Coronavirus replication-transcription complex: qhov tseem ceeb thiab xaiv NMPylation ntawm NiRAN-RdRp subunits rau cov chaw khaws cia hauv nsp9

Hloov kho los ntawm Peter Sarnow, Stanford University Tsev Kawm Ntawv Tshuaj Kho Mob, Stanford University, California, tau pom zoo rau lub Kaum Ob Hlis 25, 2020 (saib xyuas thaum Lub Kaum Hli 25, 2020)

Peb tshaj tawm kev sib cuam tshuam ntawm subunits nyob rau hauv replication ntawm coronaviruses-transcription complexes, uas yog ib qho tseem ceeb rau replication thiab evolutionary kev txuag.Peb muab pov thawj tias NiRAN domain txuam nrog nsp12 muaj nucleoside monophosphate (NMP) kev ua haujlwm hloov pauv hauv trans, thiab txheeb xyuas nsp9 (ib qho RNA binding protein) raws li nws lub hom phiaj.NiRAN catalyzes covalent txuas ntawm NMP moiety rau kev txuag nsp9 amino terminus nyob rau hauv cov tshuaj tiv thaiv uas tso siab rau Mn2+ ions thiab nyob ib sab khaws cia Asn residues.Nws tau pom tias kev ua haujlwm NiRAN thiab nsp9 NMPylation yog qhov tseem ceeb rau tus kabmob coronavirus.Cov ntaub ntawv tso cai rau peb los txuas cov haujlwm no ntawm cov kab mob zes qe menyuam rau cov kev soj ntsuam yav dhau los hauv kev xav tias kev pib ntawm RNA synthesis hauv chav kawm ntawm RNA cov kab mob ua haujlwm thiab hloov pauv tau zoo ib yam.

RNA-dependent RNA polymerase (RdRps) ntawm Nidovirales (Coronaviridae, Arterioviridae, thiab 12 lwm tsev neeg) yog txuas mus rau cov amino-terminal (N-terminal) domain nyob rau hauv lub non-structural protein (nsp) tso tawm los ntawm polyprotein, hu ua NiRAN. 1ab yog tsim los ntawm cov kab mob tseem ceeb protease (Mpro).Yav dhau los, tus kab mob hlab ntsha NiRAN-RdRp nsp tus kheej GMPylation / UMPylation kev ua haujlwm tau tshaj tawm, thiab nws tau hais kom tsim kom muaj kev hloov pauv ntawm nucleoside monophosphate (NMP) mus rau tus kab mob (tam sim no tsis paub) thiab / lossis cell biopolymerization Yam.Ntawm no, peb qhia tias tus mob coronavirus (Human Coronavirus [HCoV]-229E thiab mob hnyav ua pa nyuaj rau tus mob Coronavirus 2) nsp12 (NiRAN-RdRp) muaj Mn2+-dependent NMPylation kev ua, uas yog muab los ntawm nsp9 los ntawm kev tsim Mpro-mediated nsp9 Tom qab. N-terminal flanking nsps yog proteolytically tso tawm, phosphoramidate yog khi rau thawj amine (N3825) ntawm N-terminal ntawm nsp9.Uridine triphosphate yog qhov nyiam nucleotide hauv cov tshuaj tiv thaiv no, tab sis adenosine triphosphate, guanosine triphosphate, thiab cytidine triphosphate kuj tsim nyog co-substrates.Kev tshawb fawb txog kev hloov pauv siv cov kab mob coronavirus recombinant nsp9 thiab nsp12 proteins thiab genetic engineered HCoV-229E mutants txiav txim siab cov seem uas tsim nyog rau NiRAN-mediated nsp9 NMPylation thiab tus kab mob replication hauv cell culture.Cov ntaub ntawv tau lees paub qhov kev kwv yees ntawm NiRAN cov chaw seem seem thiab txiav txim siab lub luag haujlwm tseem ceeb ntawm nsp9 N3826 cov seem hauv nsp9 NMPylation thiab tus kab mob rov ua dua hauv vitro.Cov seem no yog ib feem ntawm kev txuag N-terminal NNE tripeptide ib ntus thiab tau ua pov thawj tias tsuas yog cov seem seem ntawm nsp9 thiab nws homologs hauv tsev neeg mob coronavirus.Txoj kev tshawb no muab lub hauv paus ruaj khov rau kev tshawb fawb txog kev ua haujlwm ntawm NMPylation kev ua ntawm lwm cov kab mob zes thiab nthuav tawm lub hom phiaj rau kev txhim kho cov tshuaj tiv thaiv kab mob.

Nidovirales positive-stranded RNA tus kab mob kis tau ntau yam kab mob vertebrates thiab invertebrates (1, 2).Qhov kev txiav txim tam sim no suav nrog 14 tsev neeg (3), uas tsev neeg Coronavirus tau kawm ntau ntxiv hauv 20 xyoo dhau los.Nyob rau lub sijhawm ntawd, peb tus kabmob zoonotic tau tshwm sim los ntawm cov tsiaj ua tswv thiab ua rau muaj kev sib kis loj ntawm kev ua pa hnyav rau tib neeg.Nrog rau cov kab mob sib kis tas mus li tshwm sim los ntawm cov kab mob sib kis loj heev.Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (4âââ7).Nidoviruses sib koom ib lub koom haum genome, thiab cov subunit ntawm cov membrane-bound replication-transcription complex (RTC) yog encoded nyob rau hauv 5-?²-terminal ob feem peb thiab lub ntsiab subunit ntawm tus kab mob particle, nrog rau ib co accessories. .Protein, encoded hauv 3??² kawg peb ntawm genome (1).Tsuas yog ib tsev neeg ntawm cov kab mob planarian (Monoviridae) (8), tag nrho cov kab mob nested encode RTC subunits nyob rau hauv ob loj qhib nyeem ntawv (ORF) ORF1a thiab ORF1b, uas yog txhais los ntawm genomic RNA ntawm.ORF1a encodes polyprotein (pp) 1a, thiab ORF1a thiab ORF1b koom ua ke encode pp1ab.Nrog rau kev koom tes ntawm lub ntsiab protease (Mpro) encoded los ntawm ORF1a, ob qho tib si pp1a thiab pp1ab yog proteolytically ua tiav rau ntau yam ntawm cov proteins uas tsis yog qauv (nsps), kuj hu ua 3CLpro, vim nws muaj homology nrog 3Cpro ntawm tus kab mob picornavirus ( 9).Cov nsps no tau xav tias yuav tsum tau sib sau ua ke rau hauv lub zog loj RTC, catalyze cov synthesis ntawm genomic RNA (replication) thiab ib txheej ntawm subgenomic RNA (transcription), thiab siv los tswj kev qhia ntawm ORF nyob rau hauv qab ntawm ORF1b (10 ?? ?12).

Cov tub ntxhais RTC suav nrog RNA-dependent RNA polymerase (RdRp) (13), superfamily 1 helicase (HEL1) (14, 15) thiab ntau RNA ua cov enzymes, uas feem ntau yog encoded hauv ORF1b thiab hauv tsev neeg mob coronavirus Nws muaj nsp12-nsp16 thiab nsp9-nsp12 hauv tsev neeg Arterioviridae (saib siv 10ââ 12).RdRp thiab HEL1 sawv cev rau ob (ib-tsib tsib) kev khaws cia ntawm tus kab mob noog lub zes thiab muaj homology ntawm lwm cov kab mob RNA.Kev hloov pauv tseem ceeb ntseeg tau tias yuav pab tau los ntawm lwm cov subunits, suav nrog ntau lub nsps me me tso tawm los ntawm carboxy-terminal (C-terminal) thaj tsam ntawm pp1a, downstream ntawm Mpro (coronavirus nsp5 thiab arterial virus nsp4, feem).Lawv muaj kev txwv rau tsev neeg tshwj xeeb kev tiv thaiv thiab ntau yam dej num (saib xyuas hauv kev siv 10ââ 12).

Tsis ntev los no, ib lub npe uas muaj cov yam ntxwv sib txawv ntawm cov qauv tsim tau pom ntawm cov amino terminus (N-terminus) nyob ib sab ntawm RdRp hauv txhua tus kab mob zes, tab sis tsis muaj lwm yam kab mob RNA (16).Raws li nws qhov chaw nyob thiab nucleotide transferase (nucleoside monophosphate [NMP] transferase), lub npe no yog hu ua NiRAN (Nestvirus RdRp-txog nucleotide transferase).Kev sib xyaw ua ke ntawm NiRAN-RdRp yog nsp12 hauv tsev neeg Coronaviridae thiab nsp9 hauv tsev neeg Arterioviridae, thiab lwm yam nestoviridae, NiRAN-RdRp yuav tsum tau tso tawm raws li kev ywj pheej nsp los ntawm tus kab mob polyprotein.Hauv tus kabmob coronavirus, NiRAN domain muaj ??1/450 residues thiab txuas nrog C-terminal RdRp domain los ntawm thaj chaw txuas (16?19).Hauv Equine Arteritis Virus (EAV) (Arteriviridae), recombinant nsp9 qhia Mn2+ ion-dependent (tus kheej) UMPylation thiab GMPylation cov dej num, uas nyob ntawm peb lub hauv paus khaws cia hauv nestovirus, AN, BN thiab CN Cov seem nyob rau hauv cov kab ke.Qhov twg N stands rau NiRAN) (16).N-terminal flanking ntawm cov motifs no yog ib tug tsawg conservative motif preAN.Ib txhia ntawm cov residues no kuj tau txais kev cawmdim nyob deb ntawm cov protein kinases, qhov uas lawv tau pom tias muaj kev koom tes hauv nucleoside triphosphate (NTP) binding thiab catalytic kev ua si (20, 21).Raws li qhov kev soj ntsuam no, ntau qhov chaw tseem ceeb ntawm qhov chaw seem hauv pseudokinase SelO los ntawm Pseudomonas syringae tuaj yeem sib sau ua ke nrog cov ntawv luam tawm tsis ntev los no SARS-CoV-2 nsp7/8/12/13 supercomplex.Conserved Coronavirus NiRAN residues superimposed hauv electron microstructure.Recombinant protein (17) .Nws tau kwv yees tias cov ntaub ntawv (tus kheej) U / GMPylation yuav tsim ib lub xeev hloov pauv hloov NMP mus rau (tam sim no tsis paub) substrate (16), thiab cov qauv zoo sib xws ntawm NiRAN thiab protein kinase (17, 19) Yog qhov kev xav tias. NiRAN hloov lwm cov proteins.

Ntau yam nta, suav nrog nws qhov tshwj xeeb thiab tshwj xeeb sib koom ua ke nrog cov kab mob zes thiab kev sib cais ntawm caj ces los ntawm RdRp, ua rau NiRAN yog ib qho tseem ceeb ntawm kev tswj hwm enzyme rau cov kab mob zes, uas yog qhov tseem ceeb rau lawv qhov tshwm sim thiab tus kheej.Yav dhau los, peb txoj haujlwm ua tau nrog NiRAN los tswj genome / subgenomic translation lossis replication / transcription raug hu.Thaum xav txog cov ntaub ntawv tsis txaus thiab tsis tiav muaj nyob rau lub sijhawm, txhua txoj haujlwm muaj nws qhov zoo thiab qhov tsis zoo (16).Hauv kev tshawb fawb no, peb tsom mus muab cov kev tshawb fawb biochemical thiab thim rov qab cov kev tshawb fawb ntawm cov kab mob coronaviruses sawv cev rau ob hom, thiab muab peb cov kev tshawb pom nyob rau hauv keeb kwm yav dhau los ntawm kev hloov pauv ntawm ntuj tsim ntawm tsev neeg tus mob coronavirus, txhawm rau kom nkag siab txog qhov tsis paub tseeb no.Peb tshaj tawm cov kev nce qib loj hauv kev nkag siab ntawm NiRAN los ntawm kev txheeb xyuas cov hom phiaj ntuj tsim hauv RTC, uas (ntawm peb qhov kev xav muaj) pab txhawb rau lub luag haujlwm ntawm lub luag haujlwm no hauv kev pib tsim cov kab mob nested RNA.Qhov kev tshawb fawb no tseem qhib qhov muaj peev xwm rau lwm lub luag haujlwm ntawm NiRAN ntawm tus kab mob tus tswv interface.

Txhawm rau ua tus yam ntxwv ntawm cov khoom muaj zog ntawm tus kab mob corona nsp12-hais txog NiRAN domain, peb tsim ib daim ntawv recombinant ntawm tib neeg tus mob coronavirus 229E (HCoV-229E) nsp12 hauv E. coli, nrog His6 tag ntawm C-terminus, thiab ua ke cov protein nrog [α32-P] Incubate ua ke nrog NTP nyob rau hauv lub xub ntiag ntawm MnCl2 raws li tau piav nyob rau hauv cov ntaub ntawv thiab txoj kev.Kev soj ntsuam ntawm cov khoom siv tshuaj tiv thaiv tau qhia tias muaj cov kab mob radiolabeled protein co-migrating nrog nsp12 (106 kDa), qhia tias tus mob coronavirus nsp12 catalyzes tsim covalent protein-NMP adducts, nyiam tsim nrog uridine monophosphate (UMP) (Daim duab 1A) Thiab B).Kev soj ntsuam ntau pom tau tias piv nrog lwm cov nucleotides, lub teeb liab siv ntawm UMP kev koom ua ke tau nce 2 mus rau 3 zaug (Daim duab 1C).Cov ntaub ntawv no tau ua raws li kev kwv yees NMP hloov pauv kev ua haujlwm ntawm NiRAN lub npe ntawm tus kabmob coronavirus (16), tab sis qhia tau hais tias qhov kev nyiam nucleotide ntawm NiRAN tus kabmob coronavirus thiab cov kab mob hauv cov hlab ntsha sib txawv.

Self-NMPylation activity of HCoV-229E nsp12.(A) HCoV-229E nsp12-His6 (106 kDa) yog incubated nrog lub npe [α-32P] NTP nyob rau hauv lub xub ntiag ntawm 6 mM MnCl2 rau 30 feeb (saib Cov Khoom Siv thiab Cov Txheej Txheem kom paub meej).Cov khoom siv tshuaj tiv thaiv tau muab cais los ntawm SDS-PAGE thiab stained nrog Coomassie ci ntsa iab xiav.(B) Radiolabeled protein yog pom los ntawm phosphorous imaging.Cov hauj lwm ntawm nsp12-His6 thiab protein molecular mass markers (hauv kilodaltons) yog qhia nyob rau hauv A thiab B. (C) Qhov kev siv ntawm lub teeb liab radioactive (txhais tau tias ± SEM) tau txiav txim siab los ntawm peb qhov kev sim ywj pheej.* P≤0.05.Lub teeb liab zog (feem pua) muaj feem xyuam rau UTP.

Txawm hais tias NiRAN-hais txog enzyme kev ua ub no tau pom tias yog qhov tseem ceeb rau kev rov ua dua ntawm EAV thiab SARS-CoV hauv kab lis kev cai ntawm tes (16), cov haujlwm tshwj xeeb NiRAN thiab cov hom phiaj tseem ceeb tseem tsis tau txiav txim siab.Tsis ntev los no tau tshaj tawm cov qauv zoo sib xws ntawm NiRAN thiab ib tsev neeg ntawm cov proteins nrog cov protein kinase-zoo li quav (17, 22) ua rau peb sim cov kev xav tias NiRAN catalyzes NMPylation ntawm lwm cov proteins.Peb tsim cov phiaj xwm homologous uas muaj peev xwm, suav nrog cov proteins uas tsis yog cov qauv uas tau muab zais los ntawm HCoV-229E ORF1a (nsps 5, 7, 8, 9, 10), txhua qhov muaj C-terminal His6 tag (SI appendix, Table S1), Thiab incubate cov proteins nrog [α32-P] uridine triphosphate ([α32-P]UTP) nyob rau hauv lub xub ntiag los yog tsis muaj nsp12.Bovine serum albumin thiab MBP-LacZα fusion protein tsim hauv E. coli tau txais kev tswj hwm (Daim duab 2A, txoj kab 1 txog 7).Lub radiolabeled protein tau soj ntsuam los ntawm sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) thiab autoradiography, thiab nws tau pom tias muaj cov teeb liab muaj zog hauv cov tshuaj tiv thaiv uas muaj nsp12 thiab nsp9.Txoj hauj lwm ntawm lub teeb liab sib haum mus rau molecular mass ntawm nsp9, qhia nsp12-mediated UMPylation ntawm nsp9 (Daim duab 2B, kab 7).Tsis muaj lwm cov tshuaj ntsuam xyuas tau pom tias yog UMPylated, uas ua rau peb txiav txim siab tias nsp9 yog ib qho tshwj xeeb substrate ntawm nsp12.Raws li tus kheej-NMPylation cov ntaub ntawv qhia hauv daim duab 1, nsp12 muaj peev xwm hloov tau tag nrho plaub NMPs rau nsp9, txawm tias qhov ua tau zoo sib txawv, UMP> adenosine monophosphate (AMP)> guanosine monophosphate (GMP)> cytidine monophosphate (CMP) ) ( Duab).3 A thiab B).Raws li cov xwm txheej siv hauv qhov kev ntsuam xyuas no (ua kom luv cov tshuaj tiv thaiv thiab lub sij hawm raug, txo qhov concentration ntawm nsp12; cov ntaub ntawv thiab txoj kev), tus kheej-NMPylation ntawm nsp12 tsis tuaj yeem kuaj pom (piv daim duab 2B, kab 7, thiab daim duab 1B), uas ua pov thawj qhov ua tau zoo (Thiab ntau qhov kev sib tw) UMP tau txav los ntawm nsp12 mus rau nsp9.UMP transferase kev ua yuav tsum muaj Mn2+ ions, raws li qhia hauv daim duab 3C, thaum tsuas yog qhov tsawg kawg nkaus UMP transferase kev ua tau pom nyob rau hauv lub xub ntiag ntawm Mg2+, thiab tsis muaj kev ua si nyob rau hauv lub xub ntiag ntawm lwm yam ob divalent cations kuaj.Cov ntaub ntawv zoo sib xws tau txais hauv NMPylation kev soj ntsuam uas muaj cytidine triphosphate (CTP), guanosine triphosphate (GTP), thiab adenosine triphosphate (ATP) (SI appendix, Figure S1).

HCoV-229E nsp12-mediated UMPylation of nsp9.Ib qho ntawm cov protein substrates (xws li bovine serum albumin, MBP-lacZα, thiab ib series ntawm HCoV-229E nsps sau nrog C-terminal His6 encoded los ntawm ORF1a) tau siv los ntsuas UMPylation kev ua ntawm HCoV-229E nsp12-His6⁺-mediated protein ntau.Incubate cov protein nrog [α-32P] UTP rau 10 feeb hauv qhov tsis muaj (A) lossis muaj (B) ntawm nsp12 raws li tau piav qhia hauv cov ntaub ntawv thiab cov txheej txheem.Nyob rau sab saum toj ntawm A thiab B, SDS-polyacrylamide gel stained nrog Coomassie Brilliant Blue yog pom, thiab nyob rau hauv qab ntawm A thiab B, cov autoradiograms coj tau pom.Txoj hauj lwm ntawm cov protein molecular mass marker (hauv kilodaltons) yog muab rau sab laug.Txoj hauj lwm ntawm nsp12-His6 (B, sab saum toj) thiab cov teeb liab xov tooj cua pom thaum lub sij hawm incubation ntawm nsp12-His6 nrog nsp9-His6 (B, kab 7) kuj qhia, uas qhia tias [α-32P] UMP rau nsp9-His6 (12.9 kDa), uas tsis tau soj ntsuam rau lwm cov proteins uas tau kuaj.

HCoV-229E NiRAN-mediated biochemical and virological characterization of nsp9 NMPylation.(A thiab B) Lub luag haujlwm ntawm nucleotide co-substrate siv hauv cov tshuaj tiv thaiv.Nsp12-His6 thiab nsp9-His6 yog tov thiab incubated nyob rau hauv lub xub ntiag ntawm txawv [α-32P] NTPs nyob rau hauv tus qauv NMPylation assay.(A, saum) Coomassie-stained nsp9-His6 cais los ntawm SDS-PAGE.(A, hauv qab) Autoradiograph ntawm tib cheeb tsam ntawm gel.(B) Tus txheeb ze kev ua ub no (txhais tau tias ± SEM) nyob rau hauv lub xub ntiag ntawm lub nucleotide cofactor raug txiav txim los ntawm peb txoj kev sim ywj pheej.* P≤0.05.(C) Lub luag haujlwm ntawm cov hlau ions.Qhia yog tus qauv NMPylation xeem nyob rau hauv lub xub ntiag ntawm [α-32P] UTP thiab sib txawv hlau ions, txhua tus nrog ib tug concentration ntawm 1 mM.Nyob rau hauv C, sab saum toj, Coomassie stained nsp9-His6 yog qhia, thiab hauv C, hauv qab, qhov sib thooj autoradiography yog qhia.Qhov loj ntawm cov ntawv sau npe protein (hauv kilodaltons) yog qhia rau sab laug ntawm A thiab C. (D) Daim ntawv mutant ntawm HCoV-229E nsp12-His6 nqa cov amino acid hloov pauv tau teev tseg hauv [α-32P]UTP, raws li tau piav qhia. hauv Cov Khoom Siv thiab Cov Txheej Txheem.Lub radiolabeled nsp9-His6 ua nyob rau hauv NMPylation cov tshuaj tiv thaiv yog kuaj pom los ntawm phosphorylation imaging (D, saum).Cov kev ua txheeb ze piv nrog cov tsiaj qus (wt) cov protein yog qhia hauv D, thiab hauv qab yog coj raws li qhov nruab nrab (±SEM) los ntawm peb qhov kev sim ywj pheej.Asterisks qhia txog kev hloov pauv ntawm cov khoom seem uas tsis tau khaws cia.(E) Tus kab mob titer nyob rau hauv kab lis kev cai supernatant ntawm p1 hlwb tau txais 24 teev tom qab kis kab mob tau txiav txim los ntawm cov quav hniav.Cov codon hloov pauv hauv NiRAN domain ntawm engineered HCoV-229E mutant yog qhia (cov lej seem yog raws li lawv txoj haujlwm hauv pp1ab).Lub replication-deficient RdRp active site mutant nsp12_DD4823/4AA tau siv los tswj.

Txhawm rau kom nkag siab tob dua ntawm qhov chaw nquag ntawm NiRAN thiab txiav txim siab cov khoom seem uas cuam tshuam nrog kev ua haujlwm ntawm nsp9-specific NMP hloov pauv, peb tau ua qhov kev hloov pauv, uas peb hloov cov khoom seem hauv NiRAN AN, BN thiab CN motifs ( 16) Nws yog Ala (SI appendix, Daim duab S2).Tsis tas li ntawd, qhov cuam tshuam ntawm kev saib xyuas Arg-to-Lys lossis Lys-to-Arg hloov pauv tau raug tshuaj xyuas hauv ob kis.Raws li kev tswj hwm (tsis zoo), cov khoom seem uas tsis yog los yog tsawg dua kev khaws cia hauv NiRAN tus kab mob coronaviruses thiab lwm yam kab mob zes tau hloov nrog Ala. Hloov K4116A (hauv motif preAN), K4135A (AN), R4178A (BN), D4188A (motif BN) thiab D4280A (CN) txo qis lossis txawm tias tshem tawm nsp9 NMPylation los ntawm nsp12, thaum cov proteins nrog kev hloov pauv kev saib xyuas (R4178K, K4116R) khaws 60% thiab 80% ntawm lawv cov haujlwm, uas qhia tau tias kev so ntawm cov kev txwv ntawm lawv sab. chains yog physicochemically rhiab heev (Daim duab 3D).Hloov ob peb lwm qhov chaw khaws cia E4145A, D4273A, F4281A thiab D4283A yog qhov teeb meem tsawg dua, thiab nsp9 UMPylation tsuas yog txo qis.Cov txiaj ntsig zoo sib xws tau txais hauv nsp9 NMPylation cov tshuaj tiv thaiv uas cuam tshuam nrog lwm cov NTPs (Daim duab 3D thiab SI appendix, Daim duab S3), lees paub tias cov txiaj ntsig pom ntawm qee cov amino acid hloov pauv yog ywj pheej ntawm hom nucleotide co-substrate siv.Tom ntej no, peb tau sim qhov muaj feem cuam tshuam ntawm cov nsp12 hloov pauv ntawm kev rov ua dua ntawm tus mob coronaviruses hauv kab lis kev cai ntawm tes.Txog rau qhov kawg no, peb tau siv cov qauv tsim kho caj ces tsim nyog ntxiv DNA (cDNA) cov qauv cloned hauv cov tshuaj tiv thaiv kab mob sib kis (23, 24) los sau 5 -7 hlwb.Titration ntawm tus kab mob kis tau tus kab mob uas tsim tawm hauv cov hlwb no tau pom tias feem ntau HCoV-229E NiRAN mutants tsis ua tau (Daim duab 3E).Ib pawg ntawm cov kab mob uas tsis tuaj yeem siv tau suav nrog lwm txoj hauv kev uas tau pom tias tshem tawm lossis txo qis NMP hloov pauv kev ua haujlwm hauv vitro (K4116A, K4135A, R4178A, D4188A, D4280A, D4283A), tab sis muaj ob txoj hauv kev (K4116R, E4805A) % tseg?Lawv cov haujlwm hauv vitro NMPylation qhia tias muaj kev txwv ntxiv.Ib yam li ntawd, ob qho kev hloov pauv (R4178K, F4281A) uas ua rau muaj kev txo qis hauv NiRAN's in vitro NMPylation kev ua haujlwm tsim cov kab mob nyob, txawm li cas los xij, cov kab mob no txo ​​qis titers los ntawm kev rov ua dua.Ua raws li cov ntaub ntawv hauv vitro ua haujlwm hauv daim duab 3D, hloov plaub lwm qhov seem uas tsis tau khaws cia hauv tus kabmob coronavirus thiab/lossis lwm yam kab mob zes (K4113A, D4180A, D4197A, D4273A) (8, 16) tsim cov kab mob uas siv tau rau cov xeeb ntxwv, txawm tias muaj ib qho kev txo qis qis dua piv rau cov kab mob qus (Daim duab 3E).

Txhawm rau kawm seb NiRAN-mediated NMP hloov pauv kev ua haujlwm nyob ntawm qhov nquag RdRp domain, ob qho kev khaws cia Asp residues koom nrog kev sib koom tes ntawm divalent hlau ions (11) hauv RdRp motif C tau hloov los ntawm Ala. Cov txiaj ntsig protein nsp12_DD4823/4AA khaws cia. nws txoj haujlwm nsp9 NMPylation, qhia tias nsp12-mediated hauv vitro nsp9 NMPylation kev ua haujlwm tsis tas yuav muaj cov haujlwm polymerase (SI Appendix, Daim duab S4).

Tom qab tsim cov nsp9 tshwj xeeb NMP hloov pauv kev ua haujlwm rau nsp12, peb tau sim ua tus yam ntxwv ntawm NMP-nsp9 adduct los ntawm huab hwm coj spectrometry (MS).Qhov ua tiav protein ntau spectrum ntawm recombinant HCoV-229E nsp9 pom qhov siab tshaj ntawm 12,045 Da (Daim duab 4A).Qhov sib ntxiv ntawm nsp12 tsis hloov qhov zoo ntawm nsp9, qhia tias nsp12 thiab nsp9 yuav tsis tsim ib qho chaw ruaj khov nyob rau hauv cov xwm txheej siv (denaturation) (Daim duab 4A).Nyob rau hauv lub xub ntiag ntawm UTP thiab GTP, qhov ntsuas loj ntawm cov tshuaj tiv thaiv uas muaj nsp9 thiab nsp12 feem pom tau hais tias cov protein ntau ntawm UTP tsiv 306 Da, thiab cov protein ntau ntawm GTP tsiv 345 Da, qhia tias txhua nsp9 molecule khi UMP lossis GMP. (Daim duab 4) C thiab D).Nws tau kwv yees tias lub zog xav tau rau NiRAN-mediated nsp9 NMPylation los ntawm NTP hydrolysis thiab pyrophosphate tso tawm.Txawm hais tias 10-fold molar ntau dhau ntawm nsp9 (lub hom phiaj) dua li nsp12 (enzyme) tau siv hauv cov tshuaj tiv thaiv no, yuav luag tiav NMPylation ntawm nsp9 tau pom, qhia tias kev sib cuam tshuam ntawm nsp12 thiab nsp9 yog luv luv, thiab nsp12 tuaj yeem NMPylate ntau nsp9. hauv vitro molecule.

Ib NMPylation ntawm nsp9 nyob rau hauv muaj nsp12 thiab UTP lossis GTP.Shown is the deconvoluted complete protein mass spectrum of HCoV-229E nsp9 (SI appendix, Table S1) (AD).(A) nsp9 ib leeg, (B) nsp9 + nsp12-His6, (C) nsp9 + nsp12-His6 nyob rau hauv lub xub ntiag ntawm UTP, (D) nsp9 + nsp12-His6 nyob rau hauv lub xub ntiag ntawm GTP.

Txhawm rau txiav txim siab nsp9 residues UMPylated los ntawm nsp12, nsp9-UMP tau cleaved nrog trypsin.Cov txiaj ntsig peptides tau sib cais los ntawm nano-high performance kua chromatography (HPLC) thiab soj ntsuam los ntawm tandem mass spectrometry (MS/MS) online.Cov ntaub ntawv tsom xam siv Byonic software pob (Protein Metrics) pom UMPylation ntawm N-terminal amino acid.Qhov no tau lees paub manually.Lub tandem loj spectrum ntawm precursor peptide [UMP] NNEIMPGK (SI appendix, daim duab S5A) qhia ib tug fragment ntawm 421 m/z, qhia tias UMP khi rau residue 1 ntawm nsp9.

Ntawm N-terminus ntawm nsp9, Asn tau khaws cia ntawm cov tswv cuab ntawm Orthocoronavirinae (SI appendix, Daim duab S6).Txawm hais tias peb ntseeg tias N-terminal thawj amine nitrogen yog qhov feem ntau yuav txais rau UMP, peb txiav txim siab kom tau txais cov pov thawj ntxiv ntawm NMP khi ntawm N-terminal.Vim li no, cov non-NMPylated thiab NMPylated N-terminal peptide nsp9 purified los ntawm HPLC tau muab los ntawm acetone thiab sodium cyanoborohydride.Raws li cov xwm txheej no, tsuas yog pub dawb thawj amines tuaj yeem hloov kho nrog propyl (25).N-terminal nsp9-derived peptide nrog cov kab ke NNEIMPGK muaj ob lub ntsiab amines, ib qho ntawm N-terminus ntawm Asn thiab lwm qhov ntawm cov saw ntawm Lys ntawm C-terminus.Yog li ntawd, propyl pawg tuaj yeem qhia tau ntawm ob qho kawg.Cov rho tawm ion chromatograms ntawm non-NMPylated peptides tau qhia hauv SI appendix, Daim duab S5B.Raws li qhov xav tau, N-terminal thiab C-terminal (mono) propylated (SI appendix, Daim duab S5B, kab sab sauv) thiab dipropylated peptides (SI appendix, Daim duab S5B, kab qis) tuaj yeem txheeb xyuas.Cov qauv no hloov nrog kev siv NMPylated N-terminal peptide ntawm nsp9.Hauv qhov no, tsuas yog C-terminal propylated peptides tuaj yeem txheeb xyuas tau, tab sis N-terminal propylated peptides thiab dipropylated peptides tsis raug txheeb xyuas (SI Appendix, Figure S5C), qhia tias UMP tau raug xa mus rau N-terminal thawj amine los tiv thaiv qhov no. pab pawg los ntawm kev hloov pauv.

Tom ntej no, peb hloov (nrog Ala lossis Ser) lossis tshem tawm cov khoom seem ntawm N-terminus ntawm nsp9 txhawm rau txheeb xyuas cov phiaj xwm tshwj xeeb.Raws li peb cov ntaub ntawv MS uas qhia tias NiRAN tsim ib qho nsp9-NMP adduct nrog cov thawj amine ntawm N-terminal residue ntawm nsp9, peb xav tias nsp9 NMPylation xav kom tus kab mob protease (Mpro, nsp5) tso tawm nsp9 N-terminal los ntawm nws cov polyprotein precursor.Txhawm rau ntsuas qhov kev xav no, peb tau tsim cov protein ua ntej nsp7-11 uas muaj nsp9 hauv E. coli thiab ua tus qauv NMPylation kuaj nyob rau hauv muaj [α-32P] UTP (cov ntaub ntawv thiab cov txheej txheem).Raws li pom nyob rau hauv daim duab 5A (txoj kab 3), lub uncut nsp7-11 precursor tsis radiolabeled nrog nsp12.Nyob rau hauv sib piv, yog nsp7-11 yog cleaved los ntawm recombinant nsp5 kom tso nsp9 (thiab lwm yam nsps) los ntawm lub precursor, ib tug radiolabeled protein uas migrate nrog nsp9 yog kuaj, paub meej tias peb xaus hais tias NiRAN thiab N- xaiv tsim covalent nsp9-NMP adducts. .Lub davhlau ya nyob twg thawj amine ntawm N-terminal Asn (txoj hauj lwm 3825 hauv pp1a/pp1ab).Qhov kev txiav txim siab no kuj tau txais kev txhawb nqa los ntawm kev sim siv nsp9 tsim, uas muaj ib lossis ob qhov seem ntxiv ntawm N-terminus.Hauv ob qhov xwm txheej, NiRAN-mediated UMPylation ntawm nsp9 raug tshem tawm (SI Appendix, Daim duab S7).Tom ntej no, peb tsim cov protein nrog ib lossis ob qho Asn residues tshem tawm los ntawm 3825-NNEIMPK-3832 peptide ntu ntawm N-terminal ntawm nsp9.Hauv ob qho xwm txheej, nsp9 UMPylation raug thaiv tag nrho (Daim duab 5B), muab pov thawj ntxiv tias qhov tseeb nsp9 N-terminus ua raws li NMP receptor.

Kev ua cov proteolytic ntawm nsp9 thiab lub luag haujlwm ntawm N-terminal residues hauv nsp12-mediated UMPylation.(A) nsp9 UMPylation yuav tsum muaj nsp9 N-terminal dawb.Nsp7-11-His6 yog pre-incubated ntawm 30 ° C nyob rau hauv NMPylation nrhiav tsis pom muaj UTP nyob rau hauv lub xub ntiag los yog tsis muaj recombinant Mpro (nsp5-His6).Tom qab 3 teev, pib qhov kev ntsuam xyuas NMPylation los ntawm kev ntxiv nsp12-His6 raws li tau piav qhia hauv Cov Khoom Siv thiab Cov Txheej Txheem.Cov tshuaj tiv thaiv uas muaj nsp5-His6 (txoj kab 1) thiab nsp9-His6 (txoj kab 2) tau siv los ua kev tswj hwm.Tom qab 10 feeb, cov tshuaj tiv thaiv raug txiav tawm thiab cov tshuaj tiv thaiv sib xyaw tau sib cais los ntawm SDS-PAGE.Cov protein tau stained nrog Coomassie Brilliant Blue (A, saum).Lub Nsp7-11-His6 precursor thiab cov khoom ua tiav los ntawm nsp5-His6 mediated cleavage yog qhia ntawm sab xis.Thov nco ntsoov (vim lawv qhov me me) uas nsp7 thiab nsp11-His6 tsis tuaj yeem kuaj pom hauv cov gel no, thiab cov tshuaj tiv thaiv ntxiv nrog nsp5-His6 (txoj kab 1 thiab 4; txoj hauj lwm ntawm nsp5-His6 yog qhia los ntawm lub voj voog ruaj khov) los yog nsp9-His6 (Txoj kab 2) muaj me me ntawm MBP (qhia los ntawm cov voj voog qhib) raws li cov khoom tsis huv vim lawv tau qhia tias yog MBP fusion proteins (SI appendix, Table S1).(B) Nsp9-His6 variant tsis muaj ib lossis ob lub N-terminal Asn residues (residue numbering raws li txoj hauj lwm hauv pp1a/pp1ab) thiab yog purified thiab incubated nrog nsp12-His6 thiab [α-32P] UTP.B, SDS-PAGE stained nrog Coomassie yog pom nyob rau sab saum toj, B, cov autoradiograph coj mus rau hauv qab.Txoj hauj lwm ntawm qhov ntsuas qhov hnyav molecular (hauv kilodaltons) yog qhia rau sab laug.(C) HCoV-229E nsp9-His6 N-terminal conserved residues tau hloov nrog Ala lossis Ser, thiab tib cov protein tau siv hauv nsp12-His6 mediated UMPylation cov tshuaj tiv thaiv.Cov khoom siv tshuaj tiv thaiv tau muab cais los ntawm SDS-PAGE thiab stained nrog Coomassie Brilliant Blue (C, saum), thiab radiolabeled nsp9-His6 tau kuaj pom los ntawm phosphorescence imaging (C, nruab nrab).Siv cov tsiaj qus (wt) protein raws li kev siv (teeb rau 100%), cov txheeb ze NMPylation kev ua (txhais tau tias ± SEM) raug xam los ntawm peb qhov kev sim ywj pheej.(D) Tus kab mob titers nyob rau hauv p1 cell kab lis kev cai supernatant ntawm Huh-7 hlwb kis nrog HCoV-229E wild-type Huh-7 cells, thiab mutants nqa cov amino acid hloov pauv hauv nsp9 tau txiav txim siab los ntawm cov tshuaj plaque.Lub replication-deficient RdRp motif C ob chav mutant DD4823/4AA tau siv los ua kev tswj tsis zoo.

Lub N-terminus ntawm nsp9 (tshwj xeeb tshaj yog txoj haujlwm 1, 2, 3, thiab 6) yog kev txuag ntawm cov tswv cuab ntawm Orthocoronavirinae subfamily (SI appendix, Daim duab S6).Txhawm rau kawm txog lub luag haujlwm ua tau ntawm cov residues hauv nsp12-mediated nsp9 NMPylation, ob qho tib si Asn residues ntawm N-terminus ntawm nsp9 tau hloov nrog Ala lossis Ser (ib leeg lossis ua ke).Piv nrog cov tsiaj qus nsp9, hloov N3825 nrog Ala lossis Ser ua rau ntau tshaj li ob npaug ntawm kev txo qis hauv nsp12-mediated UMPylation (Daim duab 5C).Raws li peb qhov kev txiav txim siab tias NMPylation tshwm sim ntawm N-terminal thawj amine es tsis txhob ntawm sab saw ntawm N-terminal residue, peb tau pom qhov tseem ceeb ntawm NMPylation nrog kev hloov ntawm N3825A thiab N3825S.Interestingly, yog tias qhov thib ob Asn hloov los ntawm Ala lossis Ser, nsp9 UMPylation raug txo qis dua (ntau dua 10 zaug), thaum qhov hloov ntawm Ala ntawm txoj haujlwm 3, 4, thiab 6 tsuas muaj qhov cuam tshuam nruab nrab ntawm nsp9 UMPylation (Daim duab 2. )5 C).Cov txiaj ntsig zoo sib xws tau txais los ntawm ATP, CTP lossis GTP (SI appendix, Figure S8).Ua ke, cov ntaub ntawv no qhia txog lub luag haujlwm tseem ceeb ntawm N2826 (txoj hauj lwm 2 hauv nsp9) hauv nsp9 NMPylation.

Txhawm rau kom tau txais cov ntaub ntawv pov thawj ntxiv ntawm kev ua haujlwm sib raug zoo ntawm N-qhov kawg ntawm nsp9 thiab NMPylation, peb tau ua ntau qhov sib xyaw ua ke (MSA) ntawm nsp9 ib ntus ntawm tsev neeg Coronavirus (sib txawv ntawm 104 thiab 113 seem) (SI Appendix, Daim duab S6).Nyob rau hauv tag nrho, nyob rau hauv 47 (paub thiab putative) hom ntawm 5 genera ntawm lub Orthocoronavirinae subfamily uas kis cov tsiaj sib txawv, noog, thiab cov tsiaj reptile hosts, tsuas yog 8 seem nyob rau hauv tag nrho tau pom tias yog invariant.Cov kev hloov pauv loj tshaj plaws, suav nrog kev tshem tawm thiab kev tso tawm, tau pom nyob rau hauv lub voj voog ntawm cov txheej txheem thib ob ntawm nsp9, raws li kev txiav txim los ntawm cov kev tshawb fawb yav dhau los (26 ??28).Tsib invariant residues tau pom nyob rau hauv β strand thiab α helix ntawm C-terminal ib feem ntawm nsp9.Peb qhov seem tsis sib xws yog NNE motif ntawm N terminus ntawm nsp9.Nws tau tshaj tawm tias qhov thib ob Asn ntawm qhov motif no tsuas yog qhov tsis sib xws, uas tseem tau sib koom los ntawm qhov kev xav ntawm nsp9 ntawm tus kab mob sib kis nyob deb, thiab sawv cev rau Microhyla letovirus 1 hom hauv subfamily Letovirinae ntawm Alphaletovirus.Kev txuag ntawm cov residues hauv nsp9 cov txheej txheem thib ob tuaj yeem ua rau muaj kev cuam tshuam los ntawm cov qauv kev txiav txim siab kom tswj tau folding lossis paub txog RNA khi cov khoom.Txawm li cas los xij, qhov laj thawj no tsis zoo li siv rau kev txuag ntawm NNE, thiab ua ntej txoj kev tshawb fawb no, qhov xwm txheej ntawm kev txwv uas txwv qhov kev hloov pauv ntawm tripeptide sib lawv liag tau pom tseeb.

Txhawm rau txiav txim siab qhov tseem ceeb ntawm nsp9-NMPylation thiab NNE kev txuag hauv tus kabmob coronavirus, peb tsim HCoV-229E mutants, uas nqa ib lossis ob npaug ntawm nsp9 N-terminal residues, qhia tias nsp9 NMPylation muaj kev phom sij hauv vitro.Ua ntej peb pib, peb sim teb cov lus nug seb puas muaj cov kev hloov pauv no (ze ntawm nsp8|9 cleavage site) cuam tshuam rau kev ua haujlwm proteolytic ntawm C-terminal pp1a cheeb tsam.Ib txheej ntawm nsp7-11 polyprotein constructs uas muaj kev hloov pauv ntawm N-terminus ntawm nsp9 tau tsim nyob rau hauv E. coli thiab txiav nrog recombinant Mpro.Lub proteolytic cleavage ntawm plaub qhov chaw (xws li nsp9 flanking site) tsis cuam tshuam los ntawm ib qho kev hloov pauv (SI appendix, daim duab S9), tsis suav nrog cov qauv kev hloov pauv hauv cov proteins uas cuam tshuam nrog Mpro-mediated nsp8|9 cleavage (lossis lwm yam) lub vev xaib.

Huh-7 hlwb tau kis nrog genome-ntev HCoV-229E RNA, encoding Ala lossis Ser hloov pauv hauv kev khaws cia NNE tripeptides (N3825, N3826, thiab E3827) ntawm nsp9 N terminus, uas qhia tias feem ntau ntawm kev hloov pauv yog tuag taus.Peb muaj peev xwm cawm tau tus kab mob los ntawm kev hloov Ser lossis Ala ntawm N-terminal Asn (N2835A lossis N2835S), tab sis ua tsis tiav tus kab mob nrog rau lwm qhov kev hloov pauv ib zaug thiab ob zaug hauv NNE ntu (N3826A, N3826S, NN3825/6AA, NN3825/6SS), E3827A) (Daim duab 5D).

Cov txiaj ntsig no qhia tau hais tias kev rov ua dua ntawm tus kabmob coronaviruses hauv cov ntaub so ntswg kab lis kev cai yog txwv (tib yam lossis zoo sib xws), txwv kev hloov pauv ntuj ntawm nsp9 NMPylation chaw hauv lub cev, thiab txhawb lub luag haujlwm tseem ceeb ntawm cov lus teb no hauv lub neej ntawm tus kabmob coronaviruses.

Hauv cov txheej txheem kawg ntawm kev sim, peb tsim C-terminal His6 sau npe SARS-CoV-2 nsp12 thiab nsp9, thiab ob hom mutant ntawm nsp12 hauv E. coli.Lub vev xaib nquag nyob hauv NiRAN thiab RdRp domains tau siv Ala hloov pauv (Daim duab 6A thiab SI appendix, Table S2).K4465 in SARS-CoV-2 nsp12 correspons to K4135 in HCoV-229E (SI Appendix, Figure S2), which proved to be required for NiRAN activity and HCoV-229E replication (Figure 3D and E).Cov residue no kuj sib xws rau cov kab mob arterial EAV nsp9 K94 residue, uas yav tas los pom tias tsim nyog rau NiRAN tus kheej-UMPylation/self-GMPylation (16).Raws li pom nyob rau hauv daim duab 6B, SARS-CoV-2 nsp12 muaj UMP transferase kev ua si siv nsp9 ua substrate, thaum lub nsp12_K4465A active site mutant yog inactive.Qhov kev hloov pauv ob zaug hauv SDD tus yam ntxwv ua ntu zus ntawm RdRp motif C tsis cuam tshuam rau UMP kev hloov pauv kev ua haujlwm (Daim duab 6B), qhia tias kev ua haujlwm RdRp tsis muaj kev cuam tshuam ncaj qha hauv nsp9 UMPylation.Cov ntaub ntawv zoo sib xws tau txais los ntawm CTP, GTP thiab ATP (SI appendix, Figure S10).Hauv cov ntsiab lus, cov ntaub ntawv no qhia tias NiRAN-mediated nsp9 NMPylation muaj kev saib xyuas kev ua haujlwm hauv tus kabmob coronaviruses sawv cev rau ntau hom kab mob orthocoronavirus subfamily.

SARS-CoV-2 nsp12-mediated NMPylation of nsp9.(A) Coomassie stained SDS-polyacrylamide gel qhia txog cov protein sib xyaw siv hauv NMPylation test.Raws li kev tswj hwm, mutant protein nrog qhov chaw hloov chaw nyob hauv NiRAN domain (K4465A) thiab RdRp domain (DD5152/3AA) ntawm SARS-CoV-2 nsp12 tau siv.Residue numbering yog nyob ntawm txoj hauj lwm hauv pp1ab.(B) Autoradiograph ntawm UMPylation nrhiav kom tau siv nsp9-His6 thiab [α-32P]UTP ua substrate ntawm nsp12-His6 (hloov hom [wt] thiab mutant).Cov molecular mass (hauv kilodaltons) ntawm cov ntawv sau npe protein yog qhia rau sab laug.

NiRAN domains feem ntau tau txais kev cawmdim hauv Nidovirales (16), qhia tias lawv catalyze enzymatic cov tshuaj tiv thaiv tseem ceeb rau Nidovirus replication.Hauv txoj kev tshawb no, peb muaj peev xwm ua pov thawj tias NiRAN domain ntawm tus kabmob coronavirus hloov NMP (tsim los ntawm NTP) mus rau nsp9, ib qho tsis paub RNA khi cov protein koom nrog cov kab mob rov ua dua tshiab (26 ?? 29), los txiav txim tias nws yog lub hom phiaj ntuj thiab tus khub ntawm tus mob coronavirus RTC.

Lub NiRAN domain sib koom peb ntu motifs (AN, BN, thiab CN), uas muaj tsawg tsawg ntawm cov seem uas tau khaws cia hauv txhua tsev neeg hauv monophyletic tab sis txawv heev Nidovirales xaj (8, 16).Cov kev tshawb fawb tsis ntev los no tau pom tias lawv muaj feem cuam tshuam nrog cov tsev neeg tsis zoo ntawm cov protein kinase-zoo li cov proteins, uas yog hu ua SelO tsev neeg (17, 19, 22, 30, 31).SelO-related proteins muaj kinase folds, tab sis tsis muaj ob peb qhov chaw nyob ruaj khov nyob rau hauv classical kinases (22, 32).Raws li qhov rov qab taw qhia ntawm ATP molecules khi rau qhov chaw nquag thiab ruaj khov los ntawm kev sib cuam tshuam tshwj xeeb, SelO tau pom zoo thiab tom qab ntawd tau lees paub kom hloov AMP (es tsis yog phosphate) mus rau cov protein substrate (22), thaum lwm cov kab mob SelO-zoo li protein YdiU muaj. Tsis ntev los no tau pom tias catalyze cov lus txuas ntawm UMP rau Tyr thiab Nws cov seem ntawm cov protein sib txawv (33).

Txhawm rau kom paub meej thiab nthuav dav qhov kev kwv yees ntawm qhov chaw ua haujlwm ntawm qhov chaw seem ntawm tus mob coronavirus NiRAN sau, peb tau siv cov tshuaj biochemical thiab rov qab cov noob caj noob ces los ua kev hloov pauv ntawm tus kabmob coronavirus nsp12 (Daim duab 3D thiab E thiab SI daim ntawv ntxiv, Daim duab S3 thiab cov lus) S1â S4).Cov ntaub ntawv qhia tau hais tias kev hloov ntawm HCoV-229E K4135, R4178 thiab D4280 nrog Ala tshem tawm hauv vitro NMP kev hloov pauv kev ua haujlwm thiab tus kab mob rov ua dua tshiab hauv kab lis kev cai ntawm tes (Daim duab 3D thiab E thiab SI appendices, Daim duab S3), txhawb nqa lawv qhov muaj nyob hauv NTP γ-phosphate. (K4135, R4178) thiab kev sib koom tes ntawm qhov chaw ua haujlwm hlau ions (D4280).Kev hloov pauv E4145A ntawm kev txuag Glu nyob rau hauv thaj tsam ntawm tus kab mob zes noog tau kwv yees kom ruaj khov K4135 (17) txoj haujlwm tau pom tias tshem tawm cov kab mob sib kis, tab sis xav tsis thoob, cov haujlwm tau khaws cia hauv vitro NMPylation assay (Daim duab 3D thiab E thiab SI appendix, Daim duab S3 Thiab Tables S1–S4).Ib qho kev soj ntsuam zoo sib xws tau ua thaum qhov sib hloov pauv tau qhia hauv YdiU homolog ntawm Salmonella typhimurium (E130A) (33).Ua ke, cov ntaub ntawv no txhawb kev tswj hwm kev ua haujlwm ntawm qhov kev khaws cia ntawm qhov seem es tsis yog lub luag haujlwm catalytic.

Hloov cov chaw khaws cia Phe residue (F4281A) nyob rau hauv thaj tsam ntawm nestovirus hauv HCoV-229E NiRAN domain (8) ua rau txo qis hauv NMPylation kev ua hauv vitro thiab txo qis ntawm tus kab mob rov ua dua tshiab hauv cell culture (Daim duab 3D, E thiab SI) daim ntawv ntxiv, daim duab S3).Cov ntaub ntawv yog ua raws li cov kev cai tseem ceeb ntawm cov residue no, xws li homologous DFG motif Phe residue tau qhia yav dhau los.Hauv classical protein kinases, nws yog ib feem ntawm Mg2 + khi lub voj thiab pab sib sau ua ke thiab tswj tus txha nraub qaum???Yuav tsum tau ua kom muaj txiaj ntsig catalytic (32, 34).Hloov Ala thiab Arg rau K4116 residues (hauv preAN motif), feem, tshem tawm cov kab mob sib kis thiab, raws li xav tau, muaj qhov sib txawv ntawm NMP transferase kev ua hauv vitro, nyob ntawm cov amino acid sab saw tau qhia (Daim duab 3D Thiab E thiab SI appendices , Daim duab S3).Cov ntaub ntawv ua haujlwm tau zoo raws li cov ntaub ntawv tsim qauv, qhia tias qhov seem no tau tsim muaj kev cuam tshuam nrog ATP phosphate (17).Hauv NiRAN domain ntawm lwm cov tsev neeg kab mob zes, txoj hauj lwm ntawm HCoV-229E pp1a/pp1ab K4116 yog nyob ntawm Lys, Arg lossis His (8), qhia tias qhov kev txwv tsis pub ua haujlwm ntawm cov khoom seem tshwj xeeb no tau so.Kev hloov pauv ntawm D4188A thiab D4283A tshem tawm lossis txo qis kev ua haujlwm enzyme thiab tshem tawm cov kab mob rov ua dua tshiab (Daim duab 3).Ob qhov seem no tau khaws cia rau hauv feem ntau (tab sis tsis yog tag nrho) cov kab mob zes (8), qhia txog tsev neeg tseem ceeb tab sis tej zaum tsis yog-catalytic muaj nuj nqi.Ala hloov ntawm ob peb lwm Lys thiab Asp residues (K4113A, D4180A, D4197A thiab D4273A) uas tsis tau khaws cia hauv Coronaviridae lossis lwm tsev neeg Nestioviridae (8) tau siv los tswj.Raws li qhov xav tau, cov kev hloov pauv no tuaj yeem zam tau ntau, nrog rau kev txo qis hauv enzyme kev ua thiab kis kab mob hauv qee kis (Daim duab 3 thiab SI appendix, Daim duab S3).Zuag qhia tag nrho, cov ntaub ntawv coronavirus mutagenesis zoo ib yam nrog tus kheej-GMP thiab cov ntaub ntawv rov qab cov noob caj noob ces ntawm EAV NiRAN-RdRp (16), uas EAV nsp9 (coronavirus nsp12 ortholog) residue K94 (suav nrog HCoV- 229E K4135) cov haujlwm tseem ceeb), R124 (xws li R4178), D132 (sib haum rau D4188), D165 (suav rau D4280), F166 (suav rau F4281).Tsis tas li ntawd, cov ntaub ntawv HCoV-229E mutagenesis yog ua raws thiab nthuav dav los ntawm yav dhau los tau tshaj tawm SARS-CoV cov ntaub ntawv rov qab cov noob caj noob ces (16), ib yam li zoo ib yam li cov uas tau pom rau qhov sib thooj CN motif Phe-to-Ala mutant SARS-CoV_nsp12. phenotype piav -F219A and HCoV-229E_F4281A (Figure 3 D and E and SI appendix, Figure S3 and Table S1-S4).

Piv nrog rau EAV orthologs (16), uas muaj qhov kev nyiam meej rau UTP thiab GTP (hauv tus kheej-NMPylation cov tshuaj tiv thaiv), peb txoj kev tshawb fawb pom tias tus mob coronavirus NiRAN domain ( sawv cev los ntawm HCoV-229E thiab SARS-CoV-2) tuaj yeem ua tau zoo. Hloov tag nrho plaub NMPs, txawm hais tias muaj kev nyiam me ntsis rau UMP (Figures 1 thiab 3).Qhov tshwj xeeb qis ntawm qhov tshwj xeeb NTP co-substrate yog raws li qhov tsis ntev los no tau tshaj tawm SARS-CoV-2 nsp7/8/12/13 supercomposite qauv, uas ADP-Mg2+ khi rau qhov chaw nquag ntawm NiRAN, tab sis tsis nrog adenine Part. ntawm kev tsim cov kev sib tshuam tshwj xeeb (17).Hauv peb txoj kev tshawb fawb, hom nucleotide siv hauv NMPylation cov tshuaj tiv thaiv tsis muaj qhov sib txawv ntawm kev ua haujlwm ntawm cov protein mutant (SI Appendix, Figure S3), qhia tias tsis muaj ib qho ntawm cov residues no cuam tshuam nrog kev khi ntawm ib qho nucleobase tshwj xeeb.Lub hauv paus txheej txheem thiab muaj peev xwm ua kom muaj roj ntsha tseem ceeb ntawm qhov sib txawv NTP co-substrate nyiam tau pom nyob rau hauv NiRAN domains ntawm tus mob coronaviruses thiab cov kab mob hlab ntsha tseem yuav tau kawm;tej zaum lawv yuav muaj tseeb lossis tej zaum yuav yog vim muaj kev txwv ntawm lawv cov kev tshawb fawb.Tam sim no, nws tsis tuaj yeem txiav txim siab tias qhov muaj peev xwm NMPylator kev ua haujlwm ntawm cov kab mob hlab ntsha NiRAN (piv rau yav dhau los tus cwj pwm ntawm tus kheej-NMPylation kev ua haujlwm) muaj qhov sib txawv co-substrate nyiam, suav nrog qhov zoo sib xws ntawm cov hlab ntsha thiab tus kabmob coronavirus. NiRAN domain yog nyob ntawm nws qhov txwv.Sib piv-raws li kev sib piv (16).Piv nrog cov pseudokinase SelO, uas siv Mg2+ ua cofactor, kev ua haujlwm ntawm tus mob coronavirus thiab kab mob arterial NiRAN yog nyob ntawm Mn2+ (16) (Daim duab 3C thiab SI appendix, Daim duab S1).Mn2 + kev vam khom thiab pom tseeb nyiam rau UTP yog qhov txawv txav ntawm cov protein NMPylators, thiab tsuas yog tsis ntev los no tau lees paub hauv YdiU protein ntawm Salmonella typhimurium, uas catalyzes nruj Mn2 +-nyob ntawm protein chaperone UMPylation los tiv thaiv hlwb los ntawm kev ntxhov siab induction Cell ATP pas dej ua ke ( 33).

Tsis ntev los no tau piav qhia txog cov qauv zoo sib xws ntawm tus mob coronavirus NiRAN domain thiab cellular protein kinases (17, 19) muab kev txhawb nqa ntxiv rau NiRAN lub peev xwm los txuas NMP rau lwm cov proteins uas peb tau tshaj tawm hauv txoj kev tshawb no.Peb tsom peb txoj kev tshawb fawb txog NiRAN lub hom phiaj ntawm cov proteins uas tau txais los ntawm HCoV-229E ORF1a, uas paub txog kev pab ncaj qha lossis ncaj qha rau RTC's ORF1b-encoded replicase (12, 35).Peb cov kev sim muab pov thawj tseeb rau qhov ua tau zoo thiab tshwj xeeb NMPylation ntawm nsp9 (Daim duab 2).Yog hais tias lub hom phiaj protein yog siv nyob rau hauv molar ntau tshaj uas yog 8 mus rau 10 lub sij hawm siab tshaj ntawm cov enzyme (nsp12), nws tau lees paub tias nsp9 yog kiag li (mono)NMPized (Daim duab 4).Peb xaus lus tias kev sib cuam tshuam ntawm nsp12 thiab nsp9 yog luv luv thiab yuav tsis tsim ib qho chaw ruaj khov nrog nsp9 (thaum tsis muaj lwm yam RTC subunits).Qhov kev txiav txim siab no tau txhawb nqa los ntawm kev tshawb fawb txog kev sib cuam tshuam ntawm cov protein ntawm SARS-CoV proteome (35).MS tsom xam pom thawj amine ntawm N-terminal residue ntawm nsp9 raws li NMPylation site (SI appendix, Daim duab S5).Kev tsim ntawm phosphoramidate daim ntawv cog lus thiab N-terminal amino pawg paub qhov txawv ntawm NiRAN-mediated NMPylation kev ua los ntawm Pseudomonas syringae SelO-mediated AMPylation cov tshuaj tiv thaiv, uas catalyzes tsim O-txuas AMP ntawm Ser, Thr, los yog Tyr residues Peptide adduct ( 22), thiab S. typhimurium YdiU cov ntaub ntawv O-txuas (nrog Tyr) thiab N-txuas (nrog Nws) peptide-UMP adducts.Cov ntaub ntawv txwv tsis pub muaj nyob ntawm SelO tsev neeg cov proteins qhia tias cov tswv cuab ntawm tsev neeg cov protein loj no txawv heev hauv kev tsim peptide-NMP adducts.Qhov no yog ib qho kev soj ntsuam nthuav dav uas tsim nyog kawm ntxiv.

Cov ntaub ntawv tau txais hauv txoj kev tshawb no coj peb mus rau kev xav tias NMPylation ntawm nsp9 yuav tsum muaj N-terminus dawb.Nyob rau hauv cov ntsiab lus ntawm tus kab mob replication, qhov no yuav muab los ntawm proteolytic cleavage ntawm nsp8|nsp9 ua chaw nyob rau hauv lub replicase polyprotein pp1a kho los ntawm Mpro thiab pp1ab.Hauv cov kab mob coronaviruses feem ntau, qhov sib txawv ntawm qhov chaw tshwj xeeb no (VKLQ | NNEI hauv HCoV-229E) thiab tag nrho lwm qhov chaw mob coronavirus Mpro cleavage yog Asn (tsis yog lwm qhov chaw me me, xws li Ala, Ser Or Gly) tuav P1â???Qhov chaw (36).Cov ntaub ntawv peptide cleavage tau txais nyob rau hauv cov kev tshawb fawb thaum ntxov qhia tau hais tias qhov cleavage efficiency ntawm nsp8|nsp9 qhov chaw yog qis dua li ntawm lwm qhov chaw, qhia tias 1) qhov chaw tshwj xeeb no yuav muaj lub luag haujlwm tswj xyuas kev ua haujlwm raws sijhawm ntawm C-terminal. pp1a cheeb tsam, los yog 2) lub luag hauj lwm ntawm tshwj xeeb conserved nsp9 N-terminus nyob rau hauv tus kab mob replication (37).Peb cov ntaub ntawv (Daim duab 5A) tau pom tias daim ntawv recombinant ntawm nsp9 nqa qhov tseeb N-terminal sib lawv liag tau zoo NMPized los ntawm nsp12.N-terminal flanking sequence raug tshem tawm los ntawm yam Xa (nsp9-His6; SI appendix, Table S1) los yog Mpro-mediated cleavage (nsp7-11-His6; Daim duab 5A thiab SI appendix, Table S1).Qhov tseem ceeb, qhov tsis raug nsp9-muaj precursor nsp7-11-His6 tau pom kev tiv thaiv rau NMPylation ntawm nsp12, uas zoo ib yam nrog peb cov ntaub ntawv, qhia tias nsp9-NMP adduct yog tsim los ntawm N-terminal thawj amine (SI appendix, Daim duab S5) .Txhawm rau kom nkag siab tob dua ntawm NiRAN substrate tshwj xeeb, peb mam li tsom mus rau N-terminal residues uas nyob ib sab ntawm nsp9.Thaum tsis muaj lwm cov proteins, lawv muaj cov qauv hloov tau yooj yim, tiv thaiv lawv los ntawm kev kuaj pom hauv daim ntawv tsis muaj npe ntawm nsp9 (26 28, 38), qhia txog lawv qhov kev hloov pauv tsis zoo no yog vim qhov tseem ceeb ntawm cov kab ke tshwj xeeb (tsis yog cov qauv thib ob ntsig txog) kev ua haujlwm ntawm nsp9 N-terminal fragment.Ala hloov pauv ntawm kev khaws cia hauv thaj av no (Daim duab 5C thiab D thiab SI daim ntawv txuas ntxiv, Daim duab S8) qhia tias N3826 yog qhov tseem ceeb rau nsp9 NMPylation hauv vitro, thaum N3825A thiab E3827A hloov pauv ua rau txo qis hauv NMPylation, thaum M3829A thiab P383 tsis hloov pauv. .Obviously cuam tshuam nsp9 NMPylation.Txawm hais tias kev hloov pauv ntawm N-terminal Asn (N3825A, N3825S) tsuas muaj qhov cuam tshuam nruab nrab ntawm nsp9 NMPylation thiab tus kab mob rov ua dua tshiab hauv kab lis kev cai ntawm tes (Daim duab 5C thiab D), kev tshem tawm ntawm Asn seem seem ntawm N-terminal 3825-NN dipeptide. ua pov thawj tias Nws yog qhov tuag taus rau cov kab mob, qhia tias yuav tsum muaj ib qho Asn residue ua ntej lwm qhov seem ntawm N-terminus, zoo dua Asn, txawm hais tias nws zoo li kev hloov pauv ntawm cov seem zoo sib xws tuaj yeem zam tau qee qhov (Daim duab 5B, C, thiab D).Peb xaus lus tias 3825-NN dipeptide, tshwj xeeb tshaj yog kev txuag thiab tseem ceeb N3826 residue nyob rau hauv cov kab mob coronavirus (SI appendix, Daim duab S6), xyuas kom meej qhov raug khi thiab taw qhia ntawm nsp9 N-terminus nyob rau hauv lub active site ntawm NiRAN.

Hloov Ala (E3827A) rau kev txuag Glu ntawm txhua tsev neeg subfamilies khaws nsp9 NMPylation hauv vitro tab sis ua rau tuag taus rau cov kab mob hauv cell kab lis kev cai (Daim duab 5C thiab D), qhia txog kev ua haujlwm ntxiv ntawm qhov seem no, piv txwv li, hauv kev sib cuam tshuam tseem ceeb (NMPylated lossis tsis hloov kho. ) nsp9 N-terminus thiab lwm yam cuam tshuam nrog tus kab mob replication.Nsp9 kev hloov pauv tsis cuam tshuam rau cov txheej txheem proteolytic ntawm nsp9 lossis ib qho nsps uas nyob ib sab (39) (SI Appendix, Daim duab S9), qhia tau hais tias kev tuag phenotypes ntawm ntau nsp9 kev hloov pauv tau pom tsis yog tshwm sim los ntawm dysregulation ntawm C proteolytic txheej txheem-terminal pp1a cheeb tsam. .

Cov ntaub ntawv saum toj no muab pov thawj tias tom qab kev kho mob kho mob Mpro ntawm nsp8|9 qhov chaw cleavage hauv pp1a/pp1ab, N-terminus ntawm nsp9 tuaj yeem UMPylated (lossis hloov ib nrab nrog lwm NMP).Tsis tas li ntawd, kev txuag tau zoo ntawm N-terminus ntawm nsp9 (xws li cov lus thiab qhov tsis sib xws ntawm Asn residues hauv tsev neeg mob coronavirus) thiab cov ntaub ntawv rov qab cov noob caj noob ces tau txais hauv txoj kev tshawb no (Figures 3E thiab 5D) coj peb los xaus lus tias cov lus piav qhia nsp9 NMPylation yog kev lom neeg muaj feem cuam tshuam thiab tseem ceeb rau tus mob coronavirus replication.Cov txiaj ntsig ua haujlwm ntawm qhov kev hloov kho no tseem yuav tau kawm, piv txwv li, hais txog cov lus piav qhia yav dhau los (tsis yog tshwj xeeb) nsp9 (tsis hloov kho) RNA binding kev ua (2628).N-terminal NMPylation kuj tseem tuaj yeem cuam tshuam rau kev sib cuam tshuam ntawm nsp9 nrog cov protein lossis RNA substrates lossis tsim ntawm plaub theem sib txawv.Cov no tau pom nyob rau hauv cov kev tshawb fawb txog kev tsim qauv thiab tau lees paub tias muaj feem cuam tshuam txog tus kabmob coronavirus, txawm hais tias tshwj xeeb tshaj yog thaum tsis muaj qhov hloov kho no (26- â 29, 40).

Txawm hais tias lub hom phiaj tshwj xeeb ntawm tus kab mob coronavirus NiRAN tseem yuav tsum tau ua kom nthuav dav ntxiv, peb cov ntaub ntawv qhia tau hais tias cov protein lub hom phiaj tshwj xeeb ntawm tus mob coronavirus NiRAN domain yog nqaim heev.Txawm hais tias kev txuag ntawm qhov chaw tseem ceeb ntawm qhov chaw seem (8, 16) hauv NiRAN domain ntawm tag nrho cov tsev neeg nidovirus txhawb nqa kev ua haujlwm ntawm kev txuag NMPylator cov proteins no, tus kheej ntawm lub substrate binding hnab tshos residues ntawm no domain Kev txuag thiab kev txuag tseem yuav tsum tau tus cwj pwm. , thiab tej zaum yuav txawv ntawm cov tsev neeg sib txawv ntawm Nidovirales lub hom phiaj.Ib yam li ntawd, cov hom phiaj tseem ceeb ntawm lwm cov kab mob zes tseem tsis tau txiav txim siab.Tej zaum lawv yuav yog tej thaj chaw deb orthologs ntawm nsp9 los yog lwm yam proteins, vim hais tias cov sequences sab nraum lub tsib replicase domains uas feem ntau tau txais kev cawmdim nyob rau hauv nested viruses yog tsawg conserved (8), nrog rau cov genome array ntawm Mpro thiab NiRAN, Ntawm lawv, nsp9 nyob rau hauv lub khau laus nas vais lav.

Tsis tas li ntawd, tam sim no peb tsis tuaj yeem txiav txim siab qhov muaj peev xwm uas NiRAN sau muaj ntxiv (suav nrog cellular) lub hom phiaj.Nyob rau hauv cov ntaub ntawv no, nws yog tsim nyog hais tias cov kab mob homologues nyob rau hauv no tshwm sim protein NMPylators (NMPylators) (30, 31) zoo li muaj "master regulators"?NMP hloov kho ntau yam cellular proteins los tswj lossis tshem tawm lawv cov dej num hauv qab no, yog li ua lub luag haujlwm hauv ntau yam txheej txheem lom neeg, xws li cov lus teb ntawm cellular stress thiab redox homeostasis (22, 33).

Nyob rau hauv txoj kev tshawb no (Daim duab 2 thiab 4 thiab SI Appendix, Figures S3 thiab S5), peb muaj peev xwm ua pov thawj tias nsp12 pauv UMP (NMP) ib feem mus rau ib qho (txhawb) txoj hauj lwm nyob rau hauv nsp9, thaum lwm yam proteins tsis tau hloov nyob rau hauv lub siv nyob rau hauv cov xwm txheej, zoo-txhais (tsis yog xoob) substrate tshwj xeeb yog txaus siab.Raws li qhov no, piv nrog N-terminal nsp9 NMPylation, nsp12's tus kheej NMPylation kev ua haujlwm tsawg heev, nws qhov kev tshawb pom yuav tsum tau siv sijhawm ntev autoradiography raug, thiab 10-fold nce hauv nsp12 concentration yog siv.Tsis tas li ntawd, peb qhov kev tshuaj ntsuam MS ua tsis tiav los muab pov thawj rau NMPylation ntawm nsp12, uas qhia tias NiRAN tus kheej-NMPylation yog (qhov zoo tshaj plaws) ib qho kev ua haujlwm thib ob.Txawm li cas los xij, nws yuav tsum tau muab sau tseg tias lwm cov kev tshawb fawb tau muab cov pov thawj ua ntej tias tus kheej-AMPylation ntawm cov kab mob NMPylator tuaj yeem tswj hwm lawv cov haujlwm NMPylation ntawm lwm cov protein substrates (22, 33).Yog li, kev tshawb fawb ntxiv yog xav tau los tshawb xyuas qhov ua tau zoo ntawm tus kheej-NMPylation cov dej num tau tshaj tawm rau EAV nsp9 (16) thiab tus mob coronavirus nsp12 (txoj kev tshawb no), suav nrog cov lus thov zoo li cov txiaj ntsig ntawm folding ntawm C-terminal RdRp domain ( 16)).

Yav dhau los, ob peb lub tswv yim hais txog qhov ua tau ntawm cov dej ntws hauv qab ntawm nidoviral NiRAN domain tau raug txiav txim siab, suav nrog RNA ligase, RNA -capped guanylate transferase thiab protein priming kev ua haujlwm (16), tab sis tsis muaj ib qho ntawm lawv tsis sib xws nrog cov haujlwm hauv qab no.Cov ntaub ntawv tau txais hauv cov haujlwm hauv qab no yog tib lub sijhawm yam tsis muaj kev xav ntxiv.Cov ntaub ntawv tau txais hauv txoj kev tshawb no feem ntau ua raws li (tab sis tsis tuaj yeem ua pov thawj) tias NiRAN sau tau koom nrog hauv kev pib ntawm protein-induced RNA synthesis.Nws yog yav dhau los ntseeg tias qhov ua haujlwm ntawm NiRAN sau hauv 5??²-RNA capping lossis RNA ligation cov tshuaj tiv thaiv tsis cuam tshuam los ntawm cov no thiab Txhawb nqa ntawm lwm cov ntaub ntawv.Yog li, piv txwv li, qhov chaw nquag ntawm NiRAN raug suav hais tias suav nrog kev khaws cia Asp ua lub hauv paus dav dav (D252 hauv Pseudomonas syringae SelO; D4271 hauv HCoV-229E pp1ab; D208 hauv SARS-CoV-2 nsp12) (SI Appendix ,daim duab 2 ).S2) (17, 22, 33), thaum lub catalysis nyob rau hauv ATP-raws li RNA ligase thiab RNA capping enzyme yog nqa tawm los ntawm covalent enzyme-(lysyl-N)-NMP intermediate, uas muaj xws li ib tug tsis-hloov Lys residue ( 41).Tsis tas li ntawd, qhov zoo tshaj plaws raws li qhov tshwj xeeb ntawm tus mob coronavirus NiRAN rau kev txuag cov hom phiaj protein thiab qhov tshwj xeeb so rau NTP co-substrates (xws li UTP) tawm tsam NiRAN-mediated capping enzyme lossis RNA ligase-zoo li cov haujlwm.

Pom tseeb, ntau txoj haujlwm ntxiv yog xav tau los txheeb xyuas thiab, yog tias muaj pov thawj, piav qhia txog lub luag haujlwm ntawm nsp9-UMP (nsp9-NMP) hauv protein-induced RNA synthesis, uas yuav txuas ntau qhov nthuav tab sis (kom deb li deb) cov ntaub ntawv yav dhau los tau tshaj tawm. .Kev soj ntsuam cais.Piv txwv li, nws tau txiav txim siab tias qhov kawg ntawm qhov tsis zoo-strand RNA ntawm tus mob coronavirus pib nrog oligo (U) strand (42, 43).Qhov kev soj ntsuam no yog ua raws li lub tswv yim hais tias kev sib txuas ntawm qhov tsis zoo-strand RNA yog pib los ntawm kev khi UMPylated daim ntawv ntawm nsp9 mus rau poly(A) tail (triggers), uas tej zaum yuav txhawb nqa los ntawm nws txoj kev khi RNA cov haujlwm thiab / lossis kev cuam tshuam nrog lwm RTC protein.UMP feem muab los ntawm nsp9 tuaj yeem siv los ua "primer" rau nsp7/8/nsp12-mediated oligouridylation, siv 3??²-poly(A) tus Tsov tus tw hauv genomic RNA lossis Lwm oligo (A)-muaj cov kab ke. ua haujlwm raws li tus qauv, zoo ib yam li cov txheej txheem tsim los rau picornavirus VPg protein (44).Yuav ua li cas yog hais tias lub tswv yim yog "non-normative"????Qhov pib ntawm (protein-induced) negative-strand RNA synthesis muab qhov txuas rau kev soj ntsuam, qhia tias tus kabmob coronavirus tsis zoo-strand RNA muaj UMP (tsis yog UTP) thaum kawg (42), uas tau txiav txim siab los qhia tias tus nucleic acid Dicer cleaves kawg phosphorylated los ntawm ib qho tsis paub uridine-specific endonuclease.Yog tias paub tseeb, qhov kev ua haujlwm ntawm nucleic acid hydrolytic tuaj yeem pab tso tawm oligomeric UMPylated daim ntawv ntawm nsp9 los ntawm 5 ² kawg ntawm cov nascent tsis zoo strand.Lub luag haujlwm ua tau ntawm nsp9 hauv cov protein priming kuj tseem ua tau raws li cov kev tshawb fawb txog keeb kwm yav dhau los, uas tau pom tias nsp9 (thiab nsp8) cuam tshuam kev cuam tshuam thiab tshwj xeeb nrog kev txuag cis-ua yeeb yam RNA nyob ze ntawm 3 kawg ntawm tus kabmob coronavirus genome.45).Raws li tsab ntawv ceeb toom no, cov kev soj ntsuam yav dhau los tam sim no tuaj yeem rov tshuaj xyuas thiab nthuav dav los ntawm kev tshawb fawb ntxiv.

Hauv cov ntsiab lus, peb cov ntaub ntawv tau txiav txim siab txog cov haujlwm tshwj xeeb ntawm tus tswv ntawm tus kab mob enzyme tag txuas rau RdRp ntawm N-terminus.Hauv kab mob coronavirus, qhov kev tshawb pom tshiab no NiRAN-mediated UMPylator / NMPylator kev ua haujlwm yog siv los tso siab rau Mn2+ thiab Asn residues uas nyob ib sab thiab ua rau tsim (tsawg-zog) phosphoramidate bonds nrog N-terminal thawj amine.Los ntawm Mpro-mediated cleavage ntawm nsp8|9 cleavage site, nsp9 lub hom phiaj tuaj yeem siv rau NMPylation, qhia txog kev ua haujlwm sib txuas ntawm cov protease thiab NiRAN domain, uas txuas mus rau RdRp.Kev txuag cov khoom tseem ceeb hauv nsp12 NiRAN lub vev xaib nquag thiab nsp9 lub hom phiaj, ua ke nrog cov ntaub ntawv tau los ntawm ob tus kab mob coronaviruses suav nrog SARS-CoV-2, muab cov pov thawj muaj zog uas nsp9 NMPylation yog tus kabmob coronavirus Conservative nta kuj yog kauj ruam tseem ceeb ntawm tus kab mob replication.Cov ntaub ntawv muaj ua rau peb txiav txim siab tias lub luag haujlwm tshwj xeeb ntawm NMPylated daim ntawv ntawm nsp9 hauv protein-induced RNA synthesis yog qhov tsim nyog rau tus mob coronavirus thiab lwm yam kab mob zes, thiab NiRAN kuj tseem tuaj yeem tsom rau lwm cov proteins uas tsis paub txog.Tswj tus kab mob.Kev sib tham tswv yim.Yog tias pom zoo, kev koom tes ntawm cov protein primers hauv viral RNA synthesis yuav ua rau muaj kev sib koom ua ke ntawm Mpro/3CLpro thiab RdRp domains ntawm yav dhau los kuaj pom tus mob coronavirus thiab picornavirus-zoo li supergroup (9), uas tam sim no tau koom ua ke hauv Pisonivirites tsis ntev los no ( 46) nyob rau hauv qeb.

Peb cov ntaub ntawv tseem qhia tau hais tias qhov yooj yim, kev xaiv thiab kev saib xyuas enzyme kev ua ub no hauv txoj kev tshawb no tuaj yeem siv los ua lub hom phiaj rau cov tshuaj tiv thaiv kab mob.Cov tshuaj uas cuam tshuam nrog kev khi (thiab kev hloov kho tom ntej) ntawm kev txuag nsp9 N-terminus nyob rau hauv lub chaw nquag ntawm NiRAN tuaj yeem tsim los ua cov tshuaj tiv thaiv kab mob zoo thiab muaj ntau yam, tsim nyog rau kev kho tsiaj thiab tib neeg tus kab mob coronaviruses los ntawm cov kab mob sib txawv (sub) kab mob. , suav nrog SARS-CoV-2 thiab Middle East Respiratory Syndrome Coronavirus.

Cov txheej txheem coding ntawm tus kabmob coronavirus uas tsim tawm hauv txoj kev tshawb no tau nthuav dav los ntawm RT-PCR siv RNA cais tawm ntawm Huh-7 kis nrog HCoV-229E lossis Vero E6 kis nrog SARS-CoV-2, thiab muab tso rau siv cov txheej txheem cloning txheem.pMAL-c2 (New England Biological Laboratory) los yog pASK3-Ub-CHis6 (47) qhia vector (SI Appendix, Tables S1 thiab S2).Tib codon hloov pauv tau qhia los ntawm PCR-based site-directed mutagenesis (48).Txhawm rau tsim cov MBP fusion protein, E. coli TB1 hlwb tau hloov nrog pMAL-c2 plasmid tsim tsim nyog (SI appendix, Table S1).Cov fusion protein tau huv los ntawm amylose affinity chromatography thiab cleaved nrog factor Xa.Tom qab ntawd, C-terminal His6-tagged protein tau ntxuav los ntawm Ni-immobilized hlau affinity chromatography (Ni-IMAC) raws li tau piav qhia yav dhau los (49).Txhawm rau tsim cov ubiquitin fusion protein, E. coli TB1 hlwb siv qhov tsim nyog pASK3-Ub-CHis6 plasmid tsim (SI Appendix, Tables S1 thiab S2) thiab pCGI plasmid DNA encoding ubiquitin-specific C-terminal hydrolase 1 (Ubp1).Kev hloov pauv (47).C-terminal His6-tagged coronavirus protein tau huv si raws li tau piav qhia yav dhau los (50).

Kev kuaj tus kheej-NMPylation ntawm HCoV-229E nsp12-His6 tau ua raws li tau piav qhia hauv EAV nsp9 (16).Hauv luv luv, nsp12-His6 (0.5 µM) muaj 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH, pH 8.0, 5 mM dithiothreitol (DTT), 6 mM MnCl2, 25 μM buffer qhov teev NTP thiab 0.17 µM matched [α32-P]NTP (3,000 Ci / mmol; Hartmann Analytic) ntawm 30 ° C rau 30 feeb.Nyob rau hauv tag nrho lwm yam (tus qauv) NMPylation kev soj ntsuam ntawm nsp12-mediated nsp9 NMPylation, cov tshuaj tiv thaiv kab mob raug kho raws li nram no: nsp12-His6 (0.05 µM) thiab nsp9-His6 (4 µM) nyob rau hauv muaj 50 mM HEPES-KOH (pH 8.0). ), 5 mM DTT, 1 mM MnCl2, 25 µM qhia NTP, thiab 0.17 µM matched [α32-P]NTP.Tom qab incubating rau 10 feeb ntawm 30 ° C, cov tshuaj tiv thaiv qauv yog tov nrog SDS-PAGE qauv tsis: 62.5 mM tris (hydroxymethyl) aminomethane HCl (pH 6.8), 100 mM DTT, 2.5% SDS, 10% glycerol thiab bromophenol 0.005%. xiav.Cov protein tau denatured los ntawm cua sov ntawm 90 ° C rau 5 feeb thiab sib cais los ntawm 12% SDS-PAGE.Cov gel yog tsau thiab stained nrog Coomassie Brilliant Blue tov (40% methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue R-250), decolorized, thiab raug rau lub phosphorescent imaging screen rau 20 teev (kom ntes nsp12 los ntawm NMPylation) los yog (qhov siab tshaj) 2 Teev (los ntsuam xyuas nsp9 NMPylation).Lub Typhoon 9200 imager (GE Healthcare) tau siv los luam theej duab lub vijtsam thiab ImageJ tau siv los tshuaj xyuas cov teeb liab siv.

Rau MS kev tshuaj xyuas, 1 µM nsp12-His6 thiab 10 µM nsp9 (tsis muaj hexahistidine tag) tau siv hauv NMPylation kev tshuaj ntsuam (SI appendix, Table S1) thiab nce siab ntawm 500 µM UTP thiab GTP.Nyob ntawm lawv cov concentration thiab xav tau cov protein zoo, Waters ACQUITY H-Class HPLC system nruab nrog MassPrep kem (Dej) tau siv los tshem tawm 1 txog 10 µL ntawm buffered protein daws hauv online.Cov protein desalted yog eluted mus rau hauv electrospray ion qhov chaw ntawm Synapt G2Si huab hwm coj spectrometer (Dej) los ntawm cov nram qab no gradient ntawm tsis A (dej / 0.05% formic acid) thiab tsis B (acetonitrile / 0.045% formic acid), thiab sab kub yog 60 ° C thiab ntws tus nqi ntawm 0.1 mL / min: elution isocratically nrog 5% A rau 2 feeb, ces ib tug linear gradient mus rau 95% B nyob rau hauv 8 feeb, thiab tuav 95% B rau lwm 4 feeb.

Cov ions zoo uas muaj ntau yam ntawm 500 txog 5000 m / z tau kuaj pom.Glu-fibrinopeptide B yog ntsuas txhua 45 vib nas this rau kev kho tsis siv neeg loj drift.Siv MassLynx ntsuas software nrog MaxEnt1 txuas ntxiv los txiav txim siab qhov nruab nrab spectrum tom qab txiav tawm hauv paus thiab smoothing.

UMPylated HCoV-229E nsp9 tau digested los ntawm kev ntxiv sequencing-qib modified trypsin (Serva) thiab incubated hmo ntuj ntawm 37 ° C.Ib tug Chromabond C18WP spin kem (ib feem naj npawb 730522; Macerey-Nagel) tau siv los desalt thiab mloog cov peptides.Thaum kawg, peptide tau yaj hauv 25 µL dej, uas muaj 5% acetonitrile thiab 0.1% formic acid.

Cov qauv no tau txheeb xyuas los ntawm MS siv lub Orbitrap Velos Pro huab hwm coj spectrometer (Thermo Scientific).Qhov kawg nanoâ HPLC system (Dionex), nruab nrog kev cai kawg-mounted 50 cm??75 μm C18 RP kem ntim nrog 2.4 μm magnetic hlaws (Dr. Albin Maisch High Performance LC GmbH) Txuas mus rau huab hwm coj spectrometer online los ntawm Proxeon nanospray qhov chaw;txhaj 6 µL ntawm trypsin digestion tov rau hauv 300 µm puab txoj kab uas hla ×??1 cm C18 PepMap pre-concentration kem (Thermo Scientific).Siv dej / 0.05% formic acid ua cov kuab tshuaj, cov qauv tau raug kaw thiab desalinated ntawm tus nqi ntws ntawm 6 µL / min.

Cov nram qab no gradients ntawm dej / 0.05% formic acid (hloov A) thiab 80% acetonitrile / 0.045% formic acid (kua B) tau siv los ua kom tiav kev sib cais ntawm tryptic peptides ntawm tus nqi ntws ntawm 300 nL / min: 4% B rau 5 feeb, ces 30 A linear gradient mus rau 45% B nyob rau hauv feeb, thiab ib tug linear nce mus rau 95% hnyav B nyob rau hauv 5 feeb.Txuas lub chromatographic kem mus rau stainless hlau nano-emitter (Proxeon), thiab tsuag lub eluent ncaj qha mus rau lub rhuab capillary ntawm huab hwm coj spectrometer siv ib tug muaj peev xwm ntawm 2,300 V. Kev soj ntsuam scan nrog ib tug daws teeb meem ntawm 60,000 nyob rau hauv lub Orbitrap mass analyzer yog txuam nrog. nrog tsawg kawg peb cov ntaub ntawv MS / MS scans, dynamically tsis suav nrog rau 30 vib nas this, siv linear ion ntxiab kev sib tsoo induced dissociation los yog siab zog sib tsoo dissociation ua ke nrog orbitrap nrhiav, qhov kev daws teeb meem yog 7,500.