa ma virus.Mankhwala omwe amasokoneza kumangirira (ndi kusinthidwa kotsatira) kwa nsp9 N-terminus yosungidwa pamalopo a NiRAN amatha kupangidwa kukhala mankhwala othandiza komanso osunthika oletsa ma virus, oyenera kuchiza ma coronavirus a nyama ndi anthu ochokera kumitundu yosiyanasiyana (sub) Matenda. , kuphatikiza SARS-CoV-2 ndi Middle East Respiratory Syndrome Coronavirus.
Kalozera wamapuloteni a coronavirus opangidwa mu kafukufukuyu adakulitsidwa ndi RT-PCR pogwiritsa ntchito RNA yotalikirana ndi Huh-7 yomwe ili ndi HCoV-229E kapena Vero E6 yomwe ili ndi kachilombo ka SARS-CoV-2, ndikuyika pogwiritsa ntchito njira zofananira.pMAL-c2 (New England Biological Laboratory) kapena pASK3-Ub-CHis6 (47) vesi la mawu (SI Zowonjezera, Matebulo S1 ndi S2).Ma codon amodzi omwe adalowetsedwa adayambitsidwa ndi mutagenesis yoyendetsedwa ndi PCR (48).Kuti apange mapuloteni osakanikirana a MBP, maselo a E. coli TB1 anasinthidwa ndi pMAL-c2 plasmid construction yoyenera (SI appendix, Table S1).Puloteni yosakanikirana idayeretsedwa ndi amylose affinity chromatography ndikumata ndi factor Xa.Pambuyo pake, puloteni ya C-terminal His6-tagged idatsukidwa ndi Ni-immobilized metal affinity chromatography (Ni-IMAC) monga tafotokozera kale (49).Kuti apange mapuloteni a ubiquitin fusion, maselo a E. coli TB1 amagwiritsa ntchito pASK3-Ub-CHIs6 plasmid construction (SI Zowonjezera, Tables S1 ndi S2) ndi pCGI plasmid DNA encoding ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Kusintha (47).Mapuloteni a C-terminal His6-tagged coronavirus adayeretsedwa monga tafotokozera kale (50).
Kuyesa kwa self-NMPylation kwa HCoV-229E nsp12-His6 kunachitika monga momwe EAV nsp9 (16) inafotokozera.Mwachidule, nsp12-His6 (0.5 µM) ili ndi 50 mM 4-(2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES)-KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25, µM buffer NTP yotchulidwa ndi 0.17 µM yofanana ndi [α32-P]NTP (3,000 Ci/mmol; Hartmann Analytic) pa 30 °C kwa mphindi 30.Muzinthu zina zonse (zokhazikika) za NMPylation za nsp12-mediated nsp9 NMPylation, momwe zimachitikira zimasinthidwa motere: nsp12-His6 (0.05 µM) ndi nsp9-His6 (4 µM) pamaso pa 50 mM HEPES-KOH (pH 8.0) ), 5 mM DTT, 1 mM MnCl2, 25 µM yosonyeza NTP, ndi 0.17 µM yofanana [α32-P]NTP.Pambuyo incubating kwa mphindi 10 pa 30 ° C, zimene chitsanzo anali wosakaniza SDS-PAGE chitsanzo bafa: 62.5 mm tris(hydroxymethyl) aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol ndi 0.005% bromophenol buluu.Puloteni idasinthidwa ndikuwotcha pa 90 ° C kwa mphindi 5 ndikulekanitsidwa ndi 12% SDS-PAGE.Gelisiyo imakhazikika komanso yodetsedwa ndi njira ya Coomassie Brilliant Blue (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), yodetsedwa, ndikuwonetsedwa pazithunzi zojambula za phosphorescent kwa maola 20 (kuti muzindikire nsp12 kuchokera ku NMPylation) kapena (pazipita) 2 Maola (kuwunika nsp9 NMPylation).Chithunzi cha Typhoon 9200 (GE Healthcare) chinagwiritsidwa ntchito kusanthula chophimba ndipo ImageJ idagwiritsidwa ntchito kusanthula kulimba kwa siginecha.
Pakusanthula kwa MS, 1 µM nsp12-His6 ndi 10 µM nsp9 (popanda tag ya hexahistidine) adagwiritsidwa ntchito pakuwunika kwa NMPylation (SI appendix, Table S1) komanso kuchuluka kwa 500 µM UTP ndi GTP kunagwiritsidwa ntchito.Kutengera kuchuluka kwawo komanso kuchuluka kwa mapuloteni omwe amayembekezeka, njira ya Waters ACQUITY H-Class HPLC yokhala ndi gawo la MassPrep (Waters) idagwiritsidwa ntchito kutulutsa 1 mpaka 10 µL ya mayankho a protein omwe amasungidwa pa intaneti.Mapuloteni osungunuka amalowetsedwa mu gwero la electrospray ion la Synapt G2Si mass spectrometer (Madzi) kudzera mumtsinje wotsatira wa buffer A (madzi/0.05% formic acid) ndi bafa B (acetonitrile/0.045% formic acid), ndipo kutentha kwa gawo ndi 60 ° C ndi kuthamanga kwa 0.1 mL / min: kutulutsa mobwerezabwereza ndi 5% A kwa mphindi ziwiri, kenako mzere wozungulira mpaka 95% B mkati mwa mphindi 8, ndikusunga 95% B kwa mphindi zina zinayi.
Ma ion abwino okhala ndi kuchuluka kwa 500 mpaka 5000 m/z amapezeka.Glu-fibrinopeptide B imayezedwa masekondi 45 aliwonse kuti akonze zongoyenda modzidzimutsa.Gwiritsani ntchito pulogalamu ya chida cha MassLynx yokhala ndi MaxEnt1 yowonjezera kuti muchepetse mawonekedwe apakati mutachotsa zoyambira ndikusalaza.
UMPylated HCoV-229E nsp9 idagayidwa powonjezera sequencing-grade modified trypsin (Serva) ndikuumirira usiku wonse pa 37 °C.Chromabond C18WP spin column (gawo nambala 730522; Macherey-Nagel) idagwiritsidwa ntchito kutsuka ndi kuyika ma peptides.Pomaliza, peptide idasungunuka mu 25 µL yamadzi, yomwe inali ndi 5% acetonitrile ndi 0.1% formic acid.
Zitsanzozi zidawunikidwa ndi MS pogwiritsa ntchito Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Makina omaliza a nanoâ HPLC (Dionex), okhala ndi chizolowezi chokwera 50 cm??75 μm C18 RP ndime yodzaza ndi 2.4 μm maginito mikanda (Dr. Albin Maisch High Performance LC GmbH) Lumikizani ku mass spectrometer pa intaneti kudzera pa gwero la Proxeon nanospray;jekeseni 6 µL wa trypsin digestion solution mu 300 µm m'mimba mwake ×??1 masentimita C18 PepMap gawo lokhazikika lachindunji (Thermo Scientific).Pogwiritsa ntchito madzi / 0.05% formic acid monga zosungunulira, chitsanzocho chinagwidwa ndi kuchotsedwa pamadzi otaya 6 µL/min.
Ma gradients otsatirawa a madzi/0.05% formic acid (solvent A) ndi 80% acetonitrile/0.045% formic acid (zosungunulira B) adagwiritsidwa ntchito kuti akwaniritse kulekanitsa kwa tryptic peptides pakuyenda kwa 300 nL/min: 4% B kwa Mphindi 5, ndiye 30 A liniya gradient kuti 45% B mkati mphindi, ndi liniya kuwonjezeka kwa 95% zosungunulira B mkati 5 Mphindi.Lumikizani ndime ya chromatographic ku nano-emitter yachitsulo chosapanga dzimbiri (Proxeon), ndikupoperani molunjika ku capillary yotenthedwa ya mass spectrometer pogwiritsa ntchito kuthekera kwa 2,300 V. Kujambula kwa kafukufukuyu ndi kusamvana kwa 60,000 mu Orbitrap mass analyzer kumalumikizidwa. ndi ma data osachepera atatu a MS/MS, osaphatikizidwa kwa masekondi a 30, pogwiritsa ntchito mzere wa ion trap collision induced dissociation kapena kugunda kwamphamvu kwamphamvu kophatikizana ndi kuzindikira kwa orbitrap, Kusamvana ndi 7,500.
Nthawi yotumiza: Aug-03-2021