Edita daga Peter Sarnow, Makarantar Magunguna ta Jami'ar Stanford, Jami'ar Stanford, California, an amince da ita a ranar 25 ga Disamba, 2020 (aka sake dubawa ranar 25 ga Oktoba, 2020)
Muna ba da rahoton hulɗar tsakanin ƙananan bayanai a cikin kwafi na coronaviruses-rufin rukunoni, waɗanda ke da mahimmanci don maimaitawa da kiyaye juyin halitta.Mun bayar da shaida cewa yankin NiRAN da ke da alaƙa da nsp12 yana da nucleoside monophosphate (NMP) transferase aiki a cikin trans, kuma an gano nsp9 (protein mai ɗaure RNA) a matsayin manufa.NiRAN yana haɓaka haɗin haɗin haɗin NMP zuwa madaidaicin nsp9 amino terminus a cikin martanin da ya dogara da ions Mn2+ da ragowar Asn da aka adana.An gano cewa ayyukan NiRAN da nsp9 NMPylation suna da mahimmanci don kwafin coronavirus.Bayanan sun ba mu damar danganta wannan aikin na alamar enzyme mai ƙwayar cuta zuwa abubuwan da suka faru a baya a cikin hasashe cewa ƙaddamar da haɗin RNA a cikin nau'in ƙwayoyin cuta na RNA yana aiki kuma daidai da juyin halitta.
RNA polymerase (RdRps) mai dogara da RNA na Nidovirales (Coronaviridae, Arterioviridae, da sauran iyalai 12) yana da alaƙa da yankin amino-terminal (N-terminal) a cikin furotin mara tsari (nsp) wanda aka saki daga polyprotein, wanda ake kira NiRAN 1ab ya ƙunshi babban kwayar cutar kwayar cuta (Mpro).A baya can, an ba da rahoton aikin GMPylation/UMPylation na NiRAN-RdRp nsp na kansa, kuma an ba da shawarar don samar da wani ɗan lokaci don canja wurin nucleoside monophosphate (NMP) zuwa ƙwayoyin cuta (wanda ba a sani ba a halin yanzu) da / ko abubuwan biopolymerization cell.Anan, mun nuna cewa coronavirus (Human Coronavirus [HCoV] -229E da Cutar Cutar Cutar Cutar Cutar Cutar Cutar Cutar ta Coronavirus 2) nsp12 (NiRAN-RdRp) tana da ayyukan NMPylation na Mn2+, wanda aka samo daga nsp9 ta hanyar samuwar Mpro-mediated nsp9 Bayan N-terminal flanking nsps an saki proteolytically, phosphoramidate an haɗa shi da amine na farko (N3825) a N-terminal na nsp9.Uridine triphosphate shine nucleotide da aka fi so a cikin wannan halayen, amma adenosine triphosphate, guanosine triphosphate, da cytidine triphosphate suma sun dace da haɗin gwiwa.Nazarin maye gurbi ta hanyar amfani da recombinant coronavirus nsp9 da nsp12 sunadaran da kwayoyin halitta HCoV-229E mutants sun ƙaddara ragowar abubuwan da suka dace don nsp9 NMPylation mai tsaka-tsakin NiRAN da kwafi a cikin al'adar tantanin halitta.Bayanan sun tabbatar da tsinkayar ragowar rukunin yanar gizon NiRAN kuma sun ƙayyade muhimmiyar rawar nsp9 N3826 a cikin nsp9 NMPylation da kwayar cutar kwafi a cikin vitro.Wannan ragowar wani bangare ne na jerin N-terminal NNE tripeptide da aka kiyaye kuma an tabbatar da cewa shine kawai ragowar nsp9 da homologs a cikin dangin coronavirus.Wannan binciken yana ba da tushe mai ƙarfi don nazarin aiki na ayyukan NMPylation na sauran ƙwayoyin cuta masu gurɓatacce kuma yana ba da shawarar yuwuwar manufa don haɓaka magungunan rigakafin cutar.
Nidovirales tabbatacce-stranded RNA virus yana cutar da nau'ikan kasusuwa da invertebrates (1, 2).A halin yanzu odar ta ƙunshi iyalai 14 (3), waɗanda aka yi nazari sosai kan dangin Coronavirus a cikin shekaru 20 da suka gabata.A wancan lokacin, coronaviruses zoonotic guda uku sun fito daga rundunonin dabbobi kuma sun haifar da barkewar manyan cututtukan cututtukan numfashi a cikin mutane.Ciki har da cututtukan cututtukan da ke ci gaba da haifar da mummunar cututtuka masu tsanani.Ciwon numfashi Coronavirus 2 (SARS-CoV-2) (4âââ7).Nidoviruses suna raba ƙungiyar genome na gama-gari, kuma ɓangaren madaidaicin madaidaicin rubutun-haɗe-haɗe (RTC) an tsara shi a cikin kashi biyu cikin uku na 5-?²-tashar da babban ɓangaren tsarin kwayar cutar, da kuma wasu kayan haɗi. .Protein, wanda aka sanya a cikin 3??² ƙarshen ukun kwayoyin halitta (1).Sai dai dangi ɗaya na ƙwayoyin cuta na planarian (Monoviridae) (8), duk ƙwayoyin cuta na gida suna ɓoye ramukan RTC a cikin manyan firam ɗin buɗe ido biyu (ORF) ORF1a da ORF1b, waɗanda aka fassara daga RNA na genomic na.ORF1a yana ɓoye polyprotein (pp) 1a, da ORF1a da ORF1b tare da ɓoye pp1ab.Tare da babban haɗin kai na babban protease (Mpro) wanda ORF1a ya tsara, duka pp1a da pp1ab ana sarrafa su ta hanyar kariya zuwa nau'ikan sunadarai marasa tsari (nsps), wanda kuma aka sani da 3CLpro, saboda yana da homology tare da 3Cpro na picornavirus ( 9).Ana tsammanin waɗannan nsps za a haɗa su cikin babban RTC mai ƙarfi, suna haifar da haɗakar halittar RNA (kwafi) da saitin RNA na ƙasa (fassara), kuma ana amfani da su don daidaita bayanin ORF da ke ƙasan ORF1b (10? ? ?12).
Babban RTC ya haɗa da RNA-dependent RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) da yawancin enzymes na sarrafa RNA, waɗanda galibi ke cikin ORF1b kuma a cikin dangin coronavirus Ya ƙunshi nsp12-nsp16 da nsp9-nsp12 a cikin dangin Arterioviridae (duba tunani 10ââ 12).RdRp da HEL1 suna wakiltar yankuna biyu (ɗaya cikin biyar) waɗanda aka kiyaye su na ƙwayar tsuntsun gida kuma suna da homology tsakanin sauran ƙwayoyin cuta na RNA.An yi imanin cewa wasu ƙananan ƙungiyoyi ne ke taimakawa, gami da ƙananan nsps da yawa waɗanda aka saki daga yankin carboxy-terminal (C-terminal) na pp1a, ƙasan Mpro (coronavirus nsp5 da ƙwayar cuta ta arterial nsp4, bi da bi).Suna da ƙayyadaddun kariyar ƙayyadaddun iyali da ayyuka daban-daban (an sake duba su a shafi 10ââ12).
Kwanan nan, an sami wani yanki mai nau'ikan halaye na musamman na jeri a madaidaicin amino (N-terminus) kusa da RdRp a cikin duk ƙwayoyin cuta na gida, amma babu wasu ƙwayoyin cuta na RNA (16).Dangane da wurin sa da kuma nucleotide transferase (nucleoside monophosphate [NMP] transferase), ana kiran wannan yanki NiRAN (Nestvirus RdRp mai alaƙa da nucleotide transferase).Haɗin yanki biyu na NiRAN-RdRp ya ƙunshi nsp12 a cikin dangin Coronaviridae da nsp9 a cikin dangin Arterioviridae, kuma a cikin sauran nestoviridae, NiRAN-RdRp ana sa ran za a sake shi azaman nsp mai zaman kanta daga polyprotein na hoto.A cikin coronavirus, yankin NiRAN ya ƙunshi ragowar ??1/450 kuma an haɗa shi zuwa yankin C-terminal RdRp ta yankin haɗin gwiwa (16?19).A cikin Equine Arteritis Virus (EAV) (Arteriviridae), recombinant nsp9 yana nuna Mn2+ ion-dogara (kai) ayyukan UMPylation da GMPylation, wanda ya dogara da tushe guda uku da aka adana a cikin nestovirus, AN, BN da CN Ragowar a cikin jerin.Inda N ke nufin NiRAN) (16).Ƙaƙwalwar N-terminal na waɗannan motifs ɗin preAN ne mai ƙarancin ra'ayin mazan jiya.Wasu daga cikin waɗannan ragowar kuma ana adana su a cikin kinases sunadaran sunadaran da ke da alaƙa, inda aka nuna su suna da hannu a cikin ɗaurin nucleoside triphosphate (NTP) da ayyukan catalytic (20, 21).Dangane da wannan lura, ragowar maɓalli masu aiki da yawa a cikin pseudokinase SelO daga Pseudomonas syringae ana iya haɗa su tare da SARS-CoV-2 nsp7/8/12/13 supercomplex da aka buga kwanan nan.Ragowar Coronavirus NiRAN da aka adana a cikin ƙananan tsarin lantarki.Recombinant protein (17).An yi hasashe cewa rubutun (kai) U/GMPylation zai haifar da yanayi na wucin gadi don canja wurin NMP zuwa (a halin yanzu ba a sani ba) substrate (16), da kuma tsarin kamance tsakanin NiRAN da protein kinase (17, 19) ) Shin hasashen cewa NiRAN tana gyara sauran sunadaran.
Siffofin da yawa, gami da keɓancewar tsarin tsarin sa na musamman tare da ƙwayoyin cuta na gida da kuma rabuwar kwayoyin halitta daga RdRp, suna sanya NiRAN ta zama madaidaicin maɓalli mai mahimmancin enzyme don ƙwayoyin cuta na gida, wanda ke da mahimmanci ga fitowar su da asalinsu.A baya can, ana kiran ayyuka uku masu yuwuwa waɗanda suka haɗa da NiRAN don daidaita fassarar kwayar halitta ko juzu'i ko kwafi.Idan aka yi la'akari da ƙarancin bayanai da ƙarancin da ake samu a lokacin, kowane aiki yana da fa'ida da rashin amfaninsa (16).A cikin wannan binciken, muna da niyyar haɗa nau'ikan sinadarai da jujjuya nazarin halittu na coronaviruses waɗanda ke wakiltar nau'ikan nau'ikan biyu, da sanya bincikenmu a cikin asalin juyin halitta na maye gurbi na dangin coronavirus, don samun haske cikin wannan daula mai ban mamaki.Muna ba da rahoton manyan ci gaba a cikin fahimtar NiRAN ta hanyar gano maƙasudin yanayi a cikin RTC, wanda (a cikin ra'ayoyin da ake da su guda uku) yana ba da gudummawa ga rawar wannan yanki wajen ƙaddamar da haɗakar ƙwayar cuta ta RNA.Wannan binciken kuma yana buɗe yuwuwar ga sauran ayyukan NiRAN akan hanyar haɗin yanar gizo.
Domin siffanta kaddarorin enzymatic na ƙwayar cuta ta corona virus nsp12 mai alaka NiRAN yankin, mun samar da wani recombinant nau'i na mutum coronavirus 229E (HCoV-229E) nsp12 a cikin E. coli, tare da His6 tag a C-terminus, da kuma hade da furotin tare da [α32-P] Haɗa tare da NTP a gaban MnCl2 kamar yadda aka bayyana a cikin Materials da Hanyoyi.Binciken samfurin amsawa ya nuna kasancewar sunadarin furotin mai alamar rediyo tare da nsp12 (106 kDa), yana nuna cewa coronavirus nsp12 yana haifar da samuwar furotin-NMP adducts, wanda aka fi so tare da uridine monophosphate (UMP) (Hoto 1A) Kuma B).Ƙididdigar ƙididdiga ya nuna cewa idan aka kwatanta da sauran nucleotides, ƙarfin siginar haɗin UMP ya karu da sau 2 zuwa 3 (Hoto 1C).Wannan bayanan ya yi daidai da aikin NMP transferase da aka annabta na yankin NiRAN na coronavirus (16), amma yana nuna cewa zaɓin nucleotide na yankin NiRAN na coronavirus da ƙwayar cuta ta arterial sun bambanta.
Ayyukan NMPylation na kai na HCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) an haɗa shi tare da keɓaɓɓen [α-32P] NTP a gaban 6 mM MnCl2 na mintuna 30 (duba Kayan aiki da Hanyoyi don cikakkun bayanai).SDS-PAGE ne ya raba samfuran amsa kuma an yi musu tabo da Coomassie shuɗi mai haske.(B) Sunan furotin da aka yi wa lakabin rediyo ana gani ta hanyar hoto na phosphorous.Matsayin nsp12-His6 da alamomin ƙwayoyin ƙwayoyin furotin (a cikin kilodaltons) an nuna su a cikin A da B. (C) An ƙayyade ƙarfin siginar rediyo (ma'anar ± SEM) daga gwaje-gwaje masu zaman kansu guda uku.*P≤0.05.Ƙarfin siginar (kashi) yana da alaƙa da UTP.
Kodayake an nuna ayyukan enzyme da ke da alaƙa da NiRAN suna da mahimmanci don kwafin EAV da SARS-CoV a cikin al'adun tantanin halitta (16), takamaiman aikin NiRAN da maƙasudin maƙasudi har yanzu ba a tantance ba.Daidaitaccen tsarin da aka ruwaito kwanan nan tsakanin NiRAN da dangin sunadaran sunadaran sunadaran kinase-kamar folds (17, 22) ya sa mu gwada hasashen cewa NiRAN yana haɓaka NMPylation na sauran sunadaran.Mun ƙirƙira saiti na yuwuwar maƙasudai masu alaƙa, gami da sunadarai marasa tsari waɗanda HCoV-229E ORF1a ke ɓoye (nsps 5, 7, 8, 9, 10), kowanne yana ɗauke da alamar C-terminal His6 (SI appendix, Table S1) , Kuma sanya waɗannan sunadaran tare da [α32-P] uridine triphosphate ([α32-P] UTP) a gaban ko rashi na nsp12.Bovine serum albumin da MBP-LacZα furotin fusion da aka samar a cikin E. coli suna aiki azaman sarrafawa (Hoto 2A, hanyoyi 1 zuwa 7).An bincika furotin da aka yi wa lakabin rediyo ta hanyar sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) da autoradiography, kuma an gano cewa akwai siginar rediyo mai ƙarfi mai ƙarfi a cikin abin da ke ɗauke da nsp12 da nsp9.Matsayin siginar ya yi daidai da yawan kwayoyin halitta na nsp9, yana nuna nsp12-mediated UMPylation na nsp9 (Hoto 2B, waƙa 7).Ba a sami wasu sunadaran gwajin da aka sami UMPylated ba, wanda ya kai mu ga ƙarshe cewa nsp9 wani takamaiman yanki ne na nsp12.Daidai da bayanan NMPylation na kai da aka nuna a cikin Hoto 1, nsp12 yana iya canja wurin duk NMPs guda hudu zuwa nsp9, kodayake yadda ya dace ya bambanta, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Hoto).3 A da B).A ƙarƙashin yanayin da aka yi amfani da shi a cikin wannan ƙididdiga (ƙaddamar da amsawa da lokacin bayyanarwa, rage ƙaddamar da nsp12; kayan aiki da hanyoyi), ba za a iya gano NMPylation na nsp12 ba (kwatanta Hoto 2B, layin 7, da Hoto 1B), wanda ya tabbatar da tasiri (Kuma dawafi da yawa) UMP ya ƙaura daga nsp12 zuwa nsp9.Ayyukan canja wuri na UMP yana buƙatar kasancewar Mn2+ ions, kamar yadda aka nuna a cikin Hoto 3C, yayin da ƙaramin aikin canja wuri na UMP kawai aka lura a gaban Mg2+, kuma babu wani aiki a gaban sauran nau'ikan cations guda biyu da aka gwada.An samo irin wannan bayanai a cikin gwaje-gwajen NMPylation wanda ya ƙunshi cytidine triphosphate (CTP), guanosine triphosphate (GTP), da adenosine triphosphate (ATP) (SI appendix, Figure S1).
HCoV-229E nsp12-matsakaici UMPylation na nsp9.An yi amfani da jerin abubuwan gina jiki (ciki har da serum albumin, MBP-lacZα, da jerin HCoV-229E nsps da aka yiwa lakabi da C-terminal His6 wanda ORF1a ya rubuta) don kimanta ayyukan UMPylation na HCoV-229E nsp12-His6⁺-matsakaici. furotin.Sanya furotin tare da [α-32P] UTP na mintuna 10 a cikin rashi (A) ko kasancewar (B) na nsp12 kamar yadda aka bayyana a cikin kayan da hanyoyin.A saman A da B, an nuna SDS-polyacrylamide gel da aka zana tare da Coomassie Brilliant Blue, kuma a kasan A da B, ana nuna madaidaicin autoradiograms.Matsayin ma'auni na ƙwayoyin furotin (a cikin kilodaltons) an ba da shi a hagu.Matsayin nsp12-His6 (B, saman) da siginar rediyo da aka lura a yayin ƙaddamar da nsp12-His6 tare da nsp9-His6 (B, layin 7) kuma ana nuna su, wanda ke nuna cewa [α-32P] UMP zuwa nsp9-His6 (12.9 kDa), wanda ba a lura da shi don wasu sunadaran da aka gwada ba.
HCoV-229E NiRAN-matsakaici biochemical da virological halayyar nsp9 NMPylation.(A da B) Matsayin haɗin gwiwar nucleotide da aka yi amfani da shi a cikin martani.Nsp12-His6 da nsp9-His6 suna gauraye kuma an haɗa su a gaban daban-daban [α-32P] NTPs a cikin daidaitattun NMPylation assay.(A, saman) Coomassie-stained nsp9-His6 ta SDS-PAGE.(A, kasa) Autoradiograph na wannan yanki na gel.(B) Ayyukan dangi (ma'anar ± SEM) a gaban mahaɗin haɗin gwiwar nucleotide an ƙaddara daga gwaje-gwaje masu zaman kansu guda uku.*P≤0.05.(C) Matsayin ions karfe.An nuna shine daidaitaccen gwajin NMPylation a gaban [α-32P] UTP da nau'ikan ions na ƙarfe daban-daban, kowanne tare da ƙaddamar da 1 mM.A cikin C, saman, an nuna Coomassie tabo nsp9-His6, kuma a cikin C, ƙasa, ana nuna madaidaicin autoradiography.Girman furotin da aka lakafta (a cikin kilodaltons) ana nunawa zuwa hagu na A da C. (D) Tsarin mutant na HCoV-229E nsp12-His6 yana ɗauke da ƙayyadadden maye gurbin amino acid yana cikin [α-32P] UTP, kamar yadda aka bayyana. a cikin Materials da Hanyoyi.Alamar rediyon nsp9-His6 da aka samar a cikin halayen NMPylation ana gano shi ta hanyar hoton phosphorylation (D, saman).Ayyukan dangi idan aka kwatanta da nau'in furotin (wt) suna nunawa a cikin D, kuma an dauki kasa a matsayin matsakaici (± SEM) daga gwaji uku masu zaman kansu.Asterisks suna nuna maye gurbin ragowar da ba a kiyaye su ba.(E) Kwayar ƙwayar cuta a cikin al'adar al'adar p1 da aka samu sa'o'i 24 bayan kamuwa da cuta an ƙaddara ta hanyar gwajin plaque.Abubuwan maye gurbin codon a cikin yankin NiRAN na injiniyoyi na HCoV-229E ana nuna su (lambobin da suka rage sun dogara ne akan matsayinsu a pp1ab).An yi amfani da mutant na RdRp-rauni mai aiki na nsp12_DD4823/4AA azaman sarrafawa.
Domin samun zurfafa fahimtar wurin aiki na NiRAN da kuma tantance ragowar da ke da alaƙa da ayyukan nsp9-takamaiman NMP transferase, mun yi nazarin maye gurbi, inda muka maye gurbin ragowar masu ra'ayin mazan jiya a cikin NiRAN AN, BN da CN motifs ( 16) Ala (SI appendix, Figure S2).Bugu da ƙari, an kimanta tasirin Arg-to-Lys masu ra'ayin mazan jiya ko Lys-to-Arg a cikin lokuta biyu.A matsayin iko (mara kyau), ragowar da ba a kiyaye su ko žasa a cikin yankin NiRAN na coronaviruses da sauran ƙwayoyin cuta na gida ana maye gurbinsu da Ala. Ana maye gurbin K4116A (a cikin motif preAN), K4135A (AN), R4178A (BN), D4188A (motif). BN) da D4280A (CN) suna ragewa ko ma kawar da nsp9 NMPylation ta hanyar nsp12, yayin da sunadaran da ke da sauye-sauye masu ra'ayin mazan jiya (R4178K) , K4116R) suna riƙe 60% da 80% na ayyukansu, wanda ke nuna cewa shakatawa na hane-hane akan bangarorin su. sarƙoƙi suna da hankali na physicochemically (Hoto 3D).Maye gurbin sauran ragowar E4145A, D4273A, F4281A da D4283A ba shi da lahani sosai, kuma nsp9 UMPylation ana rage shi kawai.An samu irin wannan sakamakon a cikin nsp9 NMPylation halayen da suka shafi wasu NTPs (Hoto 3D da SI appendix, Figure S3), yana mai tabbatar da cewa tasirin da aka gani akan takamaiman maye gurbin amino acid sun kasance masu zaman kansu daga nau'in haɗin gwiwar nucleotide da aka yi amfani da su.Bayan haka, mun gwada tasirin tasirin waɗannan nsp12 akan kwafi na coronaviruses a cikin al'adun tantanin halitta.Don wannan, mun yi amfani da samfuran DNA (cDNA) da suka dace da injiniyoyin da suka dace waɗanda aka kulle su a cikin ƙwayar cuta ta sake haɗawa (23, 24) don rubuta sel 5-7.Rarraba zuriyar ƙwayoyin cuta da aka samar a cikin waɗannan sel sun nuna cewa yawancin maye gurbi na HCoV-229E NiRAN ba su yiwuwa (Hoto 3E).Ƙungiya na ƙwayoyin cuta marasa amfani sun haɗa da hanyoyin da aka nuna don kawar da ko rage yawan ayyukan NMP a cikin vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), amma akwai wasu hanyoyi guda biyu (K4116R, E48045A) . % tanadi?Ayyukan su na in vitro NMPylation yana ba da shawarar cewa ƙarin hani yana cikin ciki.Hakazalika, wasu maye gurbi guda biyu (R4178K, F4281A) waɗanda suka haifar da raguwar matsakaici a cikin ayyukan NiRAN na in vitro NMPylation sun haifar da ƙwayoyin cuta masu rai, duk da haka, waɗannan ƙwayoyin cuta sun rage mahimmancin titers ta hanyar maimaitawa.Daidai da bayanan ayyukan in vitro da aka nuna a cikin Hoto na 3D, maye gurbin sauran ragowar guda huɗu waɗanda ba a adana su a cikin coronavirus da/ko wasu ƙwayoyin cuta (K4113A, D4180A, D4197A, D4273A) (8, 16) sun haifar da ƙwayoyin cuta masu amfani. matsakaicin raguwar titer idan aka kwatanta da kwayar cutar daji (Hoto 3E).
Don yin nazarin ko aikin NMP mai tsaka-tsaki na NiRAN ya dogara da yankin RdRp mai aiki, ragowar Asp guda biyu da aka adana a cikin daidaitawar ion ƙarfe na divalent (11) a cikin RdRp motif C an maye gurbinsu da Ala. Sakamakon furotin nsp12_DD4823/4AA yana riƙe. Ayyukan nsp9 NMPylation na nsp9, yana nuna cewa nsp12-matsakaici a cikin vitro nsp9 Ayyukan NMPylation ba ya buƙatar aikin polymerase (SI Appendix, Figure S4).
Bayan kafa aikin nsp9-takamaiman NMP transferase na nsp12, mun yi ƙoƙarin siffanta NMP-nsp9 adduct ta hanyar siffantawa (MS).Cikakken nau'in nau'in furotin na recombinant HCoV-229E nsp9 ya nuna kololuwa a 12,045 Da (Hoto 4A).Ƙarin nsp12 bai canza ingancin nsp9 ba, yana nuna cewa nsp12 da nsp9 ba za su samar da tsayayyen hadaddun ba a ƙarƙashin yanayin da ake amfani da su (denaturation) (Figure 4A).A gaban UTP da GTP, yawan ma'auni na amsawar da ke dauke da nsp9 da nsp12 bi da bi ya nuna cewa yawan furotin na UTP ya motsa 306 Da, kuma yawan furotin na GTP ya motsa 345 Da, yana nuna cewa kowane nsp9 kwayoyin yana ɗaure UMP ko GMP. (Hoto na 4) C da D).Ana hasashen cewa makamashin da ake buƙata don tsaka-tsaki na NSP9 NMPylation na NiRAN ya fito ne daga NTP hydrolysis da sakin pyrophosphate.Ko da yake an yi amfani da yawan molar molar sau 10 na nsp9 (manufa) fiye da nsp12 (enzyme) a cikin wannan yanayin, kusan an lura da NMPylation na nsp9, wanda ke nuna cewa hulɗar da ke tsakanin nsp12 da nsp9 ba ta da tsawo, kuma nsp12 na iya NMPylate ƙarin nsp9. in vitro kwayoyin halitta.
NMPylation guda ɗaya na nsp9 a gaban nsp12 da UTP ko GTP.An nuna shi shine ƙayyadadden nau'in nau'in nau'in furotin na HCoV-229E nsp9 (SI appendix, Table S1) (AD).(A) nsp9 kadai, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 a gaban UTP, (D) nsp9 + nsp12-His6 a gaban GTP.
Don tantance ragowar nsp9 UMPylated ta nsp12, nsp9-UMP an matse shi da trypsin.Sakamakon peptides an raba su ta hanyar nano-high performance liquid chromatography (HPLC) kuma an bincika ta hanyar tandem mass spectrometry (MS/MS) akan layi.Binciken bayanai ta amfani da fakitin software na Byonic (Protein Metrics) ya nuna UMPylation na amino acid N-terminal.An tabbatar da wannan da hannu.Matsakaicin adadin tandem na peptide precursor [UMP] NNEIMPGK (SI appendix, Figure S5A) ya bayyana guntu a 421 m/z, yana nuna cewa UMP yana ɗaure zuwa ragowar 1 na nsp9.
A N-terminus na nsp9, An adana Asn a tsakanin membobin Orthocoronavirinae (SI appendix, Figure S6).Kodayake mun yi imanin cewa N-terminal primary amine nitrogen shine mafi yuwuwar mai karɓar UMP, mun yanke shawarar samun ƙarin shaidar daurin NMP a N-terminal.Saboda wannan dalili, wanda ba NMPylated da NMPylated N-terminal peptide nsp9 tsarkakewa ta HPLC an samu a gaban acetone da sodium cyanoborohydride.A ƙarƙashin waɗannan sharuɗɗan, amines na farko na kyauta ne kawai za a iya canza su tare da propyl (25).N-terminal nsp9-derived peptide tare da jerin NNEIMPGK ya ƙunshi amines na farko guda biyu, ɗaya a N-terminus na Asn da ɗayan a sashin gefen Lys a C-terminus.Don haka, ana iya gabatar da ƙungiyoyin propyl a ƙarshen duka.Ana nuna chromatogram ion da aka fitar na peptides marasa NMPylated a cikin SI, Hoto S5B.Kamar yadda aka zata, N-terminal da C-terminal (mono) propylated (SI appendix, Figure S5B, babba layi) da kuma dipropylated peptides (SI appendix, Figure S5B, ƙananan layi).Wannan tsarin yana canzawa tare da amfani da NMPylated N-terminal peptide na nsp9.A wannan yanayin, kawai C-terminal propylated peptides za a iya gano, amma N-terminal propylated peptides da dipropylated peptides ba a gano (SI Appendix, Figure S5C), yana nuna cewa an canza UMP zuwa N-terminal primary amine Don hana wannan. kungiyar daga yin canje-canje.
Na gaba, muna maye gurbin (tare da Ala ko Ser) ko share ragowar da aka adana a N-terminus na nsp9 don ayyana ƙayyadaddun ƙuntatawa.Dangane da bayanan MS ɗinmu da ke nuna cewa NiRAN ta ƙirƙira wani nsp9-NMP adduct tare da amine na farko na ragowar N-terminal na nsp9, mun ɗauka cewa nsp9 NMPylation yana buƙatar protease mai hoto na hoto (Mpro, nsp5) don sakin nsp9 N-terminal daga precursor ta polyprotein.Don gwada wannan hasashe, mun samar da furotin na precursor nsp7-11 dauke da nsp9 a cikin E. coli kuma mun yi gwajin gwajin NMPylation a gaban [α-32P] UTP (kayan aiki da hanyoyin).Kamar yadda aka nuna a cikin Hoto 5A (layi na 3), ba a yiwa maƙasudin nsp7-11 wanda ba a yanke ba tare da nsp12.Sabanin haka, idan nsp7-11 ya fashe ta hanyar recombinant nsp5 don saki nsp9 (da sauran nsps) daga mafarin, an gano furotin mai lakabin rediyo wanda ke ƙaura tare da nsp9, yana tabbatar da ƙaddamarwarmu cewa NiRAN da N- Zaɓin samuwar nsp9-NMP na haɗin gwiwa. .Aminin farko na ƙarshe na N-terminal Asn (matsayi 3825 a cikin pp1a/pp1ab).Hakanan ana samun goyan bayan wannan ƙarshe ta gwaji ta amfani da ginin nsp9, wanda ya ƙunshi ƙarin rago ɗaya ko biyu a N-terminus.A cikin duka biyun, NiRAN-matsakaici UMPylation na nsp9 an soke (SI Appendix, Figure S7).Bayan haka, mun samar da furotin tare da ragowar Asn ɗaya ko biyu da aka goge daga jerin 3825-NNEIMPK-3832 peptide a N-terminal na nsp9.A cikin lokuta biyu, nsp9 UMPylation an katange gaba ɗaya (Hoto 5B), yana ba da ƙarin shaida cewa ainihin nsp9 N-terminus yana aiki azaman mai karɓar NMP.
Ayyukan proteolytic na nsp9 da kuma rawar N-terminal ragowar a cikin nsp12-matsakaicin UMPylation.(A) nsp9 UMPylation yana buƙatar nsp9 N-terminal kyauta.Nsp7-11-His6 an riga an haɗa shi a 30 ° C a cikin buffer ganowar NMPylation mai ƙunshe da UTP a gaban ko babu recombinant Mpro (nsp5-His6).Bayan awanni 3, fara gwajin NMPylation ta ƙara nsp12-His6 kamar yadda aka bayyana a cikin Materials da Hanyoyi.An yi amfani da abin da ya ƙunshi nsp5-His6 (layin 1) da nsp9-His6 (layin 2) azaman sarrafawa.Bayan mintuna 10, an dakatar da martanin kuma an raba cakudawar amsa ta SDS-PAGE.An lalata furotin da Coomassie Brilliant Blue (A, saman).Nsp7-11-His6 precursor da samfurin da aka sarrafa sakamakon nsp5-His6 tsaka-tsakin tsagewa ana nuna su a hannun dama.Da fatan za a lura (saboda ƙananan girman su) cewa nsp7 da nsp11-His6 ba a iya gano su a cikin wannan gel ɗin, kuma ana ƙara amsawa tare da nsp5-His6 (hanyoyi 1 da 4; Matsayin nsp5-His6 yana nunawa ta hanyar da'irar m). ko nsp9-His6 (Lane 2) yana ƙunshe da ƙaramin adadin MBP (wanda aka nuna ta buɗaɗɗen da'irori) azaman gurɓataccen gurɓataccen abu saboda an bayyana su azaman sunadaran haɗin MBP (SI appendix, Table S1).(B) Bambancin Nsp9-His6 ba shi da ragowar N-terminal Asn guda ɗaya ko biyu (lambobin saura bisa ga matsayi a cikin pp1a / pp1ab) kuma an tsarkake shi kuma an haɗa shi tare da nsp12-His6 da [α-32P] UTP.B, SDS-PAGE mai tabo tare da Coomassie ana nuna shi a saman, B, ana nuna madaidaicin autoradiograph a ƙasa.Matsayin alamar nauyin kwayoyin halitta (a cikin kilodaltons) ana nunawa a hagu.(C) HCoV-229E nsp9-His6 N-terminal kiyaye ragowar ragowar an maye gurbinsu da Ala ko Ser, kuma an yi amfani da adadin furotin iri ɗaya a cikin nsp12-His6 tsaka-tsakin UMPylation.An raba samfuran amsawa ta SDS-PAGE kuma an lalata su tare da Coomassie Brilliant Blue (C, saman), kuma an gano nsp9-His6 mai radiyo ta hanyar hoton phosphorescence (C, tsakiya).Yin amfani da furotin na daji (wt) a matsayin tunani (saitin zuwa 100%), aikin NMPylation dangi (ma'anar ± SEM) an ƙididdige shi daga gwaje-gwaje masu zaman kansu guda uku.(D) Titers ƙwayoyin cuta a cikin al'adun cell p1 mafi girma na ƙwayoyin Huh-7 da suka kamu da nau'in HCoV-229E nau'in daji na Huh-7, da kuma mutants ɗauke da abubuwan maye gurbin amino acid a cikin nsp9 an ƙaddara ta hanyar tantancewar plaque.RdRp motif C sau biyu DD4823/4AA an yi amfani da shi azaman iko mara kyau.
N-terminus na nsp9 (musamman matsayi 1, 2, 3, da 6) ana kiyaye su sosai a tsakanin membobin ƙungiyar Orthocoronavirinae (SI appendix, Figure S6).Domin yin nazarin yuwuwar rawar waɗannan ragowar a cikin nsp12-mediated nsp9 NMPylation, ragowar Asn guda biyu a jere a N-terminus na nsp9 an maye gurbinsu da Ala ko Ser (kaɗai ko a hade).Idan aka kwatanta da nau'in daji na nsp9, maye gurbin N3825 tare da Ala ko Ser ya haifar da raguwa fiye da sau biyu a cikin nsp12-mediated UMPylation (Figure 5C).Daidai da ƙaddamarwarmu cewa NMPylation yana faruwa a N-terminal primary amine maimakon sashin gefe na ragowar N-terminal, mun lura da ragowar NMPylation tare da maye gurbin N3825A da N3825S.Abin sha'awa, idan aka maye gurbin Asn na biyu da Ala ko Ser, nsp9 UMPylation yana raguwa da ƙarfi (fiye da sau 10), yayin da maye gurbin Ala a matsayi na 3, 4, da 6 yana da matsakaicin matsakaici kawai akan nsp9 UMPylation (Hoto 2). ) .5C ku).An samu irin wannan sakamakon ta amfani da ATP, CTP ko GTP (SI appendix, Figure S8).Gabaɗaya, waɗannan bayanan suna nuna muhimmiyar rawar N2826 (matsayi na 2 a cikin nsp9) a cikin nsp9 NMPylation.
Domin samun ƙarin shaida na alaƙar aiki tsakanin N-terminus na nsp9 da NMPylation, mun yi jeri da yawa (MSA) na jerin nsp9 na dangin Coronavirus (wanda ya bambanta tsakanin ragowar 104 da 113) (SI Appendix, Figure) S6).Gabaɗaya, a cikin nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan 47 (wanda aka sani da sanyawa) na 5 na dangin Orthocoronavirinae waɗanda ke cutar da dabbobi masu shayarwa, tsuntsaye, da runduna masu rarrafe daban-daban, ragowar 8 ne kawai aka gano ba su da bambanci.Mafi yawan sauye-sauyen canje-canje, gami da gogewa da shigarwa, an lura da su a cikin hawan keke tsakanin abubuwan tsarin na biyu na nsp9, kamar yadda binciken tsarin da ya gabata ya ƙaddara (26 ??28).An sami ragowa guda biyar masu bambanta a cikin β strand da α helix na ɓangaren C-terminal na nsp9.Ragowar maɓalli guda uku sun haɗa da NNE motif na N tasha na nsp9.An bayyana cewa Asn na biyu na wannan motif shine kawai ragowar da ba za a iya canzawa ba, wanda kuma aka raba shi ta hanyar hasashen nsp9 na coronavirus mai alaƙa da nesa, kuma yana wakiltar nau'in Microhyla letovirus 1 a cikin dangin Letovirinae na Alphaletovirus.Kiyaye ragowar abubuwan da ke cikin nsp9 na biyu za a iya daidaita su ta hanyar la'akari da tsarin don kiyaye nadawa ko sanannun kaddarorin RNA.Duk da haka, wannan dalili ba ze shafi kiyayewa na NNE ba, kuma kafin wannan binciken, yanayin matsalolin da ke ƙayyade bambancin tsarin tripeptide ya kasance gaba daya.
Domin sanin mahimmancin nsp9-NMPylation da NNE kiyayewa a cikin kwafin coronavirus, mun samar da mutants na HCoV-229E, waɗanda ke ɗaukar maye guda ɗaya ko biyu na ragowar nsp9 N-terminal, yana nuna cewa nsp9 NMPylation yana da illa a cikin vitro.Kafin mu fara, muna ƙoƙari mu amsa tambayar ko waɗannan maye gurbin (kusa da nsp8|9 cleavage site) sun shafi aikin sarrafa furotin na yankin C-terminal pp1a.Saitin ginin polyprotein nsp7-11 wanda ya ƙunshi sauye-sauye masu dacewa a N-terminus na nsp9 an samar da su a cikin E. coli kuma an yanke tare da recombinant Mpro.Ragewar proteolytic na rukunin yanar gizo guda huɗu (ciki har da shafin nsp9 flanking) baya tasiri sosai ga duk wani maye gurbin da aka gabatar (SI appendix, Figure S9), ban da canje-canjen tsarin a cikin waɗannan sunadaran da ke tsoma baki tare da Mpro-mediated nsp8 | 9 cleavage (Ko wani) gidan yanar gizo.
Kwayoyin Huh-7 an canza su tare da tsawon HCoV-229E RNA, suna sanya Ala ko Ser maye gurbi a cikin NNE tripeptides da aka adana (N3825, N3826, da E3827) a tashar nsp9 N, yana nuna cewa yawancin maye gurbi suna da mutuwa.Mun sami damar ceton kwayar cutar ta hanyar maye gurbin Ser ko Ala na N-terminal Asn (N2835A ko N2835S), amma mun kasa dawo da kwayar cutar tare da wasu maye gurbi guda daya da sau biyu a cikin jerin NNE (N3826A, N3826S, NN3825/6AA, NN3825/6SS), E3827A) (Hoto 5D).
Waɗannan sakamakon suna nuna cewa an taƙaita maimaitawar coronaviruses a cikin al'adun nama (daya ko makamancin haka), iyakance maye gurbi na nsp9 NMPylation shafukan a cikin jiki, da goyan bayan babbar rawar wannan martani a cikin tsarin rayuwar coronaviruses.
A cikin saitin gwaji na ƙarshe, mun samar da C-terminal His6 mai lakabin SARS-CoV-2 nsp12 da nsp9, da nau'ikan mutant guda biyu na nsp12 a cikin E. coli.Ragowar rukunin yanar gizon da ke cikin NiRAN da RdRp an yi amfani da Ala maimakon (Hoto 6A da SI kari, Tebura S2).K4465 a cikin SARS-CoV-2 nsp12 yayi daidai da K4135 a cikin HCoV-229E (SI Appendix, Figure S2), wanda ya tabbatar da buƙatar aikin NiRAN da kwafi na HCoV-229E (Hoto 3D da E).Wannan ragowar kuma yayi daidai da ragowar ƙwayar cuta ta arterial EAV nsp9 K94, wanda a baya aka nuna ya zama dole don NiRAN kai UMPylation/GMPylation (16).Kamar yadda aka nuna a cikin Hoto 6B, SARS-CoV-2 nsp12 yana da ayyukan canja wuri na UMP ta amfani da nsp9 a matsayin ma'auni, yayin da nsp12_K4465A mutant site ba aiki.Sauya sau biyu a cikin jerin halayen SDD na RdRp motif C baya shafar ayyukan canja wuri na UMP (Hoto 6B), yana nuna cewa aikin RdRp ba shi da tasiri kai tsaye a cikin nsp9 UMPylation.An samo irin wannan bayanai ta amfani da CTP, GTP da ATP (SI appendix, Figure S10).A taƙaice, waɗannan bayanan suna nuna cewa NSP9 NMPylation mai tsaka-tsaki na NiRAN yana da ayyukan ra'ayin mazan jiya a cikin coronaviruses da ke wakiltar nau'i daban-daban na dangin orthocoronavirus.
SARS-CoV-2 nsp12-matsakaici NMPylation na nsp9.(A) Coomassie tabon SDS-polyacrylamide gel yana nuna furotin mai sake haɗawa da aka yi amfani da shi a gwajin NMPylation.A matsayin sarrafawa, an yi amfani da furotin mutant tare da sauya wurin aiki a cikin yankin NiRAN (K4465A) da yankin RdRp (DD5152/3AA) na SARS-CoV-2 nsp12.Lambobin ragowar sun dogara ne akan matsayi a pp1ab.(B) Autoradiograph na gano UMPylation ta amfani da nsp9-His6 da [α-32P] UTP a matsayin substrate na nsp12-His6 (nau'in daji [wt] da mutant).Ana nuna yawan adadin kwayoyin halitta (a cikin kilodaltons) na furotin da aka yiwa lakabin a hagu.
Yankin NiRAN gabaɗaya ana kiyaye su a cikin Nidovirales (16), yana nuna cewa suna haɓaka halayen enzymatic masu mahimmanci don maimaita Nidovirus.A cikin wannan binciken, mun sami damar tabbatar da cewa yankin NiRAN na coronavirus yana canja wurin NMP (wanda aka kirkira daga NTP) zuwa nsp9, wani nau'in furotin mai ɗaure RNA mai ban mamaki wanda ke da hannu cikin kwafin ƙwayar cuta (26 ?? 29), don ƙayyade shi azaman manufa ta halitta kuma abokin tarayya na coronavirus RTC.
Yankin NiRAN yana raba abubuwa masu jeri guda uku (AN, BN, da CN), waɗanda ke ƙunshe da ƙaramin adadin ragowar da aka adana a cikin duk iyalai a cikin tsari na monophyletic amma bambance-bambancen Nidovirales tsari (8, 16).Binciken da aka yi kwanan nan ya nuna cewa suna da alaƙa da tsarin da ba a san su ba na furotin kinase-kamar sunadaran gina jiki, wanda aka kira asalin SelO iyali (17, 19, 22, 30, 31).Sunadaran da ke da alaƙa da SelO suna da folds kinase, amma ba su da ragowar wuraren aiki da yawa da aka adana a cikin kinases na gargajiya (22, 32).Dangane da jujjuyawar kwayoyin ATP da ke daure zuwa wurin aiki kuma an daidaita su ta takamaiman ma'amala, an yi hasashen SelO kuma daga baya an tabbatar da canja wurin AMP (maimakon phosphate) zuwa ma'aunin furotin (22), yayin da wani furotin mai kama da SelO na kwayan cuta YdiU yana da kwanan nan an nuna don haɓaka haɗin haɗin UMP zuwa Tyr da ragowar abubuwan gina jiki daban-daban (33).
Don tabbatarwa da faɗaɗa tsinkayar ragowar wuraren da ke aiki na yankin coronavirus NiRAN, mun yi amfani da sinadarai da kuma jujjuya hanyoyin kwayoyin halitta don yin nazarin maye gurbi akan coronavirus nsp12 (Hoto 3D da E da SI appendix, Hoto S3 da tebur) S1â S4).Bayanan sun nuna cewa maye gurbin HCoV-229E K4135, R4178 da D4280 tare da Ala yana kawar da aikin NMP transferase a cikin vitro da kwayar cutar kwafi a cikin al'adun tantanin halitta (Hoto 3D da E da SI appendices, Figure S3), yana goyan bayan kasancewar su a cikin NTP γ-phosphate. (K4135, R4178) da daidaitawar ions ƙarfe masu aiki (D4280).Canjin E4145A na Glu da aka adana a cikin kewayon ƙwayar tsuntsun tsuntsun da aka annabta don daidaita matsayin K4135 (17) an nuna don kawar da kwafin kwayar cuta, amma abin mamaki, an ci gaba da aikin a cikin in vitro NMPylation assay (Hoto 3D da E da SI appendix, Hoto S3 Da Tables S1-S4).An yi irin wannan lura lokacin da aka gabatar da canjin daidai a cikin YdiU homolog na Salmonella typhimurium (E130A) (33).Haɗe tare, waɗannan bayanan suna goyan bayan aikin ka'ida na wannan ragowar da aka adana maimakon aikin catalytic.
Sauya ragowar Phe da aka adana (F4281A) a cikin kewayon nestovirus a cikin HCoV-229E NiRAN yankin (8) ya haifar da raguwa a cikin ayyukan NMPylation a cikin vitro da raguwa mai yawa a cikin kwafin ƙwayoyin cuta a cikin al'adun tantanin halitta (Hoto 3D, E da SI) shafi, Hoto S3).Bayanan sun yi daidai da muhimmin aikin tsari na wannan saura, kamar DFG motif ɗin Phe ragowar da aka nuna a baya.A cikin kinases na furotin na gargajiya, yana cikin ɓangaren madauki na Mg2+ kuma yana taimakawa wajen tarawa da daidaita kashin baya???Da ake buƙata don ingantaccen aikin catalytic (32, 34).Sauya Ala da Arg don ragowar K4116 (a cikin motif na preAN), bi da bi, kawar da kwafin kwayar cuta kuma, kamar yadda aka zata, yana da tasiri daban-daban akan ayyukan NMP transferase a cikin vitro, dangane da sarkar gefen amino acid da aka gabatar (Hoto 3D Da E da SI appendices. , Hoto S3).Bayanan aiki sun yi daidai da bayanin tsarin, yana nuna cewa wannan ragowar ya kafa hulɗa tare da ATP phosphate (17).A cikin yankin NiRAN na sauran dangin ƙwayar cuta, matsayin HCoV-229E pp1a/pp1ab K4116 yana mamaye Lys, Arg ko nasa (8), yana nuna cewa an sassauta ƙuntatawar aikin wannan takamaiman ragowar.Sauya D4188A da D4283A yana kawar da ko da ƙarfi yana rage ayyukan enzyme kuma yana kawar da kwafin ƙwayoyin cuta (Hoto 3).Waɗannan ragowar biyun ana adana su a yawancin (amma ba duka ba) ƙwayoyin cuta na gida (8), suna nuna wani muhimmin aiki na musamman na dangi amma mai yuwuwa mara kuzari.Sauyin Ala na sauran ragowar Lys da Asp (K4113A, D4180A, D4197A da D4273A) waɗanda ba a kiyaye su a cikin Coronaviridae ko wasu dangin Nestioviridae (8) an yi amfani da su azaman sarrafawa.Kamar yadda aka zata, waɗannan sauye-sauyen suna da jurewa, tare da raguwa kaɗan a cikin ayyukan enzyme da kwafi a wasu lokuta (Hoto 3 da SI appendix, Figure S3).Gabaɗaya, bayanan mutagenesis na coronavirus sun yi daidai da GMP na kai da juyar da bayanan gado na EAV NiRAN-RdRp (16), wanda EAV nsp9 (coronavirus nsp12 ortholog) ragowar K94 (daidai da HCoV-229E K4135) ayyuka masu mahimmanci). R124 (daidai da R4178), D132 (daidai da D4188), D165 (daidai da D4280), F166 (daidai da F4281).Bugu da kari, bayanan mutagenesis na HCoV-229E sun yi daidai da kuma fadada daga bayanan da aka bayar na baya-bayan nan na SARS-CoV (16), kamar dai yadda aka yi kama da wadanda aka lura da madaidaicin CN motif Phe-to-Ala mutant SARS-CoV_nsp12 The phenotype da aka bayyana -F219A da HCoV-229E_F4281A (Hoto 3 D da E da SI appendix, Hoto S3 da Table S1-S4).
Idan aka kwatanta da EAV orthologs (16), waɗanda ke da fifikon fifiko ga UTP da GTP (a cikin amsawar NMPylation), bincikenmu ya nuna cewa yankin coronavirus NiRAN (wanda HCoV-229E da SARS-CoV-2 ke wakilta) na iya zama mai inganci. canja wurin Duk NMPs guda huɗu, kodayake akwai ɗan zaɓi don UMP (Hoto na 1 da 3).Ingantacciyar ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun kayan haɗin gwiwar NTP ya yi daidai da rahoton kwanan nan SARS-CoV-2 nsp7/8/12/13 babban tsari, wanda ADP-Mg2+ ya ɗaure zuwa wurin aiki na NiRAN, amma ba tare da sashin adenine ba. na samuwar takamaiman mu’amala (17).A cikin bincikenmu, nau'in nucleotide da aka yi amfani da shi a cikin amsawar NMPylation ba shi da wani tasiri mai ban sha'awa akan ayyukan gina jiki na mutant (SI Appendix, Figure S3), yana nuna cewa babu ɗayan waɗannan ragowar da ke da alaƙa da haɗin kai na musamman nucleobase.Tushen tsarin da yuwuwar mahimmancin ilimin halitta na zaɓin zaɓin haɗin gwiwa na NTP daban-daban da aka lura a cikin sassan NiRAN na coronaviruses da ƙwayoyin cuta na jijiyoyin jijiya ya kasance don yin nazari;suna iya zama gaskiya ko kuma saboda gazawar karatunsu.A halin yanzu, ba za a iya yanke hukuncin cewa yuwuwar aikin NMPylator na ƙwayar cuta ta NiRAN yankin (idan aka kwatanta da aikin NMPylation na baya) yana da fifikon haɗin gwiwa na daban, la'akari da cewa kamanceceniya tsakanin jijiya da coronavirus. Yankin NiRAN yana kan iyakar sa.Kwatanta na tushen jeri (16).Idan aka kwatanta da pseudokinase SelO, wanda ke amfani da Mg2+ a matsayin mai haɗin gwiwa, ayyukan coronavirus da ƙwayoyin jijiya NiRAN sun dogara da Mn2+ (16) (Hoto 3C da SI appendix, Hoto S1).Dogaro da Mn2+ da kuma fifikon fifiko ga UTP wani sabon abu ne na furotin NMPylators, kuma kwanan nan an tabbatar da shi a cikin furotin YdiU na Salmonella typhimurium, wanda ke haifar da tsayayyen furotin mai dogaro da Mn2 + mai dogaro da UMPylation don kare sel daga shigar da damuwa Cell ATP pool ( 33).
Tsarin kamanni na kwanan nan da aka kwatanta tsakanin yankin NiRAN na coronavirus da kinases sunadaran salula (17, 19) yana ba da ƙarin tallafi ga ikon NiRAN na haɗin gwiwa tare da NMP zuwa sauran sunadaran da muka ba da rahoto a cikin wannan binciken.Mun mayar da hankali kan bincikenmu don yuwuwar maƙasudin NiRAN akan sunadaran da HCoV-229E ORF1a ke ɓoye, waɗanda aka san su kai tsaye ko a kaikaice suna taimakawa RTC's ORF1b-encoded replicate (12, 35).Gwaje-gwajenmu suna ba da tabbataccen shaida don tasiri da takamaiman NMPylation na nsp9 (Hoto 2).Idan ana amfani da furotin da aka yi niyya a cikin abin da ya wuce sau 8 zuwa 10 fiye da na enzyme (nsp12), an tabbatar da cewa nsp9 gaba ɗaya (mono) NMPized (Hoto 4).Mun kammala cewa hulɗar tsakanin nsp12 da nsp9 ba ta daɗe ba kuma ba za ta samar da tsayayyen hadaddun tare da nsp9 (idan babu sauran sassan RTC).Wannan ƙarshe yana goyan bayan nazarin hulɗar furotin akan ƙwayar cuta ta SARS-CoV (35).Binciken MS ya gano aminin farko na ragowar N-terminal na nsp9 a matsayin wurin NMPylation (SI appendix, Figure S5).Samar da haɗin phosphoramidate da ƙungiyar amino ɗin ta N-terminal ta bambanta ayyukan NMPylation na NiRAN-matsakaici daga Pseudomonas syringae SelO-mediated AMPylation reaction, wanda ke haifar da samuwar O-linked AMP a Ser, Thr, ko Tyr ragowar Peptide adduct ( 22), da S. typhimurium YdiU suna samar da O-linked (tare da Tyr) da N-linked (tare da sa) peptide-UMP adducts.Iyakantaccen bayanin da ake samu akan dangin SelO na sunadaran suna nuna cewa membobin wannan babban dangin furotin sun bambanta sosai a cikin samuwar peptide-NMP adducts.Wannan abin lura ne mai ban sha'awa wanda ya cancanci ƙarin nazari.
Bayanan da aka samu a cikin wannan binciken ya sa mu yi tunanin cewa NMPylation na nsp9 yana buƙatar N-terminus kyauta.A cikin mahallin kwafin ƙwayar cuta, wannan za a samar da shi ta hanyar tsagewar sinadari na wurin sarrafa nsp8|nsp9 a cikin kwafin polyprotein pp1a wanda Mpro da pp1ab suka shiga.A cikin mafi yawan coronaviruses, bambanci tsakanin wannan takamaiman rukunin yanar gizon (VKLQ | NNEI a cikin HCoV-229E) da duk sauran rukunin yanar gizon Mpro coronavirus shine Asn (maimakon wani ƙaramin ragowar, kamar Ala, Ser Ko Gly) sun mamaye P1â???Wuri (36).Bayanan cleavage peptide da aka samu a cikin binciken farko ya nuna cewa ingantaccen aikin nsp8|nsp9 ya kasance ƙasa da na sauran rukunin yanar gizon, yana nuna cewa 1) wannan takamaiman rukunin yanar gizon na iya samun rawar da ya dace a cikin ingantaccen aiki na C-terminal akan lokaci. pp1a yankin, ko 2) Matsayin na musamman na nsp9 N-terminus da aka kiyaye a cikin kwafin ƙwayoyin cuta (37).Bayananmu (Hoto 5A) sun nuna cewa tsarin sake haɗawa na nsp9 yana ɗauke da ainihin jerin N-terminal NMPized da kyau ta nsp12.An cire jerin flanking N-terminal ta hanyar factor Xa (nsp9-His6; SI appendix, Table S1) ko Mpro-mediated cleavage (nsp7-11-His6; Hoto 5A da SI appendix, Table S1).Mahimmanci, wanda ba a yanke ba nsp9-dauke da precursor nsp7-11-His6 ya nuna juriya ga NMPylation na nsp12, wanda ya dace da bayanan mu, yana nuna cewa nsp9-NMP adduct an samo shi ta hanyar N-terminal primary amine (SI appendix, Figure S5) .Don samun zurfafa fahimtar ƙayyadaddun ƙayyadaddun kayan aikin NiRAN, sannan mu mai da hankali kan ragowar N-terminal na nsp9.Idan babu wasu sunadaran, suna da sassauƙa na tsari, suna hana su daga gano su a cikin nau'i na nsp9 (26 28, 38), wanda ke nuna iyakacin bambancin yanayin su Wannan ya faru ne saboda ƙayyadaddun ƙayyadaddun tsari mai mahimmanci (ba tsarin sakandare ba) aikin nsp9 N-terminal guntu.Ala canza ragowar abubuwan da aka adana a cikin wannan yanki (Figures 5C da D da SI appendix, Figure S8) sun nuna cewa N3826 yana da mahimmanci don nsp9 NMPylation a cikin vitro, yayin da N3825A da E3827A maye gurbin ya haifar da raguwa a cikin NMPylation, yayin da M3829A da P3830A ba su maye gurbin ba. .Babu shakka yana shafar nsp9 NMPylation.Kodayake maye gurbin N-terminal Asn (N3825A, N3825S) yana da matsakaicin tasiri kawai akan nsp9 NMPylation da kwafin ƙwayoyin cuta a cikin al'adar tantanin halitta (Hoto 5C da D), gogewar sauran ragowar Asn daga N-terminal 3825-NN dipeptide. An tabbatar da cewa Yana da cutarwa ga ƙwayoyin cuta, yana nuna cewa ana buƙatar ragowar Asn ɗaya kafin sauran ragowar a N-terminus, zai fi dacewa Asn, kodayake ana iya jurewa da juzu'in maye gurbin sauran ragowar (Hoto 5B, C, da D).Mun kammala cewa 3825-NN dipeptide, musamman ma kiyayewa da mahimmancin ragowar N3826 a cikin kewayon coronavirus (SI appendix, Figure S6), yana tabbatar da daidaitaccen ɗauri da daidaitawa na nsp9 N-terminus a cikin wurin aiki na NiRAN.
Sauya Ala (E3827A) don Glu da aka adana na duk dangi yana riƙe da nsp9 NMPylation a cikin vitro amma yana kashe ƙwayoyin cuta a cikin al'adar tantanin halitta (Hoto 5C da D), yana nuna ƙarin aikin wannan ragowar, alal misali, a cikin maɓalli mai mahimmanci (NMPylated ko ba a canza ba). ) nsp9 N-terminus da sauran abubuwan da ke tattare da kwayar cutar.Maye gurbin Nsp9 bai shafi tsarin proteolytic na nsp9 ko kowane nsps na kusa ba (39) (SI Appendix, Figure S9), yana nuna cewa abubuwan da ke haifar da mutuwa na yawancin maye gurbi na nsp9 da aka lura ba su haifar da dysregulation na C proteolytic process-terminal pp1a yankin ba. .
Bayanan da ke sama suna ba da shaida cewa bayan Mpro-matsakaici magani na nsp8|9 cleavage site a cikin pp1a/pp1ab, N-terminus na nsp9 na iya zama UMPylated (ko an canza shi da wani NMP).Bugu da ƙari, kyakkyawan kiyayewa na N-terminus na nsp9 (ciki har da raƙuman Asn guda ɗaya da bambance-bambance a cikin dangin coronavirus) da kuma bayanan kwayoyin halittar da aka samu a cikin wannan binciken (Figures 3E da 5D) ya sa mu kammala cewa nsp9 NMPylation da aka kwatanta. yana da alaƙa da ilimin halitta kuma yana da mahimmanci don kwafin coronavirus.Sakamakon aikin wannan gyare-gyare ya kasance da za a yi nazari, alal misali, game da abin da aka bayyana a baya (wanda ba takamaiman ba) nsp9 (nau'i wanda ba a canza shi ba) Ayyukan ɗaurin RNA (2628).N-terminal NMPylation na iya shafar hulɗar nsp9 tare da furotin ko RNA substrates ko samuwar taruka masu matakai huɗu daban-daban.An lura da waɗannan a cikin nazarin tsarin kuma an tabbatar da cewa suna da alaƙa da aikin kwafi na coronavirus, kodayake musamman idan babu A cikin yanayin wannan gyara (26-ââ29, 40).
Kodayake takamaiman takamaiman yanki na yankin NiRAN na coronavirus har yanzu yana buƙatar siffanta shi dalla dalla, bayananmu sun nuna cewa keɓancewar maƙasudin furotin na yankin NiRAN na coronavirus yana da kunkuntar sosai.Ko da yake kiyaye maɓalli na ragowar rukunin yanar gizo (8, 16) a cikin yankin NiRAN na duk iyalai na nidovirus suna goyan bayan ayyukan NMPylator da aka kiyaye waɗannan sunadaran, asalin abin da ke ɗaure ragowar aljihun wannan yanki ya kasance a siffanta shi da kiyayewa da kiyayewa. , kuma yana iya bambanta tsakanin iyalai daban-daban na dalilan Nidovirales.Hakazalika, har yanzu ba a tantance abubuwan da suka dace na sauran ƙwayoyin cuta na gida ba.Za su iya zama m orthologs na nsp9 ko wasu sunadaran, saboda jerin a waje da biyar replicate domains cewa yawanci kiyaye su a cikin gida ƙwayoyin cuta ba su da kariya (8), ciki har da genome array tsakanin Mpro da NiRAN, Daga cikinsu, nsp9 yana cikin coronavirus.
Bugu da ƙari, a halin yanzu ba za mu iya yin watsi da yiwuwar cewa yankin NiRAN yana da ƙarin (ciki har da salon salula) hari.A wannan yanayin, yana da kyau a ambaci cewa homologues na kwayan cuta a cikin wannan sunadaran sunadaran NMPylators (NMPylators) (30, 31) suna da alama suna da "masu tsarawa"?NMP yana daidaita nau'ikan sunadaran salula don daidaitawa ko kawar da ayyukansu na ƙasa, ta haka suna taka rawa a cikin matakai daban-daban na rayuwa, kamar amsa damuwa ta salula da redox homeostasis (22, 33).
A cikin wannan binciken (Figures 2 da 4 da SI Appendix, Figures S3 da S5), mun sami damar tabbatar da cewa nsp12 ya canza sashin UMP (NMP) zuwa matsayi guda (wanda aka kiyaye) a cikin nsp9, yayin da sauran sunadaran ba a canza su ba a cikin An yi amfani da shi A ƙarƙashin sharuɗɗan, ingantaccen ma'anar (maimakon sako-sako) ana samun goyan bayan ƙayyadaddun ƙayyadaddun kayan aikin.Daidai da wannan, idan aka kwatanta da N-terminal nsp9 NMPylation, nsp12's kansa NMPylation ayyukan yana da ƙasa sosai, ganowarsa yana buƙatar tsawon lokacin bayyanar autoradiography, kuma ana amfani da karuwa sau 10 a cikin nsp12 maida hankali.Bugu da ƙari, nazarin MS ɗin mu ya kasa samar da shaida don NMPylation na nsp12, wanda ke nuna cewa yankin NiRAN kai-NMPylation ne (a mafi kyau) aiki na biyu.Duk da haka, ya kamata a lura cewa wasu nazarin sun ba da shaida na farko cewa matsayin AMPylation na NMPylator na kwayan cuta na iya sarrafa ayyukan NMPylation na su akan wasu abubuwan gina jiki (22, 33).Don haka, ana buƙatar ƙarin bincike don bincika yuwuwar tasirin ayyukan ayyukan NMPylation na kai da aka ruwaito don EAV nsp9 (16) da coronavirus nsp12 (wannan binciken), gami da tasirin chaperone mai kama da nadawa na yankin C-terminal RdRp ( 16)).
A baya can, an yi la'akari da ra'ayoyin da yawa game da yiwuwar ayyuka na ƙasa na yankin nidoviral NiRAN, ciki har da RNA ligase, RNA -capped guanylate transferase da kuma furotin priming aiki (16), amma babu ɗayansu da ya dace da ayyukan da ake samuwa a ƙasa.Bayanan da aka samu a cikin matsayi masu zuwa daidai lokaci ɗaya ne ba tare da yin ƙarin zato ba.Bayanan da aka samu a cikin wannan binciken ya fi dacewa da (amma ba zai iya tabbatarwa ba) cewa yankin NiRAN yana da hannu a cikin ƙaddamar da ƙwayar RNA mai gina jiki.An yi imani da cewa aikin yankin NiRAN a cikin 5 ??²-RNA capping ko RNA ligation halayen waɗannan ba su da tasiri da Taimakon wasu bayanai.Don haka, alal misali, rukunin yanar gizon NiRAN ana ɗaukarsa ya haɗa da Asp ɗin da aka adana a matsayin babban tushe (D252 a cikin Pseudomonas syringae SelO; D4271 a cikin HCoV-229E pp1ab; D208 a cikin SARS-CoV-2 nsp12) (SI Appendix, adadi 2). ).S2) (17, 22, 33), yayin da catalysis a cikin ATP-dogara RNA ligase da RNA capping enzyme ana aiwatar da shi ta hanyar covalent enzyme (lysyl-N) - matsakaicin NMP, wanda ya ƙunshi ragowar Lys da ba Canja ba ( 41).Bugu da ƙari, ƙayyadaddun ƙayyadaddun tushen tsarin na coronavirus NiRAN don maƙasudin furotin da aka kiyaye da kuma annashuwa ƙayyadaddun ƙayyadaddun abubuwan haɗin gwiwa na NTP (ya fi son UTP) yana adawa da NiRAN-mediated capping enzyme ko RNA ligase-like ayyuka.
A bayyane yake, ana buƙatar ƙarin ƙarin aiki don tabbatarwa kuma, idan an tabbatar da hakan, ƙarin bayani kan yuwuwar rawar nsp9-UMP (nsp9-NMP) a cikin haɗin RNA da ke haifar da furotin, wanda zai haɗu da abubuwa masu ban sha'awa da yawa amma (zuwa yanzu) rahotanni da aka ruwaito a baya. .Keɓaɓɓun abubuwan lura.Misali, an ƙaddara cewa ƙarshen RNA mara kyau na coronavirus yana farawa da igiyar oligo (U) (42, 43).Wannan abin lura ya yi daidai da ra'ayin cewa haɗin RNA mara kyau ya fara ne ta hanyar ɗaure nau'in UMPylated na nsp9 zuwa wutsiya ta poly(A), wanda za'a iya inganta shi ta hanyar ɗaurin RNA Ayyukan da / ko hulɗa tare da wani furotin RTC.Za'a iya amfani da sashin UMP da nsp9 ya bayar azaman "primer" don nsp7/8/nsp12-mediated oligouridylation, ta amfani da wutsiya ta 3??²-poly(A) a cikin kwayar halittar RNA ko Wani oligo (A) mai ƙunshe da jeri. yana aiki azaman samfuri, kama da tsarin da aka kafa don furotin VPg na picornavirus (44).Idan shawarar ba ta kasance "mara al'ada ba" fa????Ƙaddamarwar (protein-induced) RNA kira mara kyau yana ba da hanyar haɗi zuwa abubuwan da aka lura, yana nuna cewa ƙwayar cuta ta RNA tana da UMP (maimakon UTP) a ƙarshensa (42), wanda aka ɗauka yana nuna cewa nucleic acid Dicer yana tsinke ƙarshen phosphorylated ta wani takamaiman endonuclease wanda ba a san shi ba.Idan an tabbatar, wannan aikin hydrolytic na nucleic acid zai iya taimakawa wajen sakin nau'in oligomeric UMPylated na nsp9 daga ƙarshen 5 ² na madaidaicin madauri.Yiwuwar rawar nsp9 a cikin furotin priming shima yayi daidai da binciken baya-bayan nan na kwayoyin halitta, wanda ya nuna cewa nsp9 (da nsp8) suna yin mu'amala mai mahimmanci kuma musamman tare da abubuwan RNA da aka kiyaye cis-aiki kusa da ƙarshen 3 na kwayar halittar coronavirus.45).A cewar wannan rahoto, waɗannan abubuwan da aka lura a baya za a iya sake gwada su tare da fadada su ta hanyar ƙarin bincike.
A taƙaice, bayananmu sun ƙayyadad da takamaiman aiki na alamar ƙwayar cuta ta ƙwayar cuta mai alaƙa da RdRp a N-terminus.A cikin coronavirus, wannan sabon aikin UMPylator/NMPylator mai shiga tsakani NiRAN da aka gano ana amfani dashi don dogara ga Mn2+ da sauran ragowar Asn kuma yana haifar da samuwar (ƙananan makamashi) phosphoramidate bond tare da N-terminal primary amine.Ta hanyar rarrabawar Mpro-mediated a nsp8|9 cleavage site, za a iya amfani da maƙasudin nsp9 don NMPylation, yana nuna haɗin aiki tsakanin protease da yankin NiRAN, wanda ya wuce zuwa RdRp.Kiyaye ragowar maɓalli a cikin nsp12 NiRAN mai aiki da maƙasudin nsp9, haɗe tare da bayanan da aka samu daga coronaviruses guda biyu ciki har da SARS-CoV-2, suna ba da shaida mai ƙarfi cewa nsp9 NMPylation fasali ne na Conservative na coronavirus shima babban mataki ne a cikin kwafin ƙwayoyin cuta.Bayanan da ake da su sun kai mu ga ƙarshe cewa takamaiman aikin NMPylated nau'in nsp9 a cikin haɗin RNA da ke haifar da furotin shine yanayin da ya dace don coronavirus da sauran ƙwayoyin cuta, kuma NiRAN na iya kaiwa wasu sunadaran da ba a tantance su ba.Daidaita kwayar cutar.hulɗar mai watsa shiri.Idan an tabbatar, shigar da furotin na furotin a cikin haɗin RNA na hoto za ta ƙara kusancin layin MPro / 3CLpro da RdRp tsakanin coronavirus da aka gano a baya da picornavirus-kamar supergroup (9), waɗanda yanzu an haɗa su cikin Pisonivirites da aka kafa kwanan nan. 46) a cikin rukuni.
Har ila yau, bayananmu sun nuna cewa ana iya amfani da mahimman ayyukan enzyme masu zaɓe da masu ra'ayin mazan jiya da aka gano a cikin wannan binciken azaman maƙasudin magungunan rigakafin cutar.Haɗin da ke yin tsangwama tare da ɗaurin (da gyare-gyare na gaba) na nsp9 N-terminus da aka kiyaye a cikin wurin aiki na NiRAN za a iya haɓaka su zuwa ingantattun magungunan rigakafin ƙwayoyin cuta, waɗanda suka dace da maganin coronaviruses na dabbobi da ɗan adam daga cututtuka daban-daban (sub) , ciki har da SARS-CoV-2 da Ciwon Gabas ta Tsakiya na numfashi Coronavirus.
Tsarin coding na furotin coronavirus da aka samar a cikin wannan binciken an haɓaka shi ta hanyar RT-PCR ta amfani da RNA keɓe daga Huh-7 mai kamuwa da HCoV-229E ko Vero E6 da ya kamu da SARS-CoV-2, kuma an saka shi ta amfani da daidaitattun hanyoyin cloning.pMAL-c2 (New England Biological Laboratory) ko pASK3-Ub-CHis6 (47) vector magana (SI Karin Bayani, Tables S1 da S2).Mutagenesis na tushen PCR ya gabatar da maye gurbin codon guda ɗaya (48).Don samar da furotin fusion na MBP, ƙwayoyin E. coli TB1 sun canza tare da tsarin pMAL-c2 da ya dace (SI appendix, Table S1).An tsarkake furotin na fusion ta amylose affinity chromatography kuma an raba shi da factor Xa.Daga baya, furotin na C-terminal His6 mai alama ya tsarkake ta Ni-immobilized metal affinity chromatography (Ni-IMAC) kamar yadda aka bayyana a baya (49).Don samar da furotin fusion na ubiquitin, ƙwayoyin E. coli TB1 sunyi amfani da pASK3-Ub-CHis6 plasmid constructed (SI Appendix, Tables S1 da S2) da pCGI plasmid DNA wanda ke ɓoye C-terminal hydrolase 1 (Ubp1).Canji (47).An tsarkake furotin na C-terminal His6 mai alamar coronavirus kamar yadda aka bayyana a baya (50).
Gwajin kai-NMPylation na HCoV-229E nsp12-His6 an yi shi kamar yadda aka bayyana a cikin EAV nsp9 (16).A takaice, nsp12-His6 (0.5 µM) ya ƙunshi 50 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) -KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 incubate buffer, µM. NTP da aka ƙayyade da 0.17 µM sun dace da [α32-P] NTP (3,000 Ci/mmol; Hartmann Analytic) a 30 °C na mintuna 30.A cikin duk sauran (misali) NMPylation na NMPylation na nsp12-mediated nsp9 NMPylation, ana daidaita yanayin halayen kamar haka: nsp12-His6 (0.05 µM) da nsp9-His6 (4 µM) a gaban 50 mM HEPES-KOH (pH 8.0) ), 5 mM DTT, 1 mM MnCl2, 25 µM ya nuna NTP, da 0.17 µM daidai [α32-P] NTP.Bayan incubating na minti 10 a 30 ° C, samfurin amsawa ya haɗu da SDS-PAGE samfurin buffer: 62.5 mM tris (hydroxymethyl) aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol da 0.005% bromophen blue.An hana furotin ta dumama a 90 ° C na mintuna 5 kuma an raba shi da 12% SDS-PAGE.An gyara gel ɗin kuma an lalata shi tare da bayani na Coomassie Brilliant Blue (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), an lalatar da shi, kuma an fallasa shi zuwa allon hoton phosphorescent na awanni 20 (don gano nsp12 daga NMPylation) ko (mafi girman) Sa'o'i 2 (don tantance nsp9 NMPylation).An yi amfani da hoton Typhoon 9200 (GE Healthcare) don duba allon kuma an yi amfani da ImageJ don nazarin ƙarfin siginar.
Don nazarin MS, 1 µM nsp12-His6 da 10 µM nsp9 (ba tare da alamar hexahistidine ba) an yi amfani da su a cikin nazarin NMPylation (SI appendix, Table S1) da kuma ƙara yawan 500 µM UTP da GTP.Dangane da tattarawarsu da ingancin furotin da ake tsammani, an yi amfani da tsarin Waters ACQUITY H-Class HPLC sanye take da ginshiƙin MassPrep (Ruwa) don lalata 1 zuwa 10 µL na hanyoyin samar da furotin a kan layi.An lalata furotin da aka lalatar a cikin tushen ion electrospray na Synapt G2Si mass spectrometer (Waters) ta hanyar madaidaicin buffer A (ruwa / 0.05% formic acid) da buffer B (acetonitrile / 0.045% formic acid), kuma zafin shafi shine. 60 ° C da ƙimar kwarara na 0.1 mL/min: elution ta ware tare da 5% A na mintuna 2, sannan layin layi zuwa 95% B a cikin mintuna 8, kuma kula da 95% B na wani mintuna 4.
Ana gano ions masu kyau tare da yawan adadin 500 zuwa 5000 m/z.Ana auna Glu-fibrinopeptide B kowane daƙiƙa 45 don gyaran ɗimbin yawa ta atomatik.Yi amfani da software na kayan aikin MassLynx tare da tsawo na MaxEnt1 don ƙaddamar da matsakaicin matsakaicin bakan bayan cire tushen tushe da santsi.
UMPylated HCoV-229E nsp9 an narkar da shi ta hanyar ƙara gyare-gyaren trypsin (Serva) da kuma sanya shi cikin dare a 37 ° C.An yi amfani da ginshiƙin jujjuyawar Chromabond C18WP (lambar sashi 730522; Macherey-Nagel) don lalata da tattara peptides.A ƙarshe, an narkar da peptide a cikin 25 µL na ruwa, wanda ya ƙunshi 5% acetonitrile da 0.1% formic acid.
MS ya bincika samfuran ta amfani da Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Tsarin nanoâ HPLC na ƙarshe (Dionex), sanye take da 50 cm na al'ada wanda aka ɗora da shi.75 μm C18 RP shafi cike da 2.4 μm Magnetic beads (Dr. Albin Maisch High Performance LC GmbH) Haɗa zuwa taro spectrometer kan layi ta hanyar Proxeon nanospray;allura 6µL na maganin narkewar trypsin cikin diamita na ciki 300 µm × ??1 cm C18 PepMap shafi na gaba-gaba (Thermo Scientific).Yin amfani da ruwa / 0.05% formic acid a matsayin mai narkewa, samfurin an kama shi ta atomatik kuma an cire shi a cikin adadin 6 µL / min.
Wadannan gradients na ruwa / 0.05% formic acid (mai narkewa A) da 80% acetonitrile / 0.045% formic acid (mai narkewa B) aka yi amfani da su cimma rabuwa da tryptic peptides a kwarara kudi na 300 nL / min: 4% B don Minti 5, sannan 30 A layin layi na gradient zuwa 45% B a cikin mintuna, da haɓaka madaidaiciya zuwa 95% ƙarfi B a cikin mintuna 5.Haɗa ginshiƙi na chromatographic zuwa nano-emitter bakin karfe (Proxeon), kuma fesa eluent kai tsaye zuwa ga zafi mai zafi na ma'aunin sigina ta amfani da yuwuwar 2,300 V. Binciken binciken tare da ƙuduri na 60,000 a cikin Orbitrap mass analyzer yana da alaƙa. tare da aƙalla bayanan MS/MS guda uku, an cire su cikin tsauri na tsawon daƙiƙa 30, ta amfani da karon tarko na linzamin linzamin kwamfuta wanda ya haifar da rarrabuwar kawuna ko haɗuwar haɗarin makamashi mafi girma hade da gano orbitrap, ƙudurin shine 7,500.
Lokacin aikawa: Agusta-03-2021