I whakatikahia e Peter Sarnow, Te Whare Wananga o Stanford School of Medicine, Te Whare Wananga o Stanford, California, i whakaaetia i te Hakihea 25, 2020 (whakaarohia i te Oketopa 25, 2020)
Ka ripoatahia e matou te pahekoheko i waenga i nga waahanga iti i roto i te tukuruatanga o nga coronaviruses-whakaahua matatini, he mea nui mo te tukurua me te tiaki kukuwhatanga.I whakaratohia e matou he taunakitanga ko te rohe NiRAN e hono ana ki te nsp12 he mahi whakawhiti nucleoside monophosphate (NMP) i roto i te whakawhiti, me te tautuhi i te nsp9 (he pūmua here RNA) hei whaainga.Ko te NiRAN e whakakorikori ana i te whakapiringa o te wahanga NMP ki te nsp9 amino terminus i roto i te tauhohenga e whakawhirinaki ana ki nga katote Mn2+ me nga toenga Asn kua tiakina.I kitea ko te mahi NiRAN me te nsp9 NMPylation he mea nui mo te tukurua coronavirus.Ko nga raraunga ka taea e tatou te hono i tenei mahi o te tohu whaariki huaketo kohanga ki nga kitenga o mua i roto i te whakapae ko te tiimata o te whakahiato RNA i roto i te karaehe o nga huaketo RNA he rite tonu te mahi me te whanaketanga.
Ko te RNA-dependent RNA polymerase (RdRps) o Nidovirales (Coronaviridae, Arterioviridae, me etahi atu hapu 12) e hono ana ki te rohe amino-terminal (N-terminal) i roto i te pūmua kore-hanganga (nsp) i tukuna mai i te polyprotein, e kiia nei ko NiRAN. Ko te 1ab kei roto i te protease matua viral (Mpro).I mua, ko te huaketo arterial NiRAN-RdRp nsp ake te mahi GMPylation / UMPylation i panuitia, a i whakaarohia kia whakaputa i te waahi mo te whakawhiti o te nucleoside monophosphate (NMP) ki te huaketo (kaore e mohiotia ana) me te / ranei nga mea biopolymerization pūtau.I konei, e whakaatu ana matou ko te coronavirus (Human Coronavirus [HCoV]-229E me te Severe Acute Respiratory Syndrome Coronavirus 2) nsp12 (NiRAN-RdRp) he Mn2+-whakawhirinaki mahi NMPylation, i ahu mai i te nsp9 na roto i te hanganga o te Mpro-mediated nsp9 I muri mai. te N-terminal flanking nsps is proteolytically released, the phosphoramidate is bonded to the primary amine (N3825) at the N-terminal of nsp9.Ko te uridine triphosphate te nucleotide pai i roto i tenei tauhohenga, engari ko te adenosine triphosphate, te guanosine triphosphate, me te cytidine triphosphate he pai ano hoki nga taapiri-rua.Ko nga rangahau irirangi ma te whakamahi i te coronavirus recombinant nsp9 me te nsp12 pūmua me nga mutants HCoV-229E i hangaia i te ira i whakatauhia nga toenga e tika ana mo te NSP9 NMPylation me te tukuruatanga huaketo i roto i te ahurea pūtau.Ko nga raraunga i whakapumau i te matapae o nga toenga pae kaha o NiRAN me te whakatau i te mahi nui o nga toenga nsp9 N3826 i roto i te nsp9 NMPylation me te tukuruatanga huaketo in vitro.Ko tenei toenga he waahanga o te raupapa tripeptide N-terminal NNE tripeptide kua tiakina, ka kitea ko ia anake te toenga o te nsp9 me ona homologs i roto i te whanau coronavirus.Ko tenei rangahau e whakarato ana i te turanga pakari mo te rangahau mahi o te mahi NMPylation o etahi atu wheori kohanga me te whakatakoto i nga whaainga ka taea mo te whanaketanga o nga raau taero antiviral.
Nidovirales positive-stranded RNA virus ka pangia e te tini o nga vertebrates me nga invertebrates (1, 2).I tenei wa kei roto i te ota nga whanau 14 (3), i akohia te whanau Coronavirus i roto i nga tau 20 kua hipa.I tera wa, e toru nga coronaviruses zoonotic i puta mai i nga kaihautu kararehe ka pa mai nga mate nui o te mate manawa ki te tangata.Tae atu ki nga mate urutomo e mau tonu ana na nga mate hopuhopu nui.Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (4ââ7).Ko nga Nidoviruses he whakahaere ira tangata noa, a ko te waahanga iti o te matatini-whakaahua-whakaahua-membrane (RTC) kua whakawaeheretia ki te 5-?²-waahanga e rua hautoru me te waahanga hanganga matua o te matūriki huaketo, me etahi taputapu. .Pūmua, kua whakawaeheretia ki te 3??² mutunga tuatoru o te genome (1).Haunga te hapu kotahi o nga wheori planarian (Monoviridae) (8), ko nga wheori kohanga katoa ka whakawaehere i nga waahanga iti RTC ki nga papa panui nui e rua (ORF) ORF1a me ORF1b, he mea whakamaori mai i te RNA genomic o.Ka whakawaeheretia e ORF1a te polyprotein (pp) 1a, ka whakawaehere tahi a ORF1a me ORF1b i te pp1ab.Na te whai waahi nui o te protease matua (Mpro) kua whakawaeheretia e ORF1a, ko te pp1a me te pp1ab ka tukatuka marumaru ki roto i nga momo momo pūmua kore-hanganga (nsps), e mohiotia ana ko 3CLpro, na te mea he homology me te 3Cpro o te picornavirus ( 9).Ko enei nsps e whakaarohia ana ka huihuia ki roto i te RTC hihiri nui, ka whakakoi i te whakahiatotanga o te genomic RNA (te tukurua) me te huinga o te RNA subgenomic (whakaahua), ka whakamahia hei whakarite i te whakaaturanga o te ORF kei raro o ORF1b (10? ? ?12).
Kei roto i te RTC matua te RNA polymerase RNA (RdRp) (13), te superfamily 1 helicase (HEL1) (14, 15) me te maha o nga enzyme tukatuka RNA, he mea whakawaehere te nuinga ki ORF1b me te whanau coronavirus Kei roto nsp12-nsp16 me nsp9-nsp12 i te whanau Arterioviridae (tirohia te tohutoro 10ââ 12).Ko te RdRp me te HEL1 e tohu ana i te rua (kotahi haurima) nga waahi tiaki o te kohanga o te manu, he homology i waenga i etahi atu wheori RNA.E whakaponohia ana ka awhinahia te tauira matua e etahi atu waahanga iti, tae atu ki etahi nsps iti i tukuna mai i te rohe carboxy-terminal (C-terminal) o pp1a, kei te awa o Mpro (coronavirus nsp5 me arterial virus nsp4, ia).He iti noa ta ratou tiaki mo te whanau me nga mahi kanorau (kua arotakehia ki te tohutoro 10ââ12).
No tata nei, i kitea he rohe me nga ahuatanga motika raupapa ahurei i te pito amino (N-terminus) e tata ana ki te RdRp i roto i nga wheori kohanga katoa, engari kaore he huaketo RNA (16).I runga i tana waahi me te whakawhitinga nucleotide (nucleoside monophosphate [NMP] transferase), ko te ingoa o tenei rohe ko NiRAN (Nestvirus RdRp-related nucleotide transferase).Ko te huinga rohe-rua o NiRAN-RdRp he nsp12 i roto i te whanau Coronaviridae me te nsp9 i te whanau Arterioviridae, me etahi atu nestoviridae, ko te NiRAN-RdRp e tika ana kia tukuna hei nsp motuhake mai i te polyprotein viral.I roto i te coronavirus, kei roto i te rohe NiRAN ??1/450 nga toenga ka hono ki te rohe C-terminal RdRp ma te rohe hono (16?19).I roto i te Equine Arteritis Virus (EAV) (Arteriviridae), ko te recombinant nsp9 e whakaatu ana i te Mn2+ katoon-tiati (whaiaro) UMPylation me nga mahi GMPylation, e whakawhirinaki ana ki nga turanga raupapa e toru kua tiakina i roto i te nestovirus, AN, BN me CN Nga toenga kei te raupapa.Ko te N e tu ana mo NiRAN) (16).Ko te taha o te pito-N o enei motif he mea iti ake te ahua preAN.Ko etahi o enei toenga ka tiakina hoki ki roto i nga kinases pūmua tata, i kitea ai he whai waahi ki te here nucleoside triphosphate (NTP) me te mahi catalytic (20, 21).E rite ana ki tenei tirohanga, he maha nga toenga o te waahi kaha i roto i te pseudokinase SelO mai i te Pseudomonas syringae ka taea te whakahiato me te SARS-CoV-2 nsp7/8/12/13 supercomplex i whakaputaina tata nei.Ko nga toenga Coronavirus NiRAN kua tiakina ki roto i te hangahanga hikohikohiko.Pūmua whakahiato (17).Kei te whakapaehia ko te U / GMPylation kua tuhia (whaiaro) ka whakaputa i te ahua whakawhiti ki te whakawhiti i te NMP ki te (16 kaore i te mohiotia i tenei wa), me te ahua o te hanganga i waenga i te NiRAN me te kinase protein (17, 19) ) Ko te whakapae e Ka whakarerekēhia e NiRAN etahi atu pūmua.
He maha nga ahuatanga, tae atu ki tana hononga nahanaha ahurei me te ahurei ki nga wheori kohanga me te wehenga ira mai i te RdRp, ka waiho a NiRAN hei whaariki ture matua tika mo nga wheori kohanga, he mea tino nui ki te puta me te tuakiri.I mua, e toru nga mahi ka taea e NiRAN ki te whakahaere i te whakamaoritanga genome/subgenomic, te tukuruatanga/whakaahua ranei.Ina whakaarohia te iti me te kore o nga raraunga e waatea ana i tera wa, kei ia mahi nga painga me nga ngoikoretanga (16).I roto i tenei rangahau, e whai ana matou ki te whakakotahi i nga rangahau ira koiora me te whakamuri o nga coronaviruses e tohu ana i nga puninga e rua, me te tuku i o maatau kitenga ki te papamuri kunenga o te whakarereketanga maori o te whanau coronavirus, kia whai matauranga ai ki tenei ao Ngaro.Ka ripoatahia e matou nga ahunga whakamua nui i roto i te maarama ki te NiRAN ma te tautuhi i nga whaainga taiao i roto i te RTC, e (i roto i nga whakapae e toru e waatea ana) ka whai waahi ki te mahi a tenei rohe ki te timata i te whakahiatotanga o te RNA huaketo kohanga.Ma tenei rangahau ano e whakatuwhera nga huarahi mo etahi atu mahi a NiRAN i runga i te atanga kaihautu huaketo.
Hei tohu i nga ahuatanga enzymatic o te rohe NiRAN e pa ana ki te huaketo corona nsp12, i hangaia e matou he momo whakahiato o te coronavirus tangata 229E (HCoV-229E) nsp12 i E. coli, me te tohu His6 i te pito C, ka whakakotahi i te pūmua ki te [α32-P ] Horohia tahi me te NTP ki te aroaro o MnCl2 e whakaahuatia ana i roto i nga Rauemi me nga Tikanga.Ko te tātaritanga o te hua tauhohenga i tohu i te aroaro o tetahi pūmua tapanga irarangi e haere tahi ana me te nsp12 (106 kDa), e tohu ana ko te coronavirus nsp12 e whakakorikori ana i te hanga o nga whakaurunga o te pūmua covalent-NMP, he mea pai ki te hanga me te uridine monophosphate (UMP) (Whakaahua 1A) B).I whakaatuhia e te tātaritanga ine i whakaritea ki etahi atu nucleotides, ko te kaha o te tohu o te kaporeihana UMP i piki ake i te 2 ki te 3 wa (Whakaahua 1C).Ko enei raraunga e rite ana ki nga mahi whakawhiti a NMP o te rohe NiRAN o te coronavirus (16), engari e tohu ana he rereke nga manakohanga nucleotide o te rohe NiRAN o te coronavirus me te huaketo arterial.
Mahi-NMPylation whaiaro o HCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) i whakakororiatia ki te [α-32P] NTP kua tohua i te aroaro o te 6 mM MnCl2 mo te 30 meneti (tirohia nga Rauemi me nga Tikanga mo nga taipitopito).Ko nga hua tauhohenga i wehea e SDS-PAGE ka pania ki te Coomassie kanapa puru.(B) Ko te pūmua tapanga irirangi ka kitea e te whakaahua phosphorous.Ko nga waahi o te nsp12-His6 me nga tohu tohu papatipu ngota pūmua (i roto i te kilodaltons) e whakaatuhia ana i roto i te A me te B. (C) Ko te kaha o te tohu radioactive (mean ± SEM) i whakatauhia mai i nga whakamatautau motuhake e toru.*P≤0.05.Ko te kaha tohu (pahekeheke) e pa ana ki te UTP.
Ahakoa kua kitea he mea tino nui nga mahi a te EAV me te SARS-CoV i roto i te ahurea pūtau (16), ko te mahi NiRAN motuhake me nga whaainga pea kaore ano kia whakatauhia.Ko te ahua o te hanganga i korerotia tata nei i waenga i te NiRAN me te whanau o nga pūmua me nga momo kinase-rite ki te poroteina (17, 22) i akiaki i a maatau ki te whakamatautau i te whakapae ko NiRAN te whakakoi i te NMPylation o etahi atu pūmua.I hangaia e matou he huinga o nga whaainga homologous pea, tae atu ki nga pūmua kore-hanganga kua whakawaeheretia e HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), kei roto ia ia he tohu C-terminal His6 (SI apiti, Ripanga S1) , Me whakakororia enei pūmua ki te [α32-P] uridine triphosphate ([α32-P]UTP) i te aroaro, i te ngaro ranei o te nsp12.Bovine serum albumin me MBP-LacZα fusion pūmua i hangaia i roto i te E. coli hei mana whakahaere (Whakaahua 2A, ara 1 ki te 7).I tātarihia te pūmua radiolabeled e te konutai dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) me te autoradiography, a ka kitea he kaha te tohu radioactive i roto i te tauhohenga kei roto te nsp12 me te nsp9.Ko te waahi o te tohu e rite ana ki te papatipu ngota o te nsp9, e tohu ana i te UMPylation o te nsp12-mediated o nsp9 (Whakaahua 2B, ara 7).Karekau i kitea he UMPylated etahi atu pūmua whakamatautau, i kii ai matou ko te nsp9 he taputapu motuhake o nsp12.E rite ana ki nga raraunga whaiaro-NMPylation e whakaatuhia ana i te Whakaahua 1, ka taea e te nsp12 te whakawhiti i nga NMP e wha ki te nsp9, ahakoa he rereke te kaha, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Pikitia).3 A me B).I raro i nga tikanga e whakamahia ana i roto i tenei whakamatautau (whakapotohia te tauhohenga me te wa whakaatu, whakaitihia te kukū o te nsp12; nga rauemi me nga tikanga), kaore i kitea te NMPylation whaiaro o te nsp12 (whakaritea te Whakaahua 2B, te huarahi 7, me te Whakaahua 1B), e i whai hua (Me nga rauna maha) I neke a UMP mai i te nsp12 ki te nsp9.Ko te mahi whakawhitinga UMP e hiahia ana kia noho mai nga katote Mn2 +, penei i te Whakaaturanga 3C, i te mea he iti noa nga mahi whakawhiti UMP i kitea i te aroaro o Mg2 +, a kaore he mahi i te aroaro o etahi atu cations divalent e rua i whakamatauria.He rite nga raraunga i whiwhi i roto i nga whakamatautau NMPylation kei roto te cytidine triphosphate (CTP), te guanosine triphosphate (GTP), me te adenosine triphosphate (ATP) (SI apiti, Whakaahua S1).
HCoV-229E nsp12-takawaenga UMPylation o nsp9.He raupapa o nga taputapu pūmua (tae atu ki te albumin serum bovine, MBP-lacZα, me te raupapa HCoV-229E nsps kua tapaina ki te C-terminal His6 kua whakawaeheretia e ORF1a) i whakamahia hei arotake i te mahi UMPylation o HCoV-229E nsp12-His6⁺-i takawaenga. pūmua.Whakanuia te pūmua ki te [α-32P] UTP mo te 10 meneti i te ngaro (A) me te noho (B) o te nsp12 i whakaahuatia i roto i nga rauemi me nga tikanga.I te tihi o A me B, ka whakaatuhia te gel SDS-polyacrylamide kua pania ki te Coomassie Brilliant Blue, kei raro o A me B, ka whakaatuhia nga autoradiograms e rite ana.Kei te taha maui te waahi o te tohu papatipu ngota ngota pūmua (i roto i te kilodaltons).Ko te waahi o te nsp12-His6 (B, runga) me te tohu radioactive i kitea i te wa o te incubation o nsp12-His6 me te nsp9-His6 (B, raina 7) e tohuhia ana, e tohu ana ko [α-32P]UMP ki nsp9-His6 ( 12.9 kDa), kaore i kitea mo etahi atu pūmua i whakamatauria.
HCoV-229E NiRAN-whakawhitinga matū koiora me te tohu virological o nsp9 NMPylation.(A me B) Te mahi a te tïpako nucleotide i whakamahia i roto i te tauhohenga.Ko te Nsp12-His6 me te nsp9-His6 ka whakauruhia, ka whakauruhia ki te aroaro o nga momo NTP [α-32P] rereke i roto i te whakamatautau NMPylation paerewa.(A, runga) Coomassie-stained nsp9-His6 i wehea e SDS-PAGE.(A, raro) Autoradiograph o te rohe kotahi o te ra.(B) Ko te mahi whanaungatanga (te tikanga ± SEM) i te aroaro o te cofactor nucleotide kua tohua ka whakatauhia mai i nga whakamatautau motuhake e toru.*P≤0.05.(C) Te mahi a nga katote whakarewa.Kei te whakaatuhia ko te whakamatautau NMPylation paerewa i te aroaro o te [α-32P] UTP me nga katote whakarewa rereke, ia me te kukū o te 1 mM.I roto i te C, te runga, Coomassie stained nsp9-His6 kua whakaatuhia, a i C, kei raro, ka whakaatuhia te autoradiography e rite ana.Ko te rahi o te pūmua kua tapaina (ki te kilodaltons) ka whakaatuhia ki te taha maui o A me C. (D) Ko te ahua mutant o HCoV-229E nsp12-His6 e kawe ana i te whakakapinga waikawa amino kua tohua kei roto i te [α-32P]UTP, pera i whakaahuatia i roto i nga Rauemi me nga Tikanga.Ko te radiolabeled nsp9-His6 i puta i te tauhohenga NMPylation ka kitea e te whakaahua phosphorylation (D, runga).Ko te ngohe whanaunga ka whakaritea ki te momo mohoao (wt) pūmua e whakaatuhia ana i D, ka tangohia te raro hei toharite (± SEM) mai i nga whakamatautau Motuhake e toru.Ko nga whetūriki e tohu ana i nga whakakapinga o nga toenga kore i tiakina.(E) Ko te titer huaketo i roto i te supernatant ahurea o nga pūtau p1 i whiwhi i te 24 haora i muri i te whakatau i te mate ma te whakamatautau tohu.Ko nga whakakapinga codon i roto i te rohe NiRAN o te mutant HCoV-229E i hangaia e tohuhia ana (kei runga i to raatau tuunga i te pp1ab).Ko te RdRp-kore-kore i te waahi kaha mutant nsp12_DD4823/4AA i whakamahia hei mana.
Kia hohonu ake ai te maaramatanga mo te waahi kaha o NiRAN me te whakatau i nga toenga e pa ana ki te mahi a te nsp9-motuhake NMP transferase, i mahia e matou he tātaritanga whakarereke, i whakakapihia e matou nga toenga tuuturu i roto i nga kaupapa NiRAN AN, BN me CN ( 16) Ko Ala (SI appendix, Figure S2).I tua atu, i arotakehia te paanga o nga whakakapinga Arg-to-Lys, Lys-to-Arg ranei i roto i nga keehi e rua.Hei mana whakahaere (kino), ka whakakapihia nga toenga karekau, iti ake ranei i te tiaki i te rohe NiRAN o nga coronaviruses me etahi atu wheori kohanga ki te Ala. Whakakapi K4116A (i roto i te motif preAN), K4135A (AN), R4178A (BN), D4188A (motif) Ko te BN) me te D4280A (CN) ka tino whakaiti, ka whakakore ranei i te nsp9 NMPylation na roto i te nsp12, i te wa e mau ana nga pūmua me nga whakakapinga tuuturu (R4178K), K4116R) 60% me te 80% o a raatau mahi, e tohu ana ko te whakangā o nga here i o raatau taha. he tairongo matū aahua nga mekameka (Whakaahua 3D).Ko te whakakapi i etahi atu toenga kua tiakina E4145A, D4273A, F4281A me D4283A he iti rawa te kino, a he iti noa te whakaheke o te nsp9 UMPylation.He rite nga hua i puta i roto i nga tauhohenga nsp9 NMPylation e pa ana ki etahi atu NTP (Whakaahua 3D me te taapiri SI, Whakaahua S3), e whakapumau ana ko nga paanga kua kitea i runga i nga whakakapinga waikawa amino motuhake he mea motuhake mai i te momo o te taapiri-a-nucleotide i whakamahia.I muri mai, i whakamatauhia e maatau te paanga o enei whakakapinga nsp12 ki te tukurua o nga coronaviruses i roto i te ahurea pūtau.I tenei mutunga, i whakamahia e matou nga tauira taapiri DNA (cDNA) i hangaia e te ira e tika ana kua katihia i roto i te huaketo kano kano (23, 24) hei tuhi 5 -7 nga pūtau.Ko te wetewetetanga o nga uri huaketo hopuhopu i puta i roto i enei pūtau i whakaatu ko te nuinga o nga mutants HCoV-229E NiRAN kaore e taea (Whakaahua 3E).Kei roto i te roopu o nga mutants viral e kore e taea e nga momo rereke kua whakaatuhia ki te whakakore, ki te whakaiti nui ranei i nga mahi whakawhiti i roto i te vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), engari e rua etahi atu rereke (K4116R, E4145A) 80 % kua rahuitia?Ko ta raatau mahi in vitro NMPylation e tohu ana he taapiri taapiri kei roto.Waihoki, e rua atu nga huringa (R4178K, F4281A) na te iti o te hekenga o te mahi NMPylation in vitro a NiRAN i hua mai i nga wheori ora, heoi, ko enei wheori i tino whakaheke i nga tohu ma te tukurua.E rite ana ki nga raraunga ngohe in vitro e whakaatuhia ana i te Whakaahua 3D, hei whakakapi i etahi atu toenga e wha kaore i te tiakina i roto i te coronavirus me/ranei etahi atu wheori kohanga (K4113A, D4180A, D4197A, D4273A) (8, 16) ka puta he wheori whaihua Ko te uri, ahakoa he he iti te taima ka whakaritea ki te huaketo momo mohoao (Whakaahua 3E).
Hei rangahau mena ka whakawhirinaki nga mahi whakawhiti NMPase a NiRAN-mediated ki te rohe RdRp kaha, ko nga toenga Asp e rua kua tiakina i uru ki te whakakotahitanga o nga katote whakarewa divalent (11) i roto i te RdRp motif C i whakakapihia e Ala. Ko tana mahi nsp9 NMPylation, e tohu ana ko te nsp12-mediated in vitro nsp9 NMPylation mahi kaore e hiahiatia he mahi polymerase (SI Appendix, Figure S4).
I muri i te whakaturanga i nga mahi whakawhiti a NMP-nsp9-motuhake mo te nsp12, ka ngana matou ki te tohu i te whakaurunga o te NMP-nsp9 ma te spectrometry papatipu (MS).Ko te tuāwhiorangi papatipu pūmua katoa o HCoV-229E nsp9 recombinant i whakaatu i te tihi i te 12,045 Da (Whakaahua 4A).Ko te taapiri o te nsp12 kaore i whakarereke i te kounga o te nsp9, e tohu ana ko te nsp12 me te nsp9 e kore e hanga he matatini pumau i raro i nga tikanga e whakamahia ana (denaturation) (Whakaahua 4A).I te aroaro o te UTP me te GTP, ko te inenga papatipu o te tauhohenga kei roto nsp9 me nsp12 i whakaatu ko te papatipu pūmua o te UTP i neke 306 Da, me te papatipu pūmua o te GTP i neke 345 Da, e tohu ana ko ia ngota nsp9 e herea ana he UMP, GMP ranei. (Whakaahua 4) C me D).E whakapaetia ana ko te kaha e hiahiatia ana mo te nsp9 NMPylation kua whakawhitia e NiRAN ka puta mai i te NTP hydrolysis me te tuku pyrophosphate.Ahakoa he 10-fold molar te taikaha o te nsp9 (target) atu i te nsp12 (enzyme) i whakamahia i roto i tenei tauhohenga, tata oti te NMPylation o nsp9 i kitea, e tohu ana he poto te tauwhitinga i waenga i te nsp12 me te nsp9, a ka taea e nsp12 te NMPylate ake nsp9 in vitro rāpoi ngota.
Ko te NMPylation kotahi o te nsp9 i te aroaro o te nsp12 me te UTP, te GTP ranei.E whakaatuhia ana ko te tuāwhiorangi papatipu pūmua kua whakakorehia o HCoV-229E nsp9 (Apitihanga SI, Ripanga S1) (AD).(A) nsp9 anake, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 i te aroaro o UTP, (D) nsp9 + nsp12-His6 i te aroaro o GTP.
Hei whakatau i nga toenga nsp9 UMPylated e nsp12, i wehea te nsp9-UMP ki te trypsin.I wehea nga peptides e te nano-high performance liquid chromatography (HPLC) ka tātarihia e te tandem mass spectrometry (MS/MS) ipurangi.Ko te rangahau raraunga ma te whakamahi i te putea rorohiko Byonic (Protein Metrics) i whakaatu i te UMPylation o te waikawa amino N-terminal.Ka whakamanahia ma te ringa.Ko te tuāwhiorangi papatipu tandem o te peptide precursor [UMP]NNEIMPGK (SI apiti, Figure S5A) i whakaatu he kongakonga i te 421 m/z, e tohu ana ka herea te UMP ki te toenga 1 o nsp9.
I te pito-N o nsp9, ka tiakina a Asn i waenga i nga mema o Orthocoronavirinae (Apitihanga SI, Whakaahua S6).Ahakoa e whakapono ana matou ko te hauota amine tuatahi N-terminal ko te tino kaikawe mo te UMP, i whakatau matou ki te tiki taunakitanga taapiri o te NMP e herea ana ki te N-terminal.Mo tenei take, ko te NMPylated me te NMPylated N-terminal peptide nsp9 kua purea e te HPLC i puta mai i te aroaro o te acetone me te konutai cyanoborohydride.I raro i enei tikanga, ko nga amine tuatahi kore utu ka taea te whakarereke me te propyl (25).Ko te N-terminal nsp9-i ahu mai i te peptide me te raupapa NNEIMPGK kei roto e rua nga amine tuatahi, ko tetahi kei te N-terminus o Asn me tetahi atu i te mekameka taha o Lys i te C-terminus.Na reira, ka taea te whakauru i nga roopu propyl ki nga pito e rua.Ko nga chromatograms katote i tangohia mai i nga peptides kore-NMPylated e whakaatuhia ana i te taapiri SI, te Whakaahua S5B.I te mea e tumanakohia ana, ka taea te tautuhi i te N-terminal me te C-terminal (mono) (te taapiri SI, te ahua S5B, te ara o runga) me nga peptides dipropylated (SI appendix, te ahua S5B, te ara o raro).Ka huri tenei tauira ma te whakamahi i te NMPylated N-terminal peptide o nsp9.I tenei keehi, ko nga peptides kua tohua te C-terminal anake ka taea te tautuhi, engari ko nga peptides propylated N-terminal me nga peptides dipropylated kaore i te tautuhia (SI Appendix, Figure S5C), e tohu ana kua whakawhitia te UMP ki te N-terminal primary amine Hei aukati i tenei. roopu mai i te whakarereketanga.
Whai muri, ka whakakapihia (me Ala, Ser ranei) ka mukua ranei nga toenga kua tiakina i te pito-N o nsp9 hei tautuhi i nga herenga-a-whaanui.I runga i o maatau raraunga MS e whakaatu ana ko NiRAN te hanga i tetahi taapiri nsp9-NMP me te amine tuatahi o te toenga N-terminal o nsp9, i whakapae matou ko te nsp9 NMPylation e hiahia ana te protease master viral (Mpro, nsp5) ki te tuku i te nsp9 N-terminal mai. tona pūmua polyprotein.Hei whakamatautau i tenei whakapae, i hangaia e matou he pūmua mua nsp7-11 kei roto i te nsp9 i roto i te E. coli ka mahia he whakamatautau NMPylation paerewa i te aroaro o te [α-32P] UTP (nga rauemi me nga tikanga).Ka rite ki te Whakaaturanga 5A (raarangi 3), kaore i tapatapahia te tohu irirangi ki te nsp12 ko te nsp7-11 kare i tapahia.He rereke, mena ka wehea te nsp7-11 e te recombinant nsp5 ki te tuku i te nsp9 (me etahi atu nsps) mai i te kaamua, ka kitea he pūmua reo irirangi e heke ana me te nsp9, e whakau ana i ta maatau whakatau ko NiRAN me te N- Ko te hanganga o te kovalent nsp9-NMP taapiri. .Ko te amine tuatahi o te N-terminal Asn (turanga 3825 i te pp1a/pp1ab).Ka tautokohia ano tenei whakatau e nga whakamatautau ma te whakamahi i te hanga nsp9, kei roto tetahi atu e rua ranei nga toenga kei te pito-N.I roto i nga take e rua, i whakakorehia te UMPylation a NiRAN-mediated o nsp9 (SI Appendix, Figure S7).I muri mai, ka whakaputahia e matou he pūmua me te kotahi, e rua ranei nga toenga Asn i mukua mai i te raupapa peptide 3825-NNEIMPK-3832 i te N-terminal o nsp9.I roto i nga take e rua, i tino aukatihia te nsp9 UMPylation (Whakaahua 5B), e whakaatu ana i etahi atu taunakitanga e mahi ana te tino nsp9 N-terminus hei kaiwhiwhi NMP.
Ko te tukatuka proteolytic o te nsp9 me te mahi o nga toenga N-terminal i roto i te nsp12-mediated UMPylation.(A) nsp9 UMPylation e hiahia ana he nsp9 N-terminal kore utu.Ko te Nsp7-11-His6 i mua i te whakauru i te 30 °C i roto i te NMPylation detection buffer kei roto UTP i te aroaro o te kore ranei o te Mpro recombinant (nsp5-His6).I muri i nga haora 3, timata te whakamatautau NMPylation ma te taapiri i te nsp12-His6 pera i te korero i roto i nga Rauemi me nga Tikanga.Ko te tauhohenga kei roto te nsp5-His6 (te huarahi 1) me te nsp9-His6 (te huarahi 2) i whakamahia hei mana whakahaere.I muri i nga meneti 10, ka mutu te tauhohenga, ka wehea te ranunga tauhohenga e te SDS-PAGE.I pokea te pūmua ki te Coomassie Brilliant Blue (A, runga).Ko te Nsp7-11-His6 tomua me te hua tukatuka i puta mai i te wehenga takawaenga nsp5-His6 ka whakaatuhia ki te taha matau.Kia mahara (na te iti o te rahi) kaore e kitea te nsp7 me te nsp11-His6 i roto i tenei reera, ka taapirihia te tauhohenga ki te nsp5-His6 (nga huarahi 1 me te 4; ko te waahi o te nsp5-His6 e tohuhia ana e te porowhita totoka) Ko te nsp9-His6 (Lane 2) kei roto he iti MBP (e tohuhia ana e nga porowhita tuwhera) hei toenga parakore na te mea kua whakahuahia hei pūmua whakakotahi MBP (te taapiri SI, Ripanga S1).(B) Ko te rereke Nsp9-His6 karekau i te kotahi, e rua ranei nga toenga N-terminal Asn (te toenga tatau i runga i te tuunga i roto i te pp1a/pp1ab) ka purea, ka whakakororia ki te nsp12-His6 me te [α-32P] UTP.B, SDS-PAGE kua pania ki a Coomassie ka whakaatuhia ki runga, B, ko te autoradiograph e whakaatuhia ana ki raro.Kei te taha maui te waahi o te tohu taumaha ngota (i nga kilodaltons).(C) HCoV-229E nsp9-His6 N-terminal toenga tiaki i whakakapi ki te Ala, Ser ranei, me te nui o te pūmua i whakamahia i roto i te tauhohenga UMPylation takawaenga nsp12-His6.Ko nga hua tauhohenga i wehea e te SDS-PAGE ka pania ki te Coomassie Brilliant Blue (C, runga), a ko te radiolabeled nsp9-His6 i kitea e te phosphorescence imaging (C, waenganui).Ma te whakamahi i te momo mohoao (wt) pūmua hei tohutoro (whakaturia ki te 100%), ko te mahi NMPylation whanaunga (te tikanga ± SEM) i tatauhia mai i nga whakamatautau motuhake e toru.(D) Ko nga tohu o te huaketo i roto i te putunga ahurea p1 o nga pūtau Huh-7 kua pangia e te HCoV-229E momo mohoao Huh-7, me nga mutants e kawe ana i nga whakakapinga waikawa amino i roto i te nsp9 i whakatauhia e te whakamatautau tohu.Ko te RdRp motif C rua mutant DD4823/4AA i whakamahia hei mana kino.
Ko te pito-N o te nsp9 (ina koa ko nga tuunga 1, 2, 3, me te 6) e tino tiakina ana i waenga i nga mema o te whanau Orthocoronavirinae (te taapiri SI, te Whakaahua S6).Hei ako i te waahi ka taea e enei toenga i roto i te nsp12-mediated nsp9 NMPylation, e rua nga toenga Asn karapīpiti i te N-terminus o te nsp9 i whakakapihia ki te Ala, ki a Ser ranei (koe, ko te whakakotahi ranei).Ka whakatauritea ki te momo mohoao nsp9, ko te whakakapi i te N3825 ki a Ala, Ser ranei i hua ake i te rua-rua whakahekenga i roto i te UMPylation-whakawaenga o nsp12 (Whakaahua 5C).E rite ana ki ta matou whakatau ka puta te NMPylation i te N-terminal primary amine hei utu mo te mekameka taha o te toenga N-terminal, i kite matou i te nui o te NMPylation toenga me te whakakapi o N3825A me N3825S.He mea whakamiharo, ki te whakakapihia te tuarua o Asn e Ala, Ser ranei, ka kaha ake te whakaheke o te nsp9 UMPylation (neke atu i te 10 nga wa), ko te whakakapi o Ala i nga waahi 3, 4, me te 6 he iti noa te paanga ki te nsp9 UMPylation (Whakaahua 2 ).5C).He rite nga hua i puta ma te whakamahi i te ATP, CTP, GTP ranei (Apitihanga SI, Whakaahua S8).Ko enei raraunga e whakaatu ana i te mahi matua o N2826 (turanga 2 i roto i te nsp9) i roto i te nsp9 NMPylation.
Kia whiwhi ai i etahi atu taunakitanga mo te hononga mahi i waenga i te N-terminus o te nsp9 me te NMPylation, i mahia e matou he raupapa raupapa maha (MSA) o te raupapa nsp9 o te whanau Coronavirus (he rereke i waenga i te 104 me te 113 toenga) (SI Appendix, Figure S6 ).Hui katoa, i roto i nga momo 47 (mohiotia me te putative) o nga puninga e 5 o te whanau-whanau Orthocoronavirinae e pangia ana nga momo whakangote, manu, me nga momo ngarara, e 8 anake nga toenga i kitea he rerekee.Ko nga huringa tino nui, tae atu ki te whakakore me te whakauru, i kitea i roto i nga huringa i waenga i nga waahanga hanganga tuarua o te nsp9, i whakatauhia e nga rangahau hanganga o mua (26 ??28).E rima nga toenga rereke i kitea i roto i te whenu β me te α helix o te wahanga C-terminal o nsp9.E toru nga toenga invariant te ahua NNE o te pito N o nsp9.Kua kitea ko te Asn tuarua o tenei kaupapa ko te toenga koretake anake, e tiritiria ana e te nsp9 whakapae o te coronavirus poroka tawhiti, me te tohu mo te momo Microhyla letovirus 1 i roto i te whanau iti Letovirinae o Alphaletovirus.Ko te tiaki i nga toenga kei roto i nga huānga hanganga tuarua o nsp9 ka taea te whakatau ma te whakaaro o te hanganga ki te pupuri i te kopa, i nga taonga here RNA e mohiotia ana.Engari, ko te ahua o tenei whakaaro kaore e pa ana ki te tiaki i te NNE, a, i mua i tenei rangahau, ko te ahua o nga here e whakaiti ana i te rereketanga o te raupapa tripeptide i tino pouri.
Hei whakatau i te hiranga o te nsp9-NMPylation me te tiaki NNE i roto i te tukurua coronavirus, i hangaia e matou nga mutants HCoV-229E, e kawe ana i nga whakakapinga kotahi, takirua ranei o nga toenga nsp9 N-terminal, e tohu ana he kino te nsp9 NMPylation in vitro.I mua i te tiimata, ka ngana taatau ki te whakautu i te patai mena ko enei whakakapinga (e tata ana ki te waahi o te nsp8|9) ka pa ki te tukatuka proteolytic o te rohe C-terminal pp1a.He huinga huinga polyprotein nsp7-11 kei roto he whakakapinga e rite ana ki te pito-N o nsp9 i hangaia i roto i te E. coli ka tapahia ki te Mpro recombinant.Ko te wehenga proteolytic o nga waahi e wha (tae atu ki te waahi o te nsp9) kaore e tino pa ana ki nga whakakapinga whakaurua (SI apiti, Whakaahua S9), haunga nga huringa hanganga i roto i enei pūmua e whakararu ana i te wehenga Mpro-mediated nsp8|9 ( Me etahi atu) paetukutuku.
Ko nga pūtau Huh-7 i whakawhitihia me te HCoV-229E RNA roa-ira, te whakawaehere i nga whakakapinga Ala, Ser ranei i roto i nga tripeptides NNE kua tiakina (N3825, N3826, me E3827) i te pito nsp9 N, e whakaatu ana he mate te nuinga o nga huringa.I taea e matou te whakaora i te huaketo ma te whakakapi i te Ser, Ala ranei o te N-terminal Asn (N2835A or N2835S), engari kaore i taea te whakaora i te huaketo me etahi atu huringa kotahi me te rua i roto i te raupapa NNE (N3826A, N3826S, NN3825 / 6AA, NN3825/6SS), E3827A) (Whakaahua 5D).
Ko enei hua e tohu ana ko te tukuruatanga o nga coronaviruses i roto i te ahurea kiko kua whakawhäitihia (he rite, he rite ranei), ka whakawhäiti i te huringa taiao o nga waahi nsp9 NMPylation i roto i te tinana, me te tautoko i te mahi matua o tenei urupare i roto i te huringa ora o nga coronaviruses.
I nga huinga whakamatautau whakamutunga, i hangaia e matou te C-terminal His6 tapanga SARS-CoV-2 nsp12 me nsp9, me nga momo mutant o nsp12 i E. coli.Ko nga toenga waahi kaha i roto i nga rohe NiRAN me te RdRp ko te whakamahi i a Ala hei utu (Whakaahua 6A me te taapiri SI, Ripanga S2).K4465 i roto i te SARS-CoV-2 nsp12 e rite ana ki te K4135 i HCoV-229E (SI Appendix, Whakaahua S2), i kitea e hiahiatia ana mo te mahi NiRAN me te tukurua HCoV-229E (Whakaahua 3D me E).Ko tenei toenga he rite ano ki te toenga o te huaketo EAV nsp9 K94, i whakaatuhia i mua e tika ana mo te NiRAN self-UMPylation/self-GMPylation (16).E whakaatuhia ana i te Whakaahua 6B, he mahi whakawhiti UMP a SARS-CoV-2 nsp12 ma te whakamahi i te nsp9 hei tïpako, i te mea ko te mutant pae kaha nsp12_K4465A kei te hohe.Ko te whakakapinga rua i roto i te raupapa ahuatanga SDD o RdRp motif C kaore e pa ki te mahi whakawhiti UMP (Whakaahua 6B), e tohu ana kaore he paanga tika o te mahi RdRp i roto i te nsp9 UMPylation.He rite nga raraunga i whiwhi ma te whakamahi i te CTP, GTP me te ATP (SI apiti, Whakaahua S10).Hei whakarāpopototanga, ko enei raraunga e tohu ana ko te NSP9 NMPylation i takawaenga a NiRAN he mahi atawhai i roto i nga coronaviruses e tohu ana i nga puninga rereke o te whanau o te orthocoronavirus.
SARS-CoV-2 nsp12-whakawaea NMPylation o nsp9.(A) Ko Coomassie i pania te gel SDS-polyacrylamide e whakaatu ana i te pūmua recombinant i whakamahia i roto i te whakamatautau NMPylation.Hei mana whakahaere, ka whakamahia he pūmua mutant me te whakakapi i te waahi kaha ki te rohe NiRAN (K4465A) me te rohe RdRp (DD5152/3AA) o SARS-CoV-2 nsp12.Ko te tatauranga toenga kei runga i te tuunga i pp1ab.(B) Autoradiograph of UMPylation detection using nsp9-His6 and [α-32P]UTP as the substrate of nsp12-His6 (momo mohoao [wt] and mutant).Kei te taha maui te papatipu ngota (i nga kirodalton) o te pūmua kua tapaina.
Ko nga rohe NiRAN te nuinga ka tiakina i roto i te Nidovirales (16), e tohu ana ka whakakorikori i nga tauhohenga enzymatic e tika ana mo te tukuruatanga Nidovirus.I roto i tenei rangahau, i taea e matou te whakaatu ko te rohe NiRAN o te coronavirus e whakawhiti ana i te NMP (i hangaia mai i te NTP) ki te nsp9, he pūmua here RNA ngaro e uru ana ki te tukuruatanga huaketo (26 ?? 29), ki te whakatau i te reira hei whainga taiao me hoa o te coronavirus RTC.
Ka tohatohahia e te rohe NiRAN nga kaupapa raupapa e toru (AN, BN, me CN), he iti rawa nga toenga e tiakina ana i roto i nga whanau katoa i roto i te raupapa Nidovirales monophyletic engari tino rerekee (8, 16).Kua whakaatuhia e nga rangahau o tata nei he hononga hangahanga ki tetahi whanau tino kore e tohuhia o nga pūmua kinase-rite, i kiia i te tuatahi ko te whanau SelO (17, 19, 22, 30, 31).Ko nga pūmua e pa ana ki te SelO he kopa kinase, engari he maha nga toenga o te waahi kua tiakina i roto i nga kinase puāwaitanga (22, 32).I runga i te tuunga whakamuri o nga ngota ngota ATP e herea ana ki te waahi hohe me te whakapumautanga e nga tauwhitinga motuhake, i whakapaehia a SelO, katahi ka whakapumautia ki te whakawhiti i te AMP (hei whakakapi i te phosphate) ki te tïpako pūmua (22), i te mea ko tetahi atu huakita SelO-rite te pūmua YdiU I whakaatu tata nei ki te whakakorikori i te taapiri o te UMP ki a Tyr me ona toenga o nga taputapu pūmua rereke (33).
Hei whakaū me te whakawhänui i te matapaetanga o nga toenga pae kaha putative o te rohe coronavirus NiRAN, i whakamahia e matou nga tikanga matū koiora me te whakamuri i nga tikanga ira hei mahi i te tātaritanga ira i runga i te coronavirus nsp12 (Whakaahua 3D me te taapiri E me te SI, Whakaahua S3 me te ripanga) S1â S4).Ko nga raraunga e whakaatu ana ko te whakakapinga o te HCoV-229E K4135, R4178 me D4280 me Ala ka whakakore i roto i te in vitro NMP te mahi whakawhiti me te tukuruatanga huaketo i roto i te ahurea pūtau (Whakaahua 3D me te E me nga taapiri SI, Whakaahua S3), e tautoko ana i to ratou aroaro i roto i te NTP γ-phosphate. ( K4135, R4178) me te ruruku o nga katote whakarewa o te waahi (D4280).Ko te whakakapinga E4145A o te Glu kua tiakina i roto i te awhe o te huaketo kohanga o te manu i tohuhia hei whakapumau i te tuunga K4135 (17) i whakaatuhia ki te whakakore i te tukurua viral, engari he mea whakamiharo, i mau tonu te mahi i roto i te in vitro NMPylation assay (Whakaahua 3D me E me SI appendix, Whakaahua S3 Me nga Ripanga S1–S4).He rite ano te tirohanga i te wa i whakauruhia te whakakapinga e rite ana ki te YdiU homolog o Salmonella typhimurium (E130A) (33).Ka huihuia, ka tautoko enei raraunga i te mahi ture o tenei toenga kua tiakina, kaua ko te mahi catalytic.
Ko te whakakapi i te toenga Phe kua tiakina (F4281A) i roto i te awhe o te nestovirus i roto i te rohe HCoV-229E NiRAN (8) ka heke te mahi NMPylation in vitro me te heke nui o te tukuruatanga huaketo i roto i te ahurea pūtau (Whakaahua 3D, E me SI) āpitihanga, Whakaahua S3).He rite tonu nga raraunga ki te mahi ture nui o tenei toenga, penei i te toenga DFG motif Phe i whakaatuhia i mua.I roto i nga kinases pūmua puāwaitanga, he wahanga o te Mg2+ here here me te awhina ki te whakahiato me te whakahaere i te tuara???E hiahiatia ana mo te mahi catalytic whai hua (32, 34).Ko te whakakapi i a Ala me Arg mo nga toenga K4116 (i roto i te kaupapa preAN), ka whakakorehia te tukuruatanga viral, me te mea i tumanakohia, he rereke nga paanga ki nga mahi whakawhiti NMPase in vitro, i runga i te mekameka taha waikawa amino i whakauruhia (Whakaahua 3D Me nga taapiri E me te SI. , Whakaahua S3).Ko nga raraunga mahi e rite ana ki nga korero hanganga, e tohu ana kua whakapumautia e tenei toenga he taunekeneke ki te ATP phosphate (17).I roto i te rohe NiRAN o etahi atu whanau wheori kohanga, ko te tuunga o HCoV-229E pp1a/pp1ab K4116 e nohoia ana e Lys, Arg, tana ranei (8), e tohu ana kua whakangawarihia te herenga mahi o tenei toenga motuhake.Ko te whakakapinga o te D4188A me te D4283A ka whakakore, ka tino whakaiti ranei i te mahi enzyme me te whakakore i te tukuruatanga huaketo (Whakaahua 3).Ko enei toenga e rua ka tiakina i roto i te nuinga (engari ehara i te katoa) nga wheori kohanga (8), e tohu ana i tetahi mahi whakahirahira mo te whanau engari kaore pea i te whakangao.Ko nga whakakapinga Ala o etahi atu toenga Lys me Asp (K4113A, D4180A, D4197A me D4273A) kaore i tiakina i roto i nga Coronaviridae me etahi atu whanau Nestioviridae (8) i whakamahia hei mana whakahaere.Ka rite ki te tumanako, he tino pai enei whakakapinga, he iti te heke o te ngohe o te enzyme me te tukurua viral i etahi wa (Whakaahua 3 me te taapiri SI, Whakaahua S3).I roto i te katoa, ko nga raraunga mutagenesis coronavirus he tino rite ki te GMP whaiaro me te raraunga ira whakamuri o EAV NiRAN-RdRp (16), kei roto i te EAV nsp9 (coronavirus nsp12 ortholog) toenga K94 (e rite ana ki te HCoV- 229E K4135) nga mahi nui), R124 (e rite ana ki te R4178), D132 (e rite ana ki te D4188), D165 (e rite ana ki te D4280), F166 (e rite ana ki te F4281).Hei taapiri, he rite tonu nga raraunga mutagenesis HCoV-229E me te whakawhänuihia mai i nga raraunga iranga whakamuri SARS-CoV i mua (16), he rite tonu ki nga mea i kitea mo te motif CN Phe-to-Ala mutant SARS-CoV_nsp12 Te tohu tohu -F219A me HCoV-229E_F4281A (Whakaahua 3 D me E me te taapiri SI, Whakaahua S3 me te Ripanga S1-S4).
Ka whakatauritea ki nga EAV orthologs (16), he maamaa te hiahia mo te UTP me te GTP (i roto i te tauhohenga whaiaro-NMPylation), ka whakaatu ta maatau rangahau ko te rohe coronavirus NiRAN (e tohuhia ana e HCoV-229E me SARS-CoV-2) ka taea te whai hua. i whakawhitia nga NMP e wha, ahakoa he iti te hiahia mo te UMP (Whakaahua 1 me te 3).Ko te tino iti o te taapapa-taapiri NTP motuhake e rite ana ki te hangahanga supercomposite SARS-CoV-2 nsp7/8/12/13 kua panuitia tata nei, i herea ai te ADP-Mg2+ ki te waahi kaha o NiRAN, engari kaua ki te waahanga adenine. o te hanganga o nga taunekeneke motuhake (17).I roto i ta maatau rangahau, ko te ahua o te nucleotide e whakamahia ana i roto i te tauhohenga NMPylation kaore he paanga rereke ki te mahi o te pūmua mutant (SI Appendix, Figure S3), e tohu ana kaore tetahi o enei toenga e hono tata ana ki te herenga o te nucleobase motuhake.Ko te turanga hanganga me te hiranga koiora pea o nga momo manakohanga takirua o te NTP e kitea ana i roto i nga rohe NiRAN o nga coronaviruses me nga wheori arterial e noho tonu ana hei ako;he pono pea, na te kaha ranei o a raatau akoranga.I tenei wa, kaore e taea te whakakorehia ko te mahi NMPylator pea o te rohe NiRAN huaketo arterial (whakaritea ki te mahi NMPylation whaiaro i tohuhia i mua) he rereke te hiahia o te taapiri, me te whakaaro ko te rite i waenga i te arterial me te coronavirus. Ko te rohe NiRAN kei tona rohe.Whakataurite-a-raupapa (16).Ka whakatauritea ki te pseudokinase SelO, e whakamahi ana i te Mg2+ hei cofactor, ko te mahi o te coronavirus me te huaketo arterial NiRAN kei te whakawhirinaki ki te Mn2+ (16) (Whakaahua 3C me te taapiri SI, Whakaahua S1).Ko te Mn2+ te ti'aturi me te tino manakohanga mo te UTP he ahuatanga rerekee o nga pūmua NMPylators, a katahi ano ka whakapumautia i roto i te pūmua YdiU o Salmonella typhimurium, e whakakorikori ana i te Mn2+-whakawhirinaki pūmua chaperone UMPylation ki te tiaki i nga pūtau mai i te ahotea ATP poka wai ( 33).
Ko te ahua o te hanganga i whakaahuahia tata nei i waenga i te rohe coronavirus NiRAN me nga kinases pūmua pūtau (17, 19) he tautoko taapiri mo te kaha o NiRAN ki te hono kovalently NMP ki etahi atu pūmua kua korerotia e matou i tenei rangahau.I arotahi matou ki te rapu i nga whaainga NiRAN ka taea ki runga i nga pūmua kua whakawaeheretia e HCoV-229E ORF1a, e mohiotia ana hei awhina tika, arataki ranei i te tauira o RTC ORF1b-whakawaeherehia (12, 35).Ka whakaratohia e a maatau whakamatautau nga taunakitanga mo te NMPylation whai hua me te motuhake o te nsp9 (Whakaahua 2).Mēnā ka whakamahia te pūmua i whāia i roto i te taikaha molar e 8 ki te 10 nga wa teitei ake i tera o te enzyme (nsp12), ka whakapumautia ko te nsp9 kua tino (mono)NMP (Whakaahua 4).I whakatauhia e matou ko te taunekeneke i waenga i te nsp12 me te nsp9 he wa poto, kaore e hanga he matatini pumau me te nsp9 (i te kore o etahi atu waahanga RTC).Ko tenei whakatau ka tautokohia e nga rangahau taunekeneke pūmua mo te proteome SARS-CoV (35).I tautuhia e te tātaritanga MS te amine tuatahi o te toenga N-terminal o nsp9 ko te waahi NMPylation (SI apiti, Whakaahua S5).Ko te hanganga o te hononga phosphoramidate me te roopu amino N-terminal e wehewehe ana i te mahi NMPylation kua whakawhitia e NiRAN mai i te Pseudomonas syringae SelO-mediated AMPylation tauhohenga, e whakatairanga ana i te hanganga o te AMP hono-O i Ser, Thr, Tyr ranei nga toenga Peptide adduct ( 22), me te S. typhimurium YdiU te hanga O-hono (me Tyr) me te N-hono (me tana) peptide-UMP taapiri.Ko te iti o nga korero e waatea ana mo te whanau SelO o nga pūmua e tohu ana he rereke nga mema o tenei whanau pūmua nui i roto i te hanganga o nga taapiri peptide-NMP.He tirohanga whakamere tenei e tika ana kia rangahau ano.
Ko nga raraunga i whiwhi i roto i tenei rangahau i arahina matou ki te whakapae ko te NMPylation o te nsp9 e hiahiatia ana he N-mutu kore utu.I roto i te horopaki o te tukurua viral, ka whakaratohia tenei e te wehenga proteolytic o te waahi tukatuka nsp8|nsp9 i roto i te tauira polyprotein pp1a i takawaenga e Mpro me pp1ab.I roto i te nuinga o nga coronaviruses, ko te rereketanga i waenga i tenei waahi motuhake (VKLQ|NNEI i HCoV-229E) me era atu waahi wehenga coronavirus Mpro ko Asn (kaore i tetahi atu toenga iti, penei i a Ala, Ser Or Gly) kei te noho P1â???Tauwāhi (36).Ko nga raraunga wehenga peptide i whiwhi i nga rangahau tuatahi i whakaatu he iti ake te pai o te wehenga o te waahi nsp8|nsp9 i tera o etahi atu waahi, e tohu ana ko 1) ka whai mana tenei waahi motuhake ki te whakahaere i te waa whakahaere o te waahi C-terminal. pp1a rohe, 2) a Ko te mahi a te nsp9 motuhake kua tiakina ki te tukuruatanga huaketo (37).Ko o maatau raraunga (Whakaahua 5A) i whakaatu ko te ahua o te recombinant o nsp9 e mau ana i te raupapa N-terminal tuuturu i whai hua te NMPized e nsp12.I tangohia te raupapa taha taha-N e te tauwehe Xa (nsp9-His6; SI appendix, Ripanga S1) ranei Mpro-mediated cleavage (nsp7-11-His6; Figure 5A me SI appendix, Ripanga S1).Ko te mea nui, ko te nsp7-11-His6 kare i tapahia ko te tohu o mua i roto i te nsp7-11-His6 i whakaatu i te aukati ki te NMPylation o te nsp12, e rite ana ki o maatau raraunga, e tohu ana ko te whakaurunga nsp9-NMP i hangaia ma te N-terminal primary amine (SI apiti, Whakaahua S5) .Kia hohonu ake te maarama mo te motuhake o te taputapu NiRAN, katahi matou ka aro ki nga toenga N-terminal o te nsp9.I te kore o etahi atu pūmua, he hangawari te hanga, e kore e kitea i roto i te ahua kore tapanga o te nsp9 (26 28, 38), e tohu ana i to raatau rerekeetanga taiao Na te mea nui te raupapa-motuhake (ehara i te hanganga tuarua) mahi o te wahanga nsp9 N-terminal.Ko nga whakakapinga Ala o nga toenga kua tiakina i tenei rohe (Whakaahua 5C me D me te taapiri SI, Whakaahua S8) e whakaatu ana he mea nui te N3826 mo te nsp9 NMPylation in vitro, i te mea ko nga whakakapinga N3825A me E3827A e arahi ana ki te whakaheke i te waahanga, engari ko te M3829A me te P3830A kaore i te whakauru i te NMPylation. .Maamaa ka pa ki te nsp9 NMPylation.Ahakoa ko te whakakapinga o te N-terminal Asn (N3825A, N3825S) he iti noa te paanga ki te nsp9 NMPylation me te tukuruatanga huaketo i roto i te ahurea pūtau (Whakaahua 5C me D), te whakakorenga o te raupapa toenga Asn mai i te N-terminal 3825-NN dipeptide i kitea he whakamate ki nga wheori, e tohu ana ka hiahiatia kia kotahi toenga Asn i mua i tetahi atu toenga i te pito-N, he pai ki a Asn, ahakoa ko te ahua ka taea te whakakapi i nga toenga rite (Whakaahua 5B, C, me D).Ka whakatauhia e matou ko te 3825-NN dipeptide, ina koa ko te toenga N3826 kua tiakina me te mea nui i roto i te awhe coronavirus (te taapiri SI, Whakaahua S6), ka whakarite kia tika te here me te whakatakotoranga o te nsp9 N-terminus i te waahi kaha o NiRAN.
Ko te whakakapi i a Ala (E3827A) mo te Glu kua tiakina o nga hapu katoa ka mau tonu te nsp9 NMPylation in vitro engari he whakamate ki nga huaketo i roto i te ahurea pūtau (Whakaahua 5C me D), e tohu ana i te mahi taapiri o tenei toenga, hei tauira, i roto i nga taunekeneke matua (NMPylated or unmodified). ) nsp9 N-terminus me etahi atu mea e pa ana ki te tukurua huaketo.Ko nga huringa Nsp9 kaore i pa ki te tukanga proteolytic o te nsp9, ki etahi atu nsps patata (39) (SI Appendix, Figure S9), e tohu ana ko nga ahuatanga whakamate o te maha o nga huringa nsp9 i kitea kaore i puta mai i te koretake o te waahanga C proteolytic process-terminal pp1a. .
Ko nga korero i runga ake nei e whakaatu ana i muri i te maimoatanga a Mpro-mediated o te waahi wehewehe nsp8|9 i roto i te pp1a/pp1ab, ka taea te UMPylated te N-terminus o te nsp9 (ka whakarereketia etahi waahanga me tetahi atu NMP).I tua atu, ko te tino tiaki i te N-terminus o te nsp9 (tae atu ki nga toenga Asn motuhake me te rerekee i roto i te whanau coronavirus) me nga raraunga ira whakamuri i whiwhi i roto i tenei rangahau (Whakaahua 3E me 5D) i arahina matou ki te whakatau ko te nsp9 NMPylation i whakaahuatia. he hononga koiora me te mea nui mo te tukurua coronavirus.Ko nga hua mahi o tenei whakarereketanga ka noho tonu ki te ako, hei tauira, mo te korero i mua ake nei (kaore i te motuhake) nsp9 (ahua kaore i whakarereketia) te mahi here RNA (2628).N-terminal NMPylation pea ka pa ki te taunekeneke o te nsp9 me te pūmua, te RNA substrates ranei te hanganga o nga huihuinga rereke e wha.Kua kitea enei i roto i nga rangahau hanganga me te mea kua whakapumautia he mahi e pa ana ki te tukuruatanga o te coronavirus, ahakoa i te kore o I te keehi o tenei whakarereketanga (26- ââ29, 40).
Ahakoa ko te tohu motuhake o te rohe coronavirus NiRAN me whakaatu ake i nga korero taipitopito, e whakaatu ana o maatau raraunga he tino whaiti te whaainga pūmua o te rohe coronavirus NiRAN.Ahakoa ko te tiaki i nga toenga pae kaha matua (8, 16) i roto i te rohe NiRAN o nga whanau nidovirus katoa e kaha tautoko ana i nga mahi a te NMPylator kua tiakina enei pūmua, ko te tuakiri o nga toenga pute e here ana i te tïpako o tenei rohe Ko te Tiaki me te tiaki ka mau tonu ki te tohu. , a ka rereke pea i waenga i nga whanau rereke o nga kaupapa Nidovirales.Waihoki, ko nga whaainga e tika ana mo etahi atu wheori kohanga kaore ano kia whakatauhia.He orthologs tawhiti pea o nsp9, etahi atu pūmua ranei, na te mea ko nga raupapa kei waho o nga rohe taapiri e rima e tiakina ana i roto i nga wheori kohanga he iti ake te tiaki (8), tae atu ki te huinga ira i waenganui i a Mpro me NiRAN, I roto i era, kei roto te nsp9 i te coronavirus.
I tua atu, kaore e taea e matou te whakakore i tenei wa ka taea e te rohe NiRAN etahi atu whaainga (tae atu ki te waea pūkoro).I roto i tenei take, he mea tika kia whakahuahia ko nga homologues huakita i roto i tenei ahua o te pūmua NMPylators (NMPylators) (30, 31) he "kaiwhakahaere rangatira"?Ko te NMP te whakarereke i nga momo momo pūmua pūtau hei whakahaere, hei whakakore ranei i a raatau mahi o raro, na reira ka whai waahi ki nga momo momo tukanga koiora, penei i te whakautu ahotea pūkoro me te homeostasis redox (22, 33).
I roto i tenei rangahau (Whakaahua 2 me 4 me SI Appendix, Whakaahua S3 me S5), i taea e matou te whakamatau i te whakawhiti a nsp12 i te waahanga UMP (NMP) ki te waahi kotahi (kua tiakina) i roto i te nsp9, ko etahi atu pūmua kaore i whakarereketia i roto i te whakamahia I raro i nga tikanga, ka tautokohia te motuhake o te tïpako kua tautuhia (kaua e wetekina).Ka rite ki tenei, ka whakatauritea ki te N-terminal nsp9 NMPylation, he iti rawa te mahi a te nsp12 ake NMPylation, he roa ake te wa whakakitenga autoradiography, a ka whakamahia te pikinga 10 o te kukū nsp12.I tua atu, kaore i taea e ta maatau rangahau MS te whakaatu taunakitanga mo te NMPylation o nsp12, e kii ana ko te NiRAN rohe whaiaro-NMPylation (i te pai) he mahi tuarua.Engari, me tohu ko etahi atu rangahau i whakarato i nga taunakitanga tuatahi ka taea e te mana whaiaro-AMPylation o te NMPylator huakita te whakahaere i a raatau mahi NMPylation i runga i etahi atu taputapu pūmua (22, 33).Na reira, me nui ake nga rangahau hei tirotiro i nga paanga mahi pea o nga mahi NMPylation-whaiaro i ripoatahia mo te EAV nsp9 (16) me te coronavirus nsp12 (tenei rangahau), tae atu ki te ahua o te chaperone e whakaarohia ana mo te kopaki o te rohe C-terminal RdRp ( 16) ).
I mua, he maha nga whakapae e pa ana ki nga mahi whakararo o te rohe nidoviral NiRAN kua whakaarohia, tae atu ki te RNA ligase, te RNA -capped guanylate transferase me te mahi priming pūmua (16), engari kaore tetahi o enei e hototahi ki nga mahi o raro e waatea ana.Ko nga korero ka riro mai i nga tuunga e whai ake nei he rite tonu te waa me te kore he whakapae taapiri.Ko nga raraunga i whiwhihia i roto i tenei rangahau e tino rite ana (engari kaore e taea te whakaatu) kei te uru te rohe NiRAN ki te whakaurunga o te RNA whakahiato i te pūmua.I whakaponohia i mua ko te mahi o te rohe NiRAN i roto i te 5 ??Ko nga tauhohenga ²-RNA taapiri, RNA here ranei e pa ana ki enei me te Tautoko o etahi atu raraunga.No reira, hei tauira, ko te waahi kaha o NiRAN e kiia ana ko te Asp kua tiakina hei turanga whanui (D252 i Pseudomonas syringae SelO; D4271 i HCoV-229E pp1ab; D208 i SARS-CoV-2 nsp12) (SI Appendix, whika 2 ).S2) (17, 22, 33), i te mea ko te catalysis i roto i te ATP-dependent RNA ligase me te RNA capping enzyme ka mahia e te covalent enzyme-(lysyl-N) -NMP takawaenga, e uru ana ki te kore- Hurihia Lys toenga ( 41).I tua atu, ko te tino motuhake o te coronavirus NiRAN mo nga whaainga pūmua kua tiakina me te maamaa mo nga taarua-tahi o te NTP (he pai ki te UTP) e whakahē ana i te NiRAN-mediated capping enzyme, RNA ligase-like ranei nga mahi.
Ma te maarama, he maha nga mahi taapiri hei manatoko, a, ki te whakamatauhia, whakamaarama i te waahi ka taea e te nsp9-UMP (nsp9-NMP) i roto i te whakahiatotanga RNA whakahiahia e te pūmua, e hono ana i nga purongo whakamere engari (i tenei wa) kua panuitia i mua. .Nga kitenga motuhake.Hei tauira, kua whakatauhia ko te mutunga o te whenu kino RNA o te coronavirus ka tiimata me te whenu oligo(U) (42, 43).Ko tenei tirohanga e rite ana ki te whakaaro ka timatahia te whakahiatotanga o te whenu-kino RNA ma te here i te ahua UMPylated o te nsp9 ki te poly(A) hiku ( triggers), tera pea e whakatairangahia e tana herenga RNA Te ngohe me te taunekeneke me te. tetahi atu pūmua RTC.Ko te wahanga UMP e whakaratohia ana e nsp9 ka taea te whakamahi hei "primer" mo te nsp7/8/nsp12-mediated oligouridylation, ma te whakamahi i te hiku 3??²-poly(A) i roto i te genomic RNA ranei tetahi atu oligo (A) -nga raupapa kei roto. he tauira, he rite ki te tikanga kua whakaritea mo te picornavirus VPg pūmua (44).Ka pehea mena he "kore-tikanga" te tono????Ko te tiimatanga o te whakahiato RNA kino-waa (whakauruhia) he hononga ki nga tirohanga, e tohu ana ko te RNA kino-kore-kore he UMP (hei utu mo te UTP) i tona mutunga (42), e kiia ana ko te Ko te waikawa nucleic ka wehea e Dicer te pito i whakapouritia e te endonuclease motuhake uridine e kore e mohiotia.Mena ka whakapumautia, ka taea e tenei ngohe hydrolytic waikawa nucleic te awhina ki te tuku i te ahua oligomeric UMPylated o nsp9 mai i te 5 ² te pito o te whenu kino kua puta.He rite ano te mahi a te nsp9 i roto i te whakangao pūmua ki nga rangahau ira whakamuri o mua, i whakaatu ko te nsp9 (me te nsp8) te taunekeneke arohaehae me te motuhake ki te huānga RNA cis-acting e tata ana ki te pito 3 o te ira coronavirus.45).E ai ki tenei purongo, ka taea te tirotiro ano i enei tirohanga o mua me te whakawhānui ake ma te rangahau ano.
Hei whakarāpopototanga, i whakatauhia e a maatau raraunga te mahi motuhake o tetahi tohu whaakini huaketo kohanga e hono ana ki te RdRp i te pito-N.I roto i te coronavirus, ka whakamahia tenei mahi hou a NiRAN-mediated UMPylator/NMPylator ki te whakawhirinaki ki te Mn2+ me nga toenga Asn e patata ana, me te hanga i nga hononga phosphoramidate (iti-kaha) ki te amine tuatahi N-terminal.Na roto i te wehewehenga a Mpro-mediated i te waahi wehewehe nsp8|9, ka taea te whakamahi i te whaainga nsp9 mo te NMPylation, e tohu ana i te hononga mahi i waenga i te protease me te rohe NiRAN, ka toro atu ki te RdRp.Ko te tiaki i nga toenga matua i roto i te waahi hohe nsp12 NiRAN me te whaainga nsp9, i honoa me nga raraunga i whiwhi mai i nga coronaviruses e rua tae atu ki te SARS-CoV-2, e whakaatu kaha ana ko te nsp9 NMPylation he coronavirus Ko nga ahuatanga Conservative he mahi matua mo te tukurua huaketo.Ko nga raraunga e waatea ana ka whakatauhia ko te mahi motuhake o te ahua NMPylated o te nsp9 i roto i te whakahiato RNA-whakaurua te pūmua he ahuatanga whaitake mo te coronavirus me etahi atu wheori kohanga, a ka taea ano e NiRAN te aro atu ki etahi atu pūmua kaore i mohiotia.Whakaritea te huaketo.Te taunekeneke a te kaihautu.Mena ka whakapumautia, ko te whakaurunga o nga kaimatai pūmua i roto i te whakahiato RNA viral ka piki ake te hononga raupapa o nga rohe Mpro/3CLpro me RdRp i waenga i te coronavirus i kitea i mua me te supergroup rite ki te picornavirus (9), kua honoa inaianei ki nga Pisonivirites tata nei ( 46) i roto i te waahanga.
E whakaatu ana hoki o taatau raraunga ko nga mahi whaariki taketake, whiriwhiri me te atawhai i kitea i roto i tenei rangahau ka taea te whakamahi hei whainga mo nga raau taero antiviral.Ko nga kohinga e whakararu ana i te herenga (me nga whakarereketanga o muri mai) o te nsp9 N-terminus kua tiakina i roto i te waahi kaha o NiRAN ka taea te hanga hei rongoa patu huaketo whaihua me te maha, e tika ana mo te maimoatanga o nga coronaviruses kararehe me te tangata mai i nga momo Infections (sub) , tae atu ki te SARS-CoV-2 me te Middle East Respiratory Syndrome Coronavirus.
Ko te raupapa tohu o te pūmua coronavirus i hangaia i roto i tenei rangahau i whakanuia e te RT-PCR ma te whakamahi i te RNA i wehea mai i te Huh-7 i pangia e te HCoV-229E, i te Vero E6 ranei i pangia e te SARS-CoV-2, ka whakauruhia ma te whakamahi i nga tikanga whakakoi paerewa.pMAL-c2 (New England Biological Laboratory) or pASK3-Ub-CHis6 (47) vector expression (SI Appendix, Ripanga S1 me S2).Ko nga whakakapinga codon kotahi i whakauruhia e te PCR-a-te-pae-whakahaere mutagenesis (48).Hei whakaputa i te pūmua fusion MBP, i hurihia nga pūtau E. coli TB1 me te hanga plasmid pMAL-c2 e tika ana (SI apiti, Ripanga S1).I purea te pūmua hanumi e te amylose affinity chromatography ka werohia ki te tauwehe Xa.I muri mai, ko te C-terminal His6-tagged pūmua i purea e Ni-immobilized metal affinity chromatography (Ni-IMAC) pera i te korero i mua (49).Hei whakaputa i te pūmua fusion ubiquitin, ka whakamahia e nga pūtau E. coli TB1 te hanga plasmid pASK3-Ub-CHis6 e tika ana (SI Appendix, Ripanga S1 me S2) me te pCGI plasmid DNA whakawaehere ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Huringa (47).Ko te C-terminal His6-tagged coronavirus pūmua i purea kia rite ki te korero o mua (50).
Ko te whakamatautau whaiaro-NMPylation o HCoV-229E nsp12-His6 i mahia pera i whakaahuatia i roto i te EAV nsp9 (16).Hei poto, kei roto i te nsp12-His6 (0.5 µM) te 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 µM buffer i taurite te NTP me te 0.17 µM ki te [α32-P]NTP (3,000 Ci/mmol; Hartmann Analytic) i te 30 °C mo te 30 meneti.I roto i era atu (paerewa) nga whakamatautau NMPylation o nsp12-mediated nsp9 NMPylation, ka whakatikahia nga tikanga tauhohenga penei: nsp12-His6 (0.05 µM) me nsp9-His6 (4 µM) i te aroaro o te 50 mM HEPES-KOH (pH 8.0) ), 5 mM DTT, 1 mM MnCl2, 25 µM i tohuhia te NTP, me te 0.17 µM ki te [α32-P]NTP.I muri i te incubating mo te 10 meneti i te 30°C, ka konatunatua te tauira tauhohenga ki te SDS-PAGE tauira parepare: 62.5 mM tris(hydroxymethyl)aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol me 0.005% bromophenol kahurangi.I whakakorehia te pūmua ma te whakamahana i te 90 °C mo te 5 meneti ka wehea e te 12% SDS-PAGE.Ko te reera ka mau, ka pania ki te otinga Coomassie Brilliant Blue (40% methanol, 10% waikawa acetic, 0.05% Coomassie Brilliant Blue R-250), kua whakakorehia te tae, ka kitea ki te mata whakaata phosphorescent mo te 20 haora (kia kitea te nsp12 mai i te NMPylation) ranei (mōrahi) 2 haora (ki te aromatawai nsp9 NMPylation).I whakamahia he whakaahua a Typhoon 9200 (GE Healthcare) ki te matawai i te mata me te ImageJ i whakamahia hei tātari i te kaha o te tohu.
Mo te tātaritanga MS, 1 µM nsp12-His6 me 10 µM nsp9 (kaore he tohu hexahistidine) i whakamahia i roto i te tātaritanga NMPylation (SI appendix, Ripanga S1) me te whakanui ake i te kukū o te 500 µM UTP me te GTP i whakamahia.I runga i to ratou kukū me te kounga o te pūmua e tumanakohia ana, ka whakamahia he punaha HPLC Waters ACQUITY H-Class me te pou MassPrep (Waters) ki te whakakore i te 1 ki te 10 µL o nga otinga pūmua parepare i runga ipurangi.Ko te pūmua kua whakakorehia ka tukuna ki roto i te puna katote electrospray o Synapt G2Si spectrometer papatipu (Waters) na roto i te rōnaki e whai ake nei o te parepare A (wai/0.05% te waikawa formic) me te parare B (acetonitrile/0.045% te waikawa formic), a ko te pāmahana o te pou he 60 ° C me te tere rere o te 0.1 mL/min: te elution isocratically ki te 5% A mo te 2 meneti, ka he rōnaki rārangi ki 95% B i roto i te 8 meneti, me te pupuri i te 95% B mo tetahi atu 4 meneti.
Ka kitea nga katote pai me te awhe papatipu o te 500 ki te 5000 m/z.Ka inehia te Glu-fibrinopeptide B ia 45 hēkona mo te whakatikatika i te paheketanga papatipu aunoa.Whakamahia te rorohiko taputapu MassLynx me te toronga MaxEnt1 ki te wetewete i te awhi toharite i muri i te tangohanga i te rarangi turanga me te whakaene.
Ko te UMPylated HCoV-229E nsp9 i kerihia ma te taapiri i te trypsin (Serva) kua whakarereketia, ka whakakororia i te po i te 37 °C.He tīwae miro Chromabond C18WP (waahanga nama 730522; Macherey-Nagel) i whakamahia hei whakakore i te tote me te whakakoi i nga peptides.Ka mutu, ka rewa te peptide ki te 25 µL o te wai, kei roto te 5% acetonitrile me te 0.1% te waikawa formic.
Ko nga tauira i tātarihia e MS ma te whakamahi i te Orbitrap Velos Pro mass spectrometer (Thermo Scientific).Ko te tino nanoâ HPLC pūnaha (Dionex), rite rawa ki te ritenga mutunga-mau 50 cm??75 μm C18 RP tīwae kikī ki te 2.4 μm pirepire autō (Dr. Albin Maisch High Performance LC GmbH) Hono ki te tūāwhiowhio papatipu ipurangi mā te Proxeon nanospray puna;werohia te 6 µL o te otinga nakunaku trypsin ki te 300 µm te diameter o roto ×??1 cm C18 PepMap tīwae i mua i te kukū (Thermo Scientific).Ma te whakamahi i te wai/0.05% te waikawa formic hei whakarewa, ka mau aunoa te tauira me te whakaheke i te reiti rere o te 6 µL/min.
Ko nga rōnaki e whai ake nei o te wai/0.05% waikawa puka (wairewa A) me te 80% acetonitrile/0.045% waikawa puka (wairewa B) i whakamahia hei whakatutuki i te wehenga o nga peptides tryptic i te tere rere o te 300 nL/min: 4% B mo 5 meneti, ka 30 A rōnaki rārangi ki te 45% B i roto i ngā meneti, me te piki rārangi ki te 95% whakarewa B i roto i te 5 meneti.Tūhonohia te tīwae chromatographic ki te kowiri tira mitanano-emitter (Proxeon), ka rehu tika i te eluent ki te capillary whakawera o te spectrometer papatipu ma te whakamahi i te kaha o te 2,300 V. Ko te karapa rangahau me te taumira o te 60,000 i roto i te Orbitrap mass analyzer e hono ana. me te iti rawa e toru nga raraunga MS/MS scans, hihiri karekau mo te 30 hēkona, te whakamahi i te tukinga mahanga katote raina i awehia te wehewehenga, te wehenga tukinga kaha teitei ake ranei me te kitenga orbitrap , Ko te taumira he 7,500.
Wā tuku: Akuhata-03-2021