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Die proefleesfunksie van Polymerases van Coronavirus

Die proefleesfunksie van Polymerases van Coronavirus


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Ek het gehoor dat die Coronavirus -familie 'n proeflees- en redigeringsfunksie in hul polimerase -ensieme het wat mutasies kan herken en uitsny. Dit is natuurlik rampspoedig vir die bevolking van besmette individue. Is dit waar, en indien wel, sou dit moontlik wees om die proeflees- en redigeersubeenheid selektief uit te skakel sodat die mutasielas geleidelik in die virale populasie sou toeneem? Kan dit dan tot iets soos Muller's Ratchet lei en die virulensie in die bevolking demp, of is daar steeds 'n meganisme om die gemuteerde virusse uit die bevolking te verwyder en die virulensie oor tyd te behou?


Baie in die wetenskaplike gemeenskap het gemobiliseer om die virus te verstaan ​​wat die wêreldwye koronavirussiekte 2019 (COVID-19) pandemie veroorsaak. Gao et al. gefokus op 'n kompleks wat 'n sleutelrol speel in die replikasie- en transkripsie-siklus van die virus. Hulle gebruik kryo-elektronmikroskopie om 'n struktuur van 2.9-angstromresolusie van die RNA-afhanklike RNA-polimerase nsp12 te bepaal, wat die sintese van virale RNA kataliseer, in kompleks met twee kofaktore, nsp7 en nsp8. nsp12 is 'n doelwit vir nukleotied analoog antivirale remmers soos remdesivir, en die struktuur kan 'n basis bied vir die ontwerp van nuwe antivirale terapieë.

'n Nuwe koronavirus [ernstige akute respiratoriese sindroom-coronavirus 2 (SARS-CoV-2)]-uitbraak het 'n wêreldwye koronavirussiekte 2019 (COVID-19) pandemie veroorsaak, wat tot tienduisende infeksies en duisende sterftes wêreldwyd gelei het. Die RNA-afhanklike RNA-polimerase [(RdRp), ook genoem nsp12] is die sentrale komponent van koronavirale replikasie- en transkripsiemasjinerie, en dit blyk 'n primêre teiken vir die antivirale geneesmiddel remdesivir te wees. Ons rapporteer die kryo-elektronmikroskopiestruktuur van nsp12 vir die volledige COVID-19-virus in kompleks met kofaktore nsp7 en nsp8 met 'n resolusie van 2,9 angstrom. Benewens die bewaarde argitektuur van die polimerasekern van die virale polimerase-familie, besit nsp12 'n nuut geïdentifiseerde β-haarnaalddomein by sy N-terminus. 'N Vergelykende ontledingsmodel toon hoe remdesivir aan hierdie polimerase bind. Die struktuur bied 'n basis vir die ontwerp van nuwe antivirale terapieë wat virale RdRp teiken.

Koronavirussiekte 2019 (COVID-19) word veroorsaak deur 'n nuwe koronavirus [ernstige akute respiratoriese sindroom – koronavirus 2 (SARS-CoV-2)] wat in Desember 2019 ontstaan ​​het (13) en het sedertdien 'n wêreldwye pandemie geword. Na berig word, word die COVID-19-virus 'n nuwe lid van die genus betacoronavirus en is nou verwant aan ernstige akute respiratoriese sindroom-koronavirus (SARS-CoV) en verskeie vlermuis-koronavirus (4). In vergelyking met SARS-CoV en respiratoriese sindroom van die Midde-Ooste-koronavirus (MERS-CoV), toon COVID-19-virus vinniger mens-tot-mens-oordrag, wat daartoe lei dat die Wêreldgesondheidsorganisasie 'n wêreldwye noodgeval vir openbare gesondheid verklaar (1, 2).

Coronaviruses (CoV's) gebruik 'n multisubunit -masjinerie vir replikasie en transkripsie. 'N Stel nie -strukturele proteïene (nsps) wat as afskeidingsprodukte van die virale poliproteïene ORF1a en ORF1ab (5) vergader om virale replikasie en transkripsie te fasiliteer. 'N Sleutelkomponent, die RNA-afhanklike RNA-polimerase [(RdRp), ook bekend as nsp12], kataliseer die sintese van virale RNA en speel dus 'n sentrale rol in die replikasie- en transkripsiesiklus van COVID-19-virus, moontlik met die hulp van nsp7 en nsp8 as kofaktore (6). Daarom word nsp12 beskou as 'n primêre teiken vir nukleotied analoog antivirale remmers soos remdesivir, wat potensiaal toon vir die behandeling van COVID-19 virale infeksies (7, 8). Om die ontwerp van die geneesmiddel in te lig, het ons die struktuur van nsp12, in kompleks met sy kofaktore nsp7 en nsp8, bepaal deur krio-elektronmikroskopie (cryo-EM) met behulp van twee verskillende protokolle: een in die afwesigheid van ditiothreitol (DTT) (datastel 1) en die ander in die teenwoordigheid van DTT (dataset 2).

Die bakteriële uitgedrukte volle lengte COVID-19-virus nsp12 (residue S1 tot Q932) is geïnkubeer met nsp7 (residue S1 tot Q83) en nsp8 (residue A1 tot Q198), en die kompleks is daarna gesuiwer (fig. S1). Cryo-EM roosters is met behulp van hierdie kompleks voorberei, en voorlopige sifting het uitstekende deeltjiesdigtheid met goeie verspreiding aan die lig gebring. Na die versameling en verwerking van 7994 mikroskopfilms het ons 'n driedimensionele rekonstruksie van 'n 2,9-Å resolusie van 'n nsp12-monomeer in kompleks verkry met een nsp7-nsp8-paar en 'n nsp8-monomeer, soos voorheen waargeneem vir SARS-CoV (9). Benewens die nsp12-nsp7-nsp8-kompleks, het ons ook enkeldeeltjieklasse waargeneem wat ooreenstem met die nsp12-nsp8-dimeer, sowel as individuele nsp12-monomere, waargeneem, maar dit produseer nie atoomresolusie-rekonstruksies nie (fig. S2). Die nsp12-nsp7-nsp8 komplekse rekonstruksie verskaf egter die strukturele inligting vir volledige strukturele analise.

Die struktuur van die COVID-19-virus nsp12 bevat 'n regterhandse RdRp-domein (residue S367 tot F920) en 'n nidovirus-spesifieke N-terminale uitbreidingsdomein (residue D60 tot R249) wat 'n nidovirus RdRp-geassosieerde nukleotidieltransferase (NiRAN) aanneem (10) argitektuur. Die polimerase-domein en NiRAN-domein word verbind deur 'n koppelvlakdomein (residu A250 tot R365) (Fig. 1, A en B). 'N Bykomende N-terminale β-haarnaald (residue D29 tot K50), gebou met die leiding van 'n ondubbelsinnige cryo-EM-kaart (fig. S3A), steek in die groef vasgeklem deur die NiRAN-domein en die palm-subdomein in die RdRp-domein (Fig. . 2). Die nsp7-nsp8-paar toon 'n bewaarde struktuur soortgelyk aan dié van die SARS-CoV nsp7-nsp8-paar (9, 11). Die oriëntasie van die N-terminale heliks van die aparte nsp8 monomeer gebind aan nsp12 word verskuif in vergelyking met die in die nsp7-nsp8 paar (fig. S4A). Die 13 addisionele aminosuurreste wat by die N-terminaal van nsp8 opgelos is, wys dat die lang skag van sy bekende gholfstokvorm gebuig is (fig. S4B).

(A) Domeinorganisasie van COVID-19-virus nsp12. Die grense tussen die domeine is gemerk met residu -getalle. Die N-terminale gedeelte met geen krio-EM kaartdigtheid en die C-terminale residue wat nie in die kaart waargeneem kan word nie, word nie by die opdrag ingesluit nie. Die polimerase-motiewe is soos volg gekleur: motief A, geel motief B, rooi motief C, groen motief D, violet motief E, siaan motief F, blou en motief G, ligbruin. (B) Lintdiagram van COVID-19-virus nsp12 polipeptiedketting in drie loodregte aansigte. Domeine is dieselfde gekleur as in (A). Die individuele nsp8 (nsp8-1) wat aan nsp12 gebind het en dat in die nsp7-nsp8-paar (nsp8-2) in grys getoon word, is die nsp7 in pienk. Die paneel links onder toon 'n oorsig van die cryo-EM-rekonstruksie van die nsp12-nsp7-nsp8-kompleks.

(A) Algehele struktuur van die N-terminale NiRAN-domein en β-haarnaald van COVID-19-virus nsp12. Die N-terminale NiRAN-domein en β-haarnaald van COVID-19-virus nsp12 word onderskeidelik as geel en siaan-tekenprente getoon, terwyl die ander streke van COVID-19-virus nsp12 as 'n molekulêre oppervlak getoon word met dieselfde kleurskema wat in Fig. 1. Die NiRAN-domein van SARS-CoV nsp12 is bo-op sy eweknie in COVID-19-virus nsp12 en word in pers vertoon. (B) Belangrike interaksies tussen die β -haarnaald en ander domeine. Die β -haarspeld word getoon as 'n siaanbuis met sy sleutelreste in die stokmodus. Dit het die naaste kontak met ander domeine van die COVID-19-virus nsp12. Die interaksie-residu in die palm- en vingersubdomein van die RdRp-domein en die NiRAN-domein word deur die etikette geïdentifiseer. Enkel-letter afkortings vir die aminosuurreste is soos volg: A, Ala C, Cys D, Asp E, Glu F, Phe G, Gly H, His I, Ile K, Lys L, Leu M, Met N, Asn P , Pro Q, Gln R, Arg S, Ser T, Thr V, Val W, Trp en Y, Tyr.

Die algehele argitektuur van die COVID-19-virus nsp12-nsp7-nsp8-kompleks is soortgelyk aan dié van SARS-CoV met 'n wortelgemiddelde vierkante afwyking (RMSD) van 0.82 vir 1078 Cɑ-atome (fig. S4C). Daar is egter belangrike kenmerke wat die twee onderskei. Die cryo-EM-kaart het ons toegelaat om die volledige struktuur van COVID-19-virus nsp12 te bou, insluitend alle oorblyfsels behalwe S1 tot D3 en G897 tot D910. Daarteenoor is die eerste 116 residue nie opgelos in SARS-CoV nsp12 (9). Die gedeelte van die NiRAN-domein wat in SARS-CoV opgelos is (residue 117 tot 249) bestaan ​​uit ses helices met 'n drie-gestrande β-vel by die N-terminus (9) (Fig. 2A). In die COVID-19-virusstruktuur het ons die residue A4 tot R118 ook opgelos. Hierdie vorm 'n strukturele blok met vyf antiparallelle β-stringe en twee helikse. Residue N215 tot D218 vorm 'n β-string in COVID-19-virus nsp12, terwyl hierdie residue minder georden is in SARS-CoV nsp12. Hierdie gebied maak kontak met die string wat reste V96 tot A100 bevat, wat bydra tot die stabilisering van die konformasie. As gevolg hiervan vorm hierdie vier stringe 'n kompakte semi -β vat -argitektuur. Daarom identifiseer ons residue A4 tot T28 en Y69 tot R249 as die volledige coronavirale NiRAN -domein. Met die resolusie van N-terminale residue is ons ook in staat om 'n N-terminale β-haarnaald te identifiseer (D29 tot K50 Fig. 1A en 2A). Hierdie β haarspeld steek in die groef vasgeklem deur die NiRAN domein en die palm subdomein in die RdRp domein en vorm 'n stel noue kontakte om die algehele struktuur te stabiliseer (Fig. 2B en fig. S5). Ons het ook waargeneem dat C301 tot C306 en C487 tot C645 disulfiedbindings vorm in die afwesigheid van DTT (dataset 1). In die teenwoordigheid van DTT (datastel 2), is gecheleerde sinkione egter teenwoordig op dieselfde plek as wat in SARS-CoV waargeneem is (fig. S3B).

Die polimerase -domein neem die bewaarde argitektuur van die virale polimerase -familie aan (12) en is saamgestel uit drie subdomeine: 'n vinger-subdomein (residu L366 tot A581 en K621 tot G679), 'n palm-subdomein (residu T582 tot P620 en T680 tot Q815), en 'n duim-subdomein (residu H2016) (Fig. ). Die katalitiese metaalione, wat waargeneem word in verskeie strukture van virale polimerases wat RNA sintetiseer (13, 14), word nie in hierdie werk waargeneem in die afwesigheid van primersjabloon-RNA en nukleosiedtrifosfate (NTP's).

Die aktiewe plek van die COVID-19-virus RdRp-domein word gevorm deur die bewaarde polimerase-motiewe A tot G in die palmdomein en gekonfigureer soos ander RNA-polimerases (Fig. 1A en 3A en fig. S6). Motief A, saamgestel uit residue 611 tot 626 (TPHLMGWDYPKCDRAM), bevat die klassieke divalente-kationbindende residu D618, wat in die meeste virale polimerases bewaar word, insluitend hepatitis C virus (HCV) ns5b (residu D220) en poliovirus (PV) 3D pol (residu D233) (13, 14) (Fig. 3, B en C). Motief C [residu 753 tot 767 (FSMMILSDDAVVCFN)] bevat die katalitiese residue [759 tot 761 (SDD)] in die draai tussen twee β-stringe. Hierdie katalitiese residue word ook bewaar in die meeste virale RdRps, byvoorbeeld 317 tot 319 (GDD) in HCV ns5b en 327 tot 329 (GDD) PV 3D pol, met die eerste residu óf serien óf glisien.

(A aan C) Strukturele vergelyking van COVID-19-virus nsp12 (A), HCV ns5b (PDB ID: 4WTG) (13) (B), en PV 3D pol (PDB ID: 3OLB) (14) (C). Die drie strukture word in dieselfde oriëntasie vertoon. Die polimerasemotiewe (motiewe A tot G) het dieselfde kleurskema as in Figuur 1A. (D) Die sjablooninskrywing, NTP-inskrywing en produkhibriede uitgangspaaie in COVID-19 virus nsp12 is onderskeidelik gemerk in leisteen, diepblou en oranje kleure. Twee katalitiese mangaanione (swart bolle), pp-sofosbuvir (donkergroen sfere vir koolstofatome) en 'n primersjabloon (oranje) uit die struktuur van HCV ns5b in komplekse pp-sofosbuvir (PDB ID: 4WTG) (13) word teenoor COVID-19-virus nsp12 geplaas om die katalitiese plek en die nukleotiedbindingsposisie aan te dui.

In hierdie struktuur, soos in ander RNA-polimerases, is die primer-sjabloon-intree-, NTP-intree- en ontluikende string-uitgangspaaie positief gelaai en oplosmiddeltoeganklik, en hulle konvergeer in 'n sentrale holte waar die RdRp-motiewe sjabloongerigte RNA-sintese bemiddel (Fig. . 3D). Die konfigurasies van die sjabloon-primer-ingangspaaie, die NTP-ingangskanaal en die ontluikende stranduitgangspad is soortgelyk aan dié wat beskryf word vir SARS-CoV en vir ander RNA-polimerases, soos HCV en PV-polimerase (14) (Fig. 3, B en C). Die NTP -ingangskanaal word gevorm deur 'n stel hidrofiliese residue, insluitend K545, R553 en R555 in motief F. Die RNA -sjabloon sal na verwagting die aktiewe terrein wat uit motiewe A en C bestaan, binnedring deur 'n groef vasgeklem deur motiewe F en G. Motief E en die duim -subdomein ondersteun die onderlaag. Die produk-sjabloonbaster verlaat die aktiewe plek deur die RNA-uitgangstunnel aan die voorkant van die polimerase.

Remdesivir, die enkele Sp-isomeer van die 2-etielbutiel L-alaninaat fosforamidaat prodrug (15) (fig. S7), word gerapporteer dat dit die verspreiding van COVID-19-virus belemmer en daarom kliniese potensiaal het (7, 8). Ons sal kortliks die moontlike bindings- en remmingsmeganisme daarvan bespreek aan die hand van die resultate van hierdie studie. Die doeltreffendheid van kettingterminerende nukleotiedanaloë vereis virale RdRps om die aktiewe vorm van die inhibeerders in die groeiende RNA-string te herken en suksesvol te inkorporeer. Sofosbuvir (2'-F-2'-C-methyluridine monofosfaat) is 'n prodrug wat op HCV ns5b gerig is en goedgekeur is vir die behandeling van chroniese HCV infeksie (16). Dit werk deur te bind aan die katalitiese plek van HCV ns5b polimerase (12, 16). Gegewe dat remdesivir en sofosbuvir beide nukleotiedanaloë is en gegewe die strukturele bewaring van die katalitiese plek tussen COVID-19 virus nsp12 en HCV ns5b polimerase (13, 16) (fig. S7), het ons remdesivirdifosfaatbinding gemodelleer aan COVID-19-virus nsp12 op grond van superposisie met sofosbuvir gebind aan HCV ns5b (fig. 4A en fig. S4D). In die algemeen het ons gevind dat die nsp12 van COVID-19-virus die hoogste ooreenkoms het met die apo-toestand van ns5b. Gegewe die konformasionele veranderinge van ns5b in apo, rek en geïnhibeerde toestande, blyk dit dat katalitiese residue D760, D761 en die klassieke D618 'n konformasionele verandering sal ondergaan om die tweewaardige katione te koördineer (Fig. 4B). Laasgenoemde sal die fosfaatgroep van die inkomende nukleotied of inhibeerders saam met die allosteriese R555 in motief F (Fig. 4C) anker. In die strukture van die HCV ns5b verlengingskompleks of sy kompleks met difosfaat sofosbuvir (pp-sofosbuvir), is 'n sleutelkenmerk dat die geïnkorporeerde pp-sofosbuvir in wisselwerking tree met N291 (gelykstaande aan N691 in COVID-19 virus). As gevolg van 'n fluoorvervanging op sy suikerdeel, is pp-sofosbuvir egter nie in staat om die waterstofbindingsnetwerk met S282 en D225 te verbind nie (Fig. 4D), wat nodig is om die inkomende natuurlike nukleotied te stabiliseer (13). Remdesivir hou egter 'n ongeskonde ribosegroep, sodat dit moontlik hierdie waterstofbindingsnetwerk kan gebruik soos 'n inheemse substraat. Daarbenewens sal T680 in COVID-19-virus nsp12 ook waarskynlik waterstofbindings vorm met die 2'-hidroksiel van remdesivir en natuurlik met inkomende natuurlike NTP (Fig. 4D). Verder, die hidrofobiese syketting van V557 in motief F sal waarskynlik stapel met en stabiliseer die +1 templaat RNA uridien basis tot basis paar met die inkomende trifosfaat remdesivir (ppp-remdesivir) (Fig. 4E).

(A) Die polimerase motiewe is gekleur soos in Fig. 3. Superposisie van die struktuur van HCV ns5b in kompleks met pp-sofosbuvir (PDB ID: 4WTG) (13) met COVID-19-virus nsp12 toon die moontlike posisies van die twee katalitiese ione (pers sfere), die priming-nukleotied (U 0), sjabloonstring en die inkomende pp-remdesivir in nsp12. (B aan E) Struktuurvergelyking van HCV apo ns5b of sy kompleks met UDP en pp-sofosbuvir met die COVID-19 virus nsp12.

Die vinnige wêreldwye verspreiding van COVID-19-virus beklemtoon die noodsaaklikheid vir die ontwikkeling van nuwe entstowwe en terapieë teen koronavirus. Die virale polimerase nsp12 blyk 'n uitstekende teiken vir nuwe terapeutiese middels te wees, veral gegewe die feit dat loodinhibeerders reeds bestaan ​​in die vorm van verbindings soos remdesivir. Met inagneming van die strukturele ooreenkoms van nukleosied -analoë, kan die bindingswyse en remmingsmeganisme wat hier bespreek word, ook van toepassing wees op ander soortgelyke middels of kandidate soos favipiravir, wat effektief bewys het in kliniese toetse (17). Hierdie doelwit, benewens ander belowende geneesmiddeldoelwitte, soos die belangrikste protease, kan die ontwikkeling van 'n skemerkel van anti-koronavirusbehandelings ondersteun wat moontlik gebruik kan word vir die ontdekking van breëspektrum antivirale middels.


Die koronavirus verander. Maar dit mag nie vir mense 'n probleem wees nie

'N Gekleurde beeld van selle van 'n pasiënt wat besmet is met die koronavirus SARS-CoV-2. Die virusdeeltjies is pienk gekleur. Die beeld is geneem uit 'n skandeerelektronmikrograaf. NIAID/Flickr steek onderskrif weg

'n Gekleurde beeld van selle van 'n pasiënt wat met die koronavirus SARS-CoV-2 besmet is. Die virusdeeltjies is pienk gekleur. Die beeld is geneem uit 'n skandeerelektronmikrograaf.

Terwyl die nuwe koronavirus steeds oor die wêreld versprei, sê navorsers dat die virus sy genetiese samestelling effens verander. Maar beteken dit dat dit gevaarliker word vir mense? En wat sou die impak op enige toekomstige entstowwe wees?

"In die letterlike sin van 'verander dit geneties', is die antwoord absoluut ja," sê Marc Lipsitch, 'n aansteeklike siekte-epidemioloog aan die Harvard Universiteit. 'Wat ter sprake is, is of daar 'n verandering is wat belangrik is vir die verloop van die siekte, die oordraagbaarheid of ander dinge waaroor ons as mense omgee.'

Tot dusver, "is daar geen geloofwaardige bewyse van 'n verandering in die biologie van die virus nie, ten goede of ten kwade," sê Lipsitch.

Koronavirusse, soos alle virusse, verander deurgaans klein dele van hul genetiese kode.

"Viruse muteer natuurlik as deel van hul lewensiklus," sê Ewan Harrison, wetenskaplike projekbestuurder vir die COVID-19 Genomics UK Consortium, 'n nuwe projek wat die virus in die Verenigde Koninkryk opspoor.

Net soos griep en masels, is die koronavirus 'n RNA -virus. Dit is 'n mikroskopiese pakket van genetiese instruksies wat in 'n proteïenskil gebundel is. Wanneer 'n virus 'n persoon besmet, stel die string genetiese instruksies die virus in staat om te versprei deur dit te vertel hoe om te repliseer sodra dit 'n sel binnedring. Die virus maak kopieë van homself en stoot dit na ander selle in die liggaam. Aansteeklike dosisse van die virus kan in druppels uitgehoes word en deur ander ingeasem word.

Vir onvermydelik maak virusse 'foute in hul genome' terwyl hulle hulself kopieer, sê Harrison. Hierdie veranderinge kan ophoop en oorgedra word na toekomstige kopieë van die virus. Navorsers gebruik hierdie klein, kumulatiewe veranderinge om die weg van die virus deur groepe mense op te spoor.

Navorsers wat die genetiese veranderinge in SARS-CoV-2-die amptelike naam vir die koronavirus-opspoor, sê tot dusver dat dit relatief stabiel lyk. Dit verkry ongeveer twee mutasies per maand tydens hierdie proses van verspreiding, sê Harrison - ongeveer een derde tot die helfte van die tempo van die griep.

Koronavirusse verskil van griepvirusse op 'n ander belangrike manier wat die aantal mutasies verminder. Hulle proeflees hul eie genome as hulle hulself kopieer, en sny dinge uit wat nie reg lyk nie. "Hulle handhaaf hierdie vermoë om hul genoom redelik ongeskonde te hou," sê Vineet Menachery, 'n viroloog aan die Universiteit van Texas Medical Branch. "Die mutasies wat hulle inkorporeer, is relatief skaars."

Hierdie bykomende proefleesfunksie beteken dat koronavirus ook een van die grootste RNA -virusse is. Hulle is ongeveer 30 000 nukleotiede lank - dubbel die grootte van griepvirusse. Maar teen 125 nanometer breed, is hulle steeds mikroskopies, 800 van hulle kan in die breedte van 'n menslike haar pas.

Hulle relatief groter grootte beteken egter dat 'hulle baie meer gereedskap in hul werktuiggordel het' in vergelyking met ander RNA -virusse, sê Menachery - met ander woorde meer vermoë om 'n gasheer se immuunstelsel af te weer en kopieë van hulself te maak.

Navorsers is op hul hoede vir veranderinge wat die gedrag van die koronavirus by mense kan beïnvloed. As die koronavirus byvoorbeeld maniere ontwikkel om dele van ons immuunstelsel te blokkeer, kan dit in ons liggame skuil en homself beter vestig. As dit ontwikkel om sterker aan menslike selle te bind, kan dit dit doeltreffender binnedring en vinniger herhaal.

Maar dit is nie asof die koronavirus kragtiger moet word om te oorleef en te floreer nie. Justin Bahl, 'n evolusionêre bioloog aan die Universiteit van Georgia, is reeds suksesvol oor die hele wêreld. 'Die virusse self is eintlik nie onder groot druk om te verander nie.'

Selektiewe druk kan ontstaan ​​as gevolg van die bekendstelling van behandelings en entstowwe wat effektief is teen 'n nou groep koronavirusstamme. As dit gebeur, sal stamme wat nie deur hierdie maatreëls geteiken word nie, waarskynlik vermeerder.

Die klein genetiese veranderinge wat navorsers tot dusver opgemerk het, blyk nie die funksie van die virus te verander nie. 'Ek dink nie ons gaan groot nuwe eienskappe sien nie, maar ek dink wel dat ons verskillende variante in die bevolking sal sien verskyn,' sê Bahl.

En die stadiger tempo van verandering is moontlik goeie nuus vir behandelings en entstowwe. Navorsers dink dat sodra 'n persoon immuniteit kry teen SARS-CoV-2, hetsy deur te herstel van 'n infeksie of deur 'n toekomstige entstof, dit waarskynlik "jare eerder as maande" teen die stamme in omloop beskerm word, voorspel Trevor Bedford, 'n evolusionêre bioloog by die Fred Hutchinson Cancer Research Center, in 'n beoordeling wat op Twitter gedeel is.

Projekte soos die COVID-19 Genomics UK Consortium sal hierdie genetiese dryf gebruik om die pad van die virus op te spoor en uit te vind of daar hospitale of gemeenskapsentrums is wat brandpunte vir besmetting is, volgens Harrison. Dit sal amptenare van openbare gesondheid 'n idee gee van waar en hoe die virus nou oorgedra word.

Sal die koronavirus toeneem as skole heropen? Sal nuwe stamme opduik wat weerstand ontwikkel teen middels of entstowwe wat bekendgestel word? Om sulke vrae te beantwoord, sê Harrison, is die langtermynplan om die virus intyds op te spoor-en te sien hoe dit verander namate dit versprei.


Coronavirus Proteases

Navorsers gebruik hierdie strukture aktief om te soek na verbindings wat die werking van die proteas blokkeer, as antivirale middels. Die diversiteit van koronavirusse bied 'n groot uitdaging met hierdie poging: koronavirusse is in vier afsonderlike genera ingedeel, en volgorde- en struktuurstudies het getoon dat die proteases van hierdie virusse baie anders kan wees, dus medisyne wat bedoel is om 'n mens te bestry, is moontlik nie effektief teen ander. Een moontlike manier om hierdie uitdaging aan te spreek, is om te probeer om 'n breëspektrum-inhibeerder te ontwerp wat gerig is teen die stamvadervlermuis-koronavirus, soos die een wat hier gewys word vanaf PDB-inskrywing 4yoi, wat dan 'n voorsprong kan bied vir die ontdekking van inhibeerders teen nuut-opkomende virusse . Die aktiewe plek sisteïen en histidien word in die illustrasie getoon, met 'n inhibeerder in turkoois. Om hierdie struktuur in meer besonderhede te ondersoek, klik op die prentjie vir 'n interaktiewe JSmol.

Onderwerpe vir verdere bespreking

  1. 'n Ongewone oktameriese vorm van die hoofprotease kan by sy rypwording betrokke wees. Jy kan dit sien in PDB-inskrywing 3iwm.
  2. U kan die voue van hoofproteases van koronavirus en serienproteases vergelyk met die instrument "Struktuurbelyning". Probeer om tripsinogeen (PDB-inskrywing 1tgs) te gebruik, sodat die hele ensiem een ​​ketting vir die belyning is.

Verwante PDB-101 Hulpbronne

Verwysings

  1. Cui, J., Li, F., Shi, Z.L. (2019) Oorsprong en evolusie van patogene koronavirusse. Nat. Eerwaarde Microbiol. 17, 181-192.
  2. 4yoi: St John, SE, Tomar, S., Stauffer, SR, Mesecar, AD (2015) Doelstelling van soönotiese virusse: Struktuurgebaseerde remming van die 3C-agtige protease van vlermuis-koronavirus HKU4-Die waarskynlike reservoirgasheer vir die menslike koronavirus veroorsaak respiratoriese sindroom in die Midde -Ooste (MERS). Bioorg.Med.Chem. 23: 6036-6048
  3. 4ow0: Baez-Santos, YM, Barraza, SJ, Wilson, MW, Agius, MP, Mielech, AM, Davis, NM, Baker, SC, Larsen, SD, Mesecar, AD (2014) X-straal strukturele en biologiese evaluering van 'n Reeks potensiële en hoogs selektiewe remmers van menslike koronavirus Papainagtige proteas. J.Med.Chem. 57: 2393-2412
  4. Hilgenfeld, R. (2014) Van SARS tot MERS: kristallografiese studies oor koronavirale proteas maak die ontwerp van antivirale middels moontlik. FEBS J. 281,4085-4096
  5. 1q2w: Pollack, A. (2003) Company sê dit het 'n deel van die SARS -virus gekarteer. New York Times, 30 Julie, afdeling C, bladsy 2.

Februarie 2020, David Goodsell

Oor PDB-101

PDB-101 help onderwysers, studente en die algemene publiek om die 3D-wêreld van proteïene en nukleïensure te verken. Om meer te leer oor hul uiteenlopende vorms en funksies, help om alle aspekte van biomedisyne en landbou te verstaan, van proteïensintese tot gesondheid en siektes tot biologiese energie.

Hoekom PDB-101? Navorsers regoor die wêreld maak hierdie 3D -strukture vrylik beskikbaar by die Protein Data Bank (PDB) argief. PDB-101 bou inleidende materiaal om beginners te help om met die vak te begin ("101", soos in 'n intreevlakkursus) sowel as hulpbronne vir uitgebreide leer.


1: DNA -replikasie, transkripsie en translasie

A. Funksie: DNA -basisvolgorde kodeer inligting vir aminosuurvolgorde van proteïene. Genetiese kode: 1 tot 1 verhouding tussen 'n kodon (spesifieke volgorde van 3 basisse) en 1 aminosuur. Sentrale Dogma van genetika/inligtingvloei in selle -Grondslagfiguur: Vloei van Genetiese Inligting bl 1. DNS sal gerepliseer word en deurgegee word aan &ldquodogterselle&rdquo

B. DNA -struktuur: figuur 8.3 Dubbelstrengs (2 stringe DNA), spiraalvormige en dubbele heliks & rdquo, antiparallel

1. Twee stringe wat bymekaar gehou word deur waterstofbindings tussen komplementêre basisse binne die heliks
2. Sterk buitenste &lquosuiker-fosfaat&rdquo ruggraatkovalente fosfodiesterbindings skakel nukleotiede
3. DNA -stringe: polimere van nukleotiede
4. Nukleotiede: 3 komponente. Suiker = deoksiribose, fosfaat, stikstofbasis
5. Stikstofbasisse van DNA

a. puriene (2 ringe) = adenien (A) en guanien (G) pirimidiene (1 ring) = timien (T) en sitosien (C)
b. Chagraff & rsquos-reëls: hoeveelheid A = T en hoeveelheid C = G, dit is as gevolg van komplementêre baseparingsreëls

A=T vorm 2 waterstofbindings
G = C vorm 3 waterstofbindings

*c. komplementêre basisparing laat die presiese replikasie van DNA toe

6. Deoksiribose: pentose 5 koolstofstowwe. C1 'kovalent gekoppel aan stikstofhoudende basis.

C3&rsquo= vry OH (stert)
C5&rsquo gekoppel aan fosfaatgroep (kop)

7. Prokariotiese chromosome sien figuur Die meeste bakterieë het 'n enkele sirkelvormige chromosoom. 1 kopie van chromosome = & ldquohaploid selle & rdquo (die meeste menslike selle het 2 kopieë van lineêre chromosome en word & ldquodiploid selle & rdquo sien & ldquoeukaryotic chromosome genoem).

8. Topoisomerases en bakteriële gyrase

-Topoisomerases Ensieme wat 'n 'supercoil' en 'DNA' ontlok of verskillende tipes toposiomerases in 'n supercoiling verlig E coli.
Tipe I/III&rdquo &ldquorelax&rdquo DNA-superspoele
Tipe II = Bakteriële Gyrase: stel negatiewe supercoils bekend
Deur die werking van topoisomerases kan die DNA -molekule afwisselend opgerol en verslap word. Omdat opwinding nodig is om DNA in die sel van 'n sel te verpak en ontspanning nodig is sodat DNA herhaal kan word (en getranskribeer kan word), speel hierdie twee komplementêre prosesse 'n belangrike rol in die gedrag van DNA in die sel. & Ldquo Brock Biologie van Mikroörganismes 8ste uitgawe p 185)

-bakteriële gyrase is betrokke by supercoiling/verligting van supercoiling van DNA

-antibiotika kinolone (bv. nalidiksiensuur) en fluorokinolone (soos ciprofloxasien) en novobiosien rem bakteriële gyrase en belemmer DNA -replikasie/transkripsie, sien bl.

C. DNA -sintese deur DNA -polimerases fig ___ Tabel _____

1. DNA -polimerase benodig sjabloonstreng (gids), primerstreng met vrye 3 & rsquoOH -groep, geaktiveerde substrate/voorlopers = nukleosiedtrifosfate

*2. DNA gerepliseer in 5&rsquo tot 3&rsquo rigting (5&rsquo->3&rsquo). Inkomende nukleotiede kan slegs bygevoeg word by 3 & rsquoOH -stert van 'n groeiende DNA -string

3. Suurstof van 3 & rsquoOH -groepe maak 'n nukleofiele aanval op die binneste fosforatoom van inkomende nukleosiedtrifosfaat. Pirofosfaat verdeel af en sal deur sellulêre fosfatases gehidroliseer word met die vrystelling van energie om sintese aan te dryf. Nukleotied is gekoppel aan primer -string deur fosfodiesterbinding (esterbinding = binding tussen alkohol en suur)

4. Indien geen 3&rsquoOH teenwoordig is nie, kan DNA-string nie verleng word nie=DNA-kettingbeëindiging. Gebruik van dideoxinucleoside trifosfate as basis analoë en in DNA volgorde reaksies.

II. Replikasie van bakteriese chromosoom vy ____

A. Herroep bakteriese chromosoom: enkelvoudige, sirkelvormige dubbelstring-DNS in sitoplasma

B. DNA -replikasie begin op 'n spesifieke plek & ldquoori & rdquo = oorsprong van replikasie

C. DNA-replikasie verloop tweerigting vanaf ori, met die vorming van replikasieborrel en 2 replikasievurke. Replikasievurke= streke waar d.s. DNA afgewikkel, vorm s.s. DNA-sjablone, DNA-polimerase maak komplementêre kopie van ouer ssDNA-sjabloon.

D. DNA-replikasie is semi-konserwatief. 1 ouer & ldquoold & rdquo DNA -string word gebruik as sjabloon of gids vir die sintese van 1 nuwe dogter DNA -string. Gevolg: 1 ouerchromosoom -& gt 2 dogterchromosome. Elke dogterchromosoom is 'n kopie van ouerchromosoom. Elke dogter -chromosoom bestaan ​​uit 1 ou ouer -DNA -string en 1 nuwe dogter -DNA -string. 1 ouerstreng word in elke nuwe dogter -chromosoom bewaar en gerdquo

E. Ensieme/proteïene betrokke by DNA -sintese. WEET VIR EKSAMEN. Fig 8___ Tabel ___

1.* Topoisomerases bv. Bakteriële Gyrase betrokke by DNA supercoiling/verligting van
supercoiling (teiken van kinolone, bv. ciprofloxacin en ldquocipro & rdquo, gebruik om te behandel/te voorkom
inaseming miltsiekte)

1. Helikase: wikkel ds DNA af, breek H-bindings tussen basisse, vorm ss DNA-sjabloon

2. Enkelstrengbindende proteïene SSBP bind, stabiliseer en beskerm ssDNA

3. RNA Primase: 'n RNA -polimerase wat nie 'n primer -string benodig om te begin nie
primer sintese. Sintetiseer 'n kort aanvullende RNA -primerstreng met gratis 3 & rsquoOH
groep met behulp van ss DNA as sjabloon. Skep RNA-primer, wat DNS-polimerase toelaat
begin met die sintese van DNA. (RNA -polimerase lees en bewys nie dat dit kan maak nie
baie foute).

4-5. DNA -polimerase: benodig onderlaag, sjabloon en geaktiveerde nukleosied
trifosfate (dATP, dTTP, DCTP, dGTP). Moet DNA-sjabloon hê. Sintetiseer komplementêre DNA
string met behulp van ouer string as sjabloon/gids. DNA -polimerase het & ldquoproofleesvermoëns & rdquo, hulle & ldquocheck & rdquo
elke nukleotied wat hulle byvoeg, verwyder indien verkeerd en voeg korrekte nukleotied by. DNA -polimerases het 'n hoë
getrouheid, hulle maak baie min foute. Oorspronklike foutkoerse 10-4 na proeflees, foutsyfer=
10-9 dws een verkeerde basis in elke 109 basisse wat bygevoeg word E coli: DNA -polimerase III voer die meeste DNA -sintese uit
DNA-polimerase I: sal RNA-primer verwyder en vervang met DNA-volgorde

6. Ligase: skakel kort rye DNA (genaamd Okazaki -fragmente) saam op en ldquolagging
strand & rdquo huiswerk sien remming van nukleïensuur sintese. Wat is nukleotied analoë? Wat is hul gebruike?

Vergelyk en kontrasteer bakteriële DNA -polimerases en RNA -polimerases
Let op: ss = enkelstreng ds = dubbeldraad P = fosfaat
Oorsig:

DNA -polimerases sintetiseer komplementêre DNA met behulp van 'n DNA -sjabloon/gids
dna
ssDNA -sjabloon -basisvolgorde: A T A G G C
Komplementêre DNA -volgorde T A T C C G dna
gesintetiseer deur DNA -polimerase


RNA -polimerases sintetiseer komplementêre RNA -rye met behulp van DNA as 'n sjabloon/gids
dna
ssDNA -sjabloon -basisvolgorde: A T A G G C
Komplementêre RNA -volgorde U A U C C G rna
gesintetiseer deur RNA -polimerase


Sintese van DNA en RNA vereis inset van energie, beide ATP en gelaaide voorlopers

Vergelyk en kontrasteer DNA-polimerase en sellulêre RNA-polimerase
------------------------------------------------------------------------------------------------------------
DNA -polimerase RNA -polimerase
Sjabloon/gids ss DNA ssDNA
Sintetiseer komplementêre DNA komplementêre RNA
Oplaaide voorlopers deoxyadenosine tri-P = dATP adenosine tri-P = ATP
deoksitimidien tri-P=dTTP uridien tri-P=UTP
deoksisitodien tri-P = dCTP sitodien tri-P = CTP
deoxyguanosine tri-P = dGTP guanosine tri-P = GTP
onderlaag benodig? ja nee
proeflees/redigeer? ja * geen


*DNA polimerase proeflees/redigering
Polymerases have a &rdquonormal&rdquo or &ldquointrinsic&rdquo mistake rate of approximately 10 -4 &ndash 10 -5 nucleotides (this means the polymerases introduce the incorrect nucleotide every 10,000 to 100, 000 nucleotides). DNA polymerases have the ability to &ldquoproofread
and edit&rdquo their mistakes. If they introduce the wrong nucleotide, they can remove or &ldquoexcise&rdquo the wrong nucleotide and try again to make a correct match. This reduces the mistake rate of DNA polymerases to approximately 10 -9 &ndash 10 -10 (or only one incorrect
nucleotide every 1,000,000,000 &ndash 10,000,000,000 nucleotides). RNA polymerase cannot proofread or edit so RNA polymerase make many mistakes (one reason many RNA viruses, for example HIV, mutate so rapidly. more later)

Transcription Prokaryotic


Review flow of information in cell
DNA--------> RNA --------->Protein

replication transcription translation

ek. Genetiese kode: one to one relationship between specific codon (specific 3 base sequence) and an amino acid

II. Bacterial Transcription: use of DNA as template/guide to synthesize complementary RNA.
DNA info is rewritten in RNA sequence. Fig ___

A. First step in gene expression

B. Products of transcription

1. messenger RNA=mRNA: will be translated into specific amino acid
sequence of a protein
2. transfer RNA=tRNA: actual &ldquotranslator&rdquo molecule, recognizes both a
specific codon and specific amino acid
3. ribosomal RNA=rRNA: combined with ribosomal proteins, will form
the ribosome, the &ldquoworkbench&rdquo at which mRNA is translated into a specific amino acid
sequence/polypeptide/protein

4. additional RNA products

III. Promoters and Bacterial RNA polymerases

A. Promotors: specific DNA sequences which signal the &ldquostart&rdquo points for gene
transkripsie. Sigma factor/subunit of RNA polymerase binds to promoters to
initiate transcription

B. Bacterial RNA polymerases: enzyme complex which recognizes DNA promoters, binds
to promoter and synthesizes complementary RNA copy using DNA as
template/guide

E coli RNA Polymerase: 2 subunits, sigma subunit and core

a. sigma subunit/factor= &ldquobrains&rdquo of RNA polymerase. Reis
along DNA until it reaches a promoter, binds promoter

b. core subunit: binds to sigma attached at promoter. &ldquoWorkhorse&rdquo
of RNA polymerase, carries out actual RNA synthesis. Vereis
activated precursors and template strand, DOES NOT REQUIRE
PRIMER (compare to DNA Polymerase). Synthesizes RNA in 5&rsquo -
to->3&rsquo , similar to DNA polymerase. No proofreading ability
therefore will make more mistakes than DNA Polymerase

c. sigma subunit will drop off after the first few ribonucleotides
have been linked together, core continues alone. Note: core would
start transcription randomly of DNA without direction of sigma
subeenheid. Polisistroniese mRNA (prok. only)

IV. Termination of transcription (over-simplified)

Terminators: DNA sequences which signal transcription stop signals. RNA
polymerase releases DNA when transcription terminator sequence encountered
Homework Describe antimicrobial drugs which bind to and inhibit function of bacterial RNA
polymerases (answer: rifampin _used to treat which pathogen?)

Bacterial Translation fig

Translation: RNA base sequence translated into amino acid sequence of protein. mRNA is template for
polypeptide synthesis. Second step in gene expression.

A.Translation of mRNA into a polypeptide chain is possible because of the genetic code:

1. genetic code: One to one relationship between a codon (specific sequence of 3 bases)
and a specific amino acid. Figure __ Genetic code table

voorbeelde
mRNAcodon=amino acid
GAC=aspartate
CCU=proline
UUG=leucine
Genetic code: Redundant (more than one codon for each amino acid) yet specific (each codon
encodes info for 1 amino acid only). Universal most cellular organisms use same genetic code
some exceptions

B. Translation requires tRNA, amino acids, ATP/GTP, ribosomes and mRNA

C. tRNA =transfer RNA. Adaptor/translator molecule. Only molecule which can "recognize" correct amino acid AND correct codon

1. structure: ss RNA, stem and loop

a. amino acid attachment site at one end
b. anticodon which "recognizes"(forms H bonds with) codon of mRNA

2. *45 different tRNA&rsquos for 20 different amino acids &ldquowobble&rdquo permits some tRNA&rsquos to bind to more than one codon (&ldquorelaxed&rdquo/improper base painring between 3 base of codon and anticodon)

D. amino acyl tRNA synthetases* : &ldquoload&rdquo proper amino acid on proper tRNA= amino acid activation. 20 different transferases for 20 different amino acids/tRNA&rsquos amino acid x+ ATP + tRNAx--> tRNAx:amino acid x + AMP + 2 P* &ldquocharged tRNA&rdquo or &ldquoactivated amino acid&rdquo

E. Ribosomes: 70S in prokaryotes. 2 subunits 30S (small subunit) + 50S (large subunit) S=Svedberg Unit, use to express sedimentation rates, ultracentrifugation

made of rRNA and ribosomal proteins. &ldquoWorkbench&rdquo at which mRNA will be translated into a polypeptide. 16s rRNA binds RBS (Ribosomal Binding Site on mRNA). 23s rRNA acts a ribozyme, forms peptide bonds between amino acids E, P and A sites.

F. Mechanics of translation: text. GTP is hydrolyzed during translation

Translation Initiation (note: tRNA-f met may first bind 30S subunit before 30S subunit binds RBS)

1. 30S subunit recognizes ribosomal binding site RBS/Shine-Dalgarno sequence. Complementary to 16s rRNA sequence of ribosome.
2. Translation begins at start codon AUG closest to ribosomal binding site
3. An initiator tRNA:methionine ( more precisely a formyl methionine in bacteria) enters the &ldquoP&rdquo or peptidyl binding site of the ribosome. A tRNA fits into the binding site when its anticodon base-pairs with the mRNA codon
4. The larger ribosomal 50S subunit then binds the complex
5. Additional proteins called initiation factors are required to bring all components together

Translation Elongation: amino acids are added one by one to first amino acid. Additional protein
elongation factors required

1. A second appropriately charged tRNA enters the &ldquoA&rdquo or aminoacyl binding site of the ribosome, bearing the next amino acid.
2. Peptide bond formation. 23s rRNA of large subunit catalyzes formation of peptide bond between amino acid at P site and amino acid at A site (rRNA acts as a &ldquoribozyme&rdquo, RNA catalyst) -amino acid of tRNA at P site is transferred to amino acid bond of tRNA at A site
3. Now ribosome moves &ldquodownstream&rdquo by one codon. tRNA carrying dipeptide is now in P site, A site is empty.
4. New appropriately amino acid charged tRNA enters A site
5. Ribosome catalyzes peptide bond formation between dipeptide and new incoming amino acid. Tripeptide is carried by tRNA at A site
6. Translocation:
7. Requires energy (GTP )

1. Ribosome reaches one of 3 nonsense codons/stop codons: UAA, UGA, UAG
2. Release factor binds A site, causes polypeptide and ribosome to be released from mRNA (by activation of ribozyme)

G. Polycistronic mRNA in prokaryotes permit coordinated gene expression in prokaryotes

mRNA encodes more than one gene so ribosomes can coordinately produce several
different proteins. For example 3 genes for proteins involved in lactose transport/metabolism in E. coli are
transcribed into a single mRNA molecule. Ribosomes translate all 3 into proteins at same time

H. Simultaneous transcription and translation in prokaryotes only. Ribosomes can bind mRNA and begin translation before transcription is finished. Very efficient. Fig ____


Abstrak

We are attempting to understand the processes required to accurately replicate the repetitive DNA sequences whose instability is associated with several human diseases. Here we test the hypothesis that the contribution of exonucleolytic proofreading to frameshift fidelity during replication of repetitive DNA sequences diminishes as the number of repeats in the sequence increases. The error rates of proofreading-proficient T7, T4, and Pyrococcus furiosis DNA polymerases are compared to their exonuclease-deficient derivatives, for +1 and −1 base errors in homopolymeric repeat sequences of three to eight base pairs. All three exonuclease-deficient polymerases produce frameshift errors during synthesis at rates that increase as a function of run length, suggesting the involvement of misaligned intermediates. Their wild-type counterparts are all much more accurate, suggesting that the majority of the intermediates are corrected by proofreading. However, the contribution of the exonuclease to fidelity decreases substantially as the length of the homopolymeric run increases. For example, the exonuclease enhances the frameshift fidelity of T7 DNA polymerase in a run of three A·T base pairs by 160-fold, similar to its contribution to base substitution fidelity. However, in a run of eight consecutive A·T base pairs, the exonuclease only enhances frameshift fidelity by 7-fold. A similar pattern was observed with T4 and Pfu DNA polymerases. Thus, both polymerase selectivity and exonucleolytic proofreading efficiency are diminished during replication of repetitive sequences. This may place an increased relative burden on post-replication repair processes to reduce rates of addition and deletion mutations in organisms whose genome contains abundant simple repeat DNA sequences.

Aan wie korrespondensie gerig moet word. Phone: (919)-541-2644. Fax: (919)-541-7613. Email: [email protected]


From Bats to Human Lungs, the Evolution of a Coronavirus

For thousands of years, a parasite with no name lived happily among horseshoe bats in southern China. The bats had evolved to the point that they did not notice they went about their nightly flights unbothered. One day, the parasite—an ancestor of the coronavirus, SARS-CoV-2—had an opportunity to expand its realm. Perhaps it was a pangolin, the scaly anteater, an endangered species that is a victim of incessant wildlife trafficking and sold, often secretly, in live-animal markets throughout Southeast Asia and China. Or not. The genetic pathway remains unclear. But to survive in a new species, whatever it was, the virus had to mutate dramatically. It might even have taken a segment of a different coronavirus strain that already inhabited its new host, and morphed into a hybrid—a better, stronger version of itself, a pathogenic Everyman capable of thriving in diverse species. More recently, the coronavirus found a new species: ours. Perhaps a weary traveller rubbed his eyes, or scratched his nose, or was anxiously, unconsciously, biting his fingernails. One tiny, invisible blob of virus. One human face. And here we are, battling a global pandemic.

The world’s confirmed cases (those with a positive lab test for COVID-19, the disease caused by SARS-CoV-2) doubled in seven days, from nearly two hundred and thirteen thousand, on March 19th, to four hundred and sixty-seven thousand, on March 26th. Nearly twenty-one thousand people have died. The United States now has more confirmed cases than any country on earth, with more than eighty thousand on March 26th. These numbers are a fraction of the real, unknown total in this country and around the world, and the numbers will keep going up. Scientists behind a new study, published earlier this month in the journal Wetenskap, have found that for every confirmed case there are likely five to ten more people in the community with an undetected infection. This will likely remain the case. “The testing is not near adequate,” one of the study’s authors, Jeffrey Shaman, an environmental-health sciences professor at Columbia University, said. Comments from emergency-room doctors have been circulating on social media like S.O.S. flares. One, from Daniele Macchini, a doctor in Bergamo, north of Milan, described the situation as a “tsunami that has overwhelmed us.”

Scientists first discovered that coronaviruses originate among bats following the outbreak of Severe Acute Respiratory Syndrome (SARS) in 2003. Jonathan Epstein, an epidemiologist at the EcoHealth Alliance in New York who studies zoonotic viruses—those that can jump from animals to people—was part of a research team that went hunting for the source in China’s Guangdong Province, where simultaneous SARS outbreaks had occurred, suggesting multiple spillovers from animals to people. At first, health officials believed palm civets, a mongoose-like species commonly eaten in parts of China, were responsible, as they were widely sold at markets connected to the SARS outbreak, and tested positive for the virus. But civets bred elsewhere in Guangdong had no antibodies for the virus, indicating that the market animals were only an intermediary, highly infectious host. Epstein and others suspected that bats, which are ubiquitous in the area’s rural, agricultural hills, and were, at the time, also sold from cages at Guangdong’s wet markets, might be the coronavirus’s natural reservoir.

The researchers travelled through the countryside, setting up field labs inside limestone caverns and taking swabs from dozens of bats through the night. After months of investigation, Epstein’s team discovered four species of horseshoe bats that carried coronaviruses similar to SARS, one of which carried a coronavirus that was, genetically, a more than ninety per cent match. “They were found in all of the locations where SARS clusters were happening,” he said.

After years of further bat surveillance, researchers eventually found the direct coronavirus antecedent to SARS, as well as hundreds of other coronaviruses circulating among some of the fourteen hundred bats species that live on six continents. Coronaviruses, and other virus families, it turns out, have been co-evolving with bats for the entire span of human civilization, and possibly much longer. As the coronavirus family grows, different strains simultaneously co-infect individual bats, turning their little bodies into virus blenders, creating new strains of every sort, some more powerful than others. This process happens without making bats sick—a phenomenon that scientists have linked to bats’ singular ability, among mammals, to fly. The feat takes a severe toll, such that their immune systems have evolved a better way to repair cell damage and to fight off viruses without provoking further inflammation. But when these viruses leap into a new species—whether a pangolin or a civet or a human—the result can be severe, sometimes deadly, sickness.

In 2013, Epstein’s main collaborator in China, Shi Zheng-Li, sequenced a coronavirus found in bats, which, in January, she discovered shares ninety-six per cent of its genome with SARS-CoV-2. The two viruses have a common ancestor that dates back thirty to fifty years, but the absence of a perfect match suggests that further mutation took place in other bat colonies, and then in an intermediate host. When forty-one severe cases of pneumonia were first announced in Wuhan, in December, many of them were connected to a wet market with a notorious wildlife section. Animals are stacked in cages—rabbits on top of civets on top of ferret-badgers. “That’s just a gravitational exchange of fecal matter and viruses,” Epstein said. Chinese authorities reported that they tested animals at the market—all of which came back negative—but they have not specified which animals they tested, information that is crucial for Epstein’s detective work. Authorities later found the virus in samples taken from the market’s tables and gutters. But, because not all of the first patients were tied to the market, nor were they connected to one another, Epstein said, “it raised the question of, well, perhaps those forty-one weren’t the first cases.”

Analyses of the SARS-CoV-2 genome indicate a single spillover event, meaning the virus jumped only once from an animal to a person, which makes it likely that the virus was circulating among people before December. Unless more information about the animals at the Wuhan market is released, the transmission chain may never be clear. There are, however, numerous possibilities. A bat hunter or a wildlife trafficker might have brought the virus to the market. Pangolins happen to carry a coronavirus, which they might have picked up from bats years ago, and which is, in one crucial part of its genome, virtually identical to SARS-CoV-2. But no one has yet found evidence that pangolins were at the Wuhan market, or even that venders there trafficked pangolins. “We’ve created circumstances in our world somehow that allows for these viruses, which would otherwise not be known to cause any problems, to get into human populations,” Mark Denison, the director of pediatric infectious diseases at Vanderbilt University Medical Center’s Institute for Infection, Immunology, and Inflammation, told me. “And this one happened to say, ‘I really like it here.’ ”

The new coronavirus is an elusive killer. Since people have never seen this strain before, there is much about it that remains a mystery. But, in just the past few weeks, genetic sleuthing, atomic-level imaging, computer modelling, and prior research on other types of coronaviruses, including SARS en MERS (Middle East Respiratory Syndrome), have helped researchers to quickly learn an extraordinary amount—particularly what might treat or eradicate it, through social-distancing measures, antiviral drugs, and, eventually, a vaccine. Since January, nearly eight hundred papers about the virus have been posted on BIORxiv, a preprint server for studies that have not yet been peer-reviewed. More than a thousand coronavirus genome sequences, from different cases around the world, have been shared in public databases. “It’s insane,” Kristian Andersen, a professor in the Department of Immunology and Microbiology at Scripps Research, told me. “Almost the entire scientific field is focussed on this virus now. We’re talking about a warlike situation.”

There are endless viruses in our midst, made either of RNA or DNA. DNA viruses, which exist in much greater abundance around the planet, are capable of causing systemic diseases that are endemic, latent, and persistent—like the herpes viruses (which includes chicken pox), hepatitis B, and the papilloma viruses that cause cancer. “DNA viruses are the ones that live with us and stay with us,” Denison said. “They’re lifelong.” Retroviruses, like H.I.V., have RNA in their genomes but behave like DNA viruses in the host. RNA viruses, on the other hand, have simpler structures and mutate rapidly. “Viruses mutate quickly, and they can retain advantageous traits,” Epstein told me. “A virus that’s more promiscuous, more generalist, that can inhabit and propagate in lots of other hosts ultimately has a better chance of surviving.” They also tend to cause epidemics—such as measles, Ebola, Zika, and a raft of respiratory infections, including influenza and coronaviruses. Paul Turner, a Rachel Carson professor of ecology and evolutionary biology at Yale University, told me, “They’re the ones that surprise us the most and do the most damage.”

Scientists discovered the coronavirus family in the nineteen-fifties, while peering through early electron microscopes at samples taken from chickens suffering from infectious bronchitis. The coronavirus’s RNA, its genetic code, is swathed in three different kinds of proteins, one of which decorates the virus’s surface with mushroom-like spikes, giving the virus the eponymous appearance of a crown. Scientists found other coronaviruses that caused disease in pigs and cows, and then, in the mid-nineteen-sixties, two more that caused a common cold in people. (Later, widespread screening identified two more human coronaviruses, responsible for colds.) These four common-cold viruses might have come, long ago, from animals, but they are now entirely human viruses, responsible for fifteen to thirty per cent of the seasonal colds in a given year. We are their natural reservoir, just as bats are the natural reservoir for hundreds of other coronaviruses. But, since they did not seem to cause severe disease, they were mostly ignored. In 2003, a conference for nidovirales (the taxonomic order under which coronaviruses fall) was nearly cancelled, due to lack of interest. Toe SARS emerged, leaping from bats to civets to people. The conference sold out.

SARS is closely related to the new virus we currently face. Whereas common-cold coronaviruses tend to infect only the upper respiratory tract (mainly the nose and throat), making them highly contagious, SARS primarily infects the lower respiratory system (the lungs), and therefore causes a much more lethal disease, with a fatality rate of approximately ten per cent. (MERS, which emerged in Saudi Arabia, in 2012, and was transmitted from bats to camels to people, also caused severe disease in the lower respiratory system, with a thirty-seven per cent fatality rate.) SARS-CoV-2 behaves like a monstrous mutant hybrid of all the human coronaviruses that came before it. It can infect and replicate throughout our airways. “That’s why it is so bad,” Stanley Perlman, a professor of microbiology and immunology who has been studying coronaviruses for more than three decades, told me. “It has the lower-respiratory severity of SARS en MERS coronaviruses, and the transmissibility of cold coronaviruses.”

Een rede daarvoor SARS-CoV-2 may be so versatile, and therefore so successful, has to do with its particular talent for binding and fusing with lung cells. All coronaviruses use their spike proteins to gain entry to human cells, through a complex, multistep process. First, if one imagines the spike’s mushroom shape, the cap acts like a molecular key, fitting into our cells’ locks. Scientists call these locks receptors. In SARS-CoV-2, the cap binds perfectly to a receptor called the ACE-2, which can be found in various parts of the human body, including the lungs and kidney cells. Coronaviruses attack the respiratory system because their ACE-2 receptors are so accessible to the outside world. “The virus just hops in,” Perlman told me, “whereas it’s not easy to get to the kidney.”

While the first SARS virus attached to the ACE-2 receptor, as well, SARS-CoV-2 binds to it ten times more efficiently, Kizzmekia Corbett, the scientific lead of the coronavirus program at the National Institutes of Health Vaccine Research Center, told me. “The binding is tighter, which could potentially mean that the beginning of the infection process is just more efficient.” SARS-CoV-2 also seems to have a unique ability, which SARS en MERS did not have, to use enzymes from our human tissue—including one, widely available in our bodies, named furin—to sever the spike protein’s cap from its stem. Only then can the stem fuse the virus membrane and the human-cell membrane together, allowing the virus to spit its RNA into the cell. According to Lisa Gralinski, an assistant professor in the Department of Epidemiology at the University of North Carolina at Chapel Hill, this supercharged ability to bind to the ACE-2 receptor, and to use human enzymes to activate fusion, “could aid a lot in the transmissibility of this new virus and in seeding infections at a higher level.”

Once a coronavirus enters a person—lodging itself in the upper respiratory system and hijacking the cell’s hardware—it rapidly replicates. When most RNA viruses replicate themselves in a host, the process is quick and dirty, as they have no proofreading mechanism. This can lead to frequent and random mutations. “But the vast majority of those mutations just kill the virus immediately,” Andersen told me. Unlike other RNA viruses, however, coronaviruses doen have some capacity to check for errors when they replicate. “They have an enzyme that actually corrects mistakes,” Denison told me.

It was Denison’s lab at Vanderbilt that first confirmed, in experiments on live viruses, the existence of this enzyme, which makes coronaviruses, in a sense, cunning mutators. The viruses can remain stable in a host when there is no selective pressure to change, but rapidly evolve when necessary. Each time they leap into a new species, for example, they are able to hastily transform in order to survive in the new environment, with its new physiology and a new immune system to battle. Once the virus is spreading easily within a species, though, its attitude is, “I’m happy, I’m good, no need to change,” Denison said. That seems to be playing out now in humans as SARS-CoV-2 circles the globe, there are slight variations among its strains, but none of them seem to affect the virus’s behavior. “This is not a virus that is rapidly adapting. It’s like the best car in the Indy 500. It’s out in front and there is no obstacle in its path. So there is no benefit to changing that car.”

A virus replicates in order to shed from its host—through mucus, snot, phlegm, and even our breath—as soon as possible, in great quantities, so that it can keep spreading. The coronavirus happens to be a brilliant shedder. A preprint study by German researchers, published earlier this month, and one of the first outside China to examine data from patients diagnosed with COVID-19, found clear evidence that infected people shed the coronavirus at significant rates before they develop symptoms. In effect—possibly due to that supercharged ability to bind and fuse to our cells—the virus wears an invisibility cloak. Scientists recently estimated that undocumented cases of COVID-19, or infected people with mild symptoms, are fifty-five per cent as contagious as severe cases. Another study found that in more severe cases (requiring hospitalization), patients shed the virus from their respiratory tracts for as long as thirty-seven days.

Outside a host, in parasitical purgatory, a virus is inert, not quite alive, but not dead, either. A hundred million coronavirus particles could fit on the head of a pin—typically, thousands or tens of thousands are necessary to infect an animal or a person—and they might remain viable for long stretches. Researchers at the Virus Ecology Unit of Rocky Mountain Laboratories, in Montana, a facility connected to the National Institute of Allergy and Infectious Diseases, have found that the virus can linger on copper for four hours, on a piece of cardboard for twenty-four hours, and on plastic or stainless steel for as long as three days. They also found that the virus can survive, for three hours, floating through the air, transmitted by the tiny respiratory droplets an infected person exhales, sneezes, or coughs out. (Other research suggests the virus might be able to exist as an aerosol, but only in very limited conditions.) Most virus particles, though, seem to lose their virulency fairly quickly. The infection window is highest in the first ten minutes. Still, the risk of infection has turned many of us, understandably, into germophobes.

All a virus wants is an endless chain of hosts. Contagion is the evolutionary end goal. Based on experiments so far, researchers estimate that COVID-19 is slightly more communicable than the common flu and less communicable than the most highly infectious viruses, like measles, with which a single sick person can infect around twelve other people. There are likely coronavirus super-spreaders—people who, for whatever reason, are almost entirely asymptomatic but transmit the disease to many other people. But pinning down an exact infection rate, at this point, is an impossible task. “We tend to focus on these absolute numbers as telling us how worried we should be,” Denison said. “Look, it’s like flooding. You know, is it up to my knees or is it up to my chin? It doesn’t matter. I need to do something to try to make sure I’m not gonna drive my car into the flood.”

In many places, we already have driven into the flood. As hundreds of people die each day, hospitals are running out of supplies, beds, and ventilators. In these severe COVID-19 cases, according to scientists’ current understanding, the disease may have more to do with a haywire immune response to the virus than anything else. Because the virus can gain a foothold in our lower respiratory system while still wearing that invisibility cloak, it “basically beats the immune system to the punch and starts replicating too rapidly,” Perlman said. When the immune system finally does register its presence, it might go into overdrive, and send everything in its arsenal to attack, since it has no specific antibodies to fight these strange new invaders. “It’s like pouring gas on the fire,” Denison told me. The lung tissue swells and fills with fluid. Breathing is restricted, as is the exchange of oxygen. “The host immune response just gets triggered to such an extreme level, and then builds on itself and builds on itself until ultimately the body kind of goes into shock,” Gralinski said. It is almost like an autoimmune disease the immune system is attacking parts of the body that it should not.

This type of response might be why the elderly are, on the whole, more vulnerable to COVID-19, just as they were to the SARS outbreak in 2003. (In that outbreak, there were almost no deaths among children under the age of thirteen, and, when kids did get sick, the disease was, on average, milder than what affected adults.) When studying SARS in mice models, Denison told me that he has observed a phenomenon known as “immune senescence,” in which older mice no longer had the capacity to respond in a balanced way to a new virus their immune systems’ overreaction then caused even more severe disease. This occurred in some of the worst cases during the first SARS outbreak, too, Denison said, and explains why antiviral drugs may be significantly more helpful at the onset of illness, before the immune system has had a chance to wreak havoc.

In the last decade, Denison’s lab and collaborators at the University of North Carolina have been researching antiviral treatments to try to find something that worked not just against SARS en MERS but for a new coronavirus which, they knew, would inevitably arrive. Together, they did much of the early research into the drug now known as Remdesivir, which is currently in development by Gilead and in studies on infected patients, and another antiviral drug compound, known as NHC. Both drugs, in animal models, were able to bypass, avoid, or block the coronavirus’s proofreading function, which helped stop the virus from replicating successfully in the body. “They worked very effectively against all the coronaviruses that we’ve tested,” Denison told me.

Coronaviruses likely have that proofreading enzyme because they are huge—one of the largest RNA viruses in existence—and they need a mechanism that maintains such a long genome’s structure. From our perspective, the benefit of such a big genome, Andersen told me, “is that the more genes and protein products a virus has, the more opportunities we have to design specific treatments against them.” For instance, the virus’s unique ability to use the human enzyme furin offers promise for antiviral drugs that act as furin inhibitors.

COVID-19, while still new to us hosts, will continue to be responsible for widespread infection and death. But, Epstein said, “Over time, as viruses evolve with their natural habitats, they tend to cause less severe disease. And that is good for both the host and the virus.” The more virulent strains might burn out (which, however, means many more awful deaths), while the remaining hosts might build up some immunity. More immediately, and urgently, the virus’s stability—how much it is thriving among us right now, and mutating only minimally—bodes well for the performance of antiviral drugs and, eventually, a vaccine. If the growing number of mitigation measures—this unprecedented national and international shutdown—are held in place for enough time, the speed at which the virus is spreading should slow, giving hospitals and health workers some relief. “The virus is our teacher,” Denison told me. It has spent thousands of years evolving to get where it is. We’re now just rushing to catch up.


The pace of evolution

Mutations may happen randomly, but the rate at which they occur depends on the virus. The enzymes that copy DNA viruses, called DNA polymerases, can proofread and fix errors in the resulting strings of genetic letters, leaving few mutations in each generation of copies.

But RNA viruses, like SARS-CoV-2, are the evolutionary gamblers of the microscopic world. The RNA polymerase that copies the virus’s genes generally lacks proofreading skills, which makes RNA viruses prone to high mutation rates—up to a million times greater than the DNA-containing cells of their hosts.

Coronaviruses have a slightly lower mutation rate than many other RNA viruses because they can do some light genetic proofreading. “But it’s not enough that it prevents these mutations from accumulating,” says virologist Louis Mansky, the director for the Institute for Molecular Virology at the University of Minnesota. So as the novel coronavirus ran amok around the world, it was inevitable that a range of variants would arise.

The true mutation rate of a virus is difficult to measure though. “Most of those mutations are going to be lethal to the virus, and you’ll never see them in the actively growing, evolving virus population,” Mansky says.

Instead, genetic surveys of sick people can help determine what’s known as the fixation rate, which is a measure of how often accumulated mutations become “fixed” within a viral population. Unlike mutation rate, this is measured over a period of time. So the more a virus spreads, the more opportunities it has to replicate, the higher its fixation rate will be, and the more the virus will evolve, Duffy says.

For SARS-CoV-2, scientists estimate that one mutation becomes established in the population every 11 days or so. But this process may not always happen at a steady pace.

In December 2020, the variant B.1.1.7 caught scientists’ attention when its 23 mutations seemed to suddenly crop up as the virus rampaged through Kent. Some scientists speculate that a chronically ill patient provided more opportunities for replication and mutation, and the use of therapies such as convalescent plasma may have pressured the virus to evolve. Not every change was necessarily useful to the virus, Duffy notes, yet some mutations that emerged allowed the variant to spread rapidly.


Meer inligting

Aansoeke

  • DNA labeling by nick translation
  • DNA end blunting of 5'- and 3'-overhangs
  • cDNA synthesis from DNA or RNA template

Bron

Berging

Unit definition

One unit is defined as the amount of enzyme that catalyzes the incorporation of 10 nmol of total nucleotides into acid-insoluble product in 30 minutes at 37°C and pH 7.4, using poly d(A-T) as the template-primer.

Konsentrasie

Product citations

Friedberg, E. C. The eureka enzyme: the discovery of DNA polymerase. Nat. Ds Mol. Sel Biol. 7, 143&ndash7 (2006).

Lehman, I. R., Bessman, M. J., Simms, E. S. & Kornberg, A. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J. Biol. Chem. 233, 163&ndash70 (1958).

Okayama, H. & Berg, P. High-efficiency cloning of full-length cDNA. Mol. Sel. Biol. 2, 161&ndash70 (1982).

Ricchetti, M. & Buc, H. E. coli DNA polymerase I as a reverse transcriptase. EMBO J. 12, 387&ndash96 (1993).

Rigby, P. W., Dieckmann, M., Rhodes, C. & Berg, P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237&ndash51 (1977).

Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular cloning : a laboratory manual. (Cold Spring Harbor Laboratory, 1989).

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