A Mechanism for the Activation of the Influenza Virus

Molecular Cell, Volume 70

Supplemental Information

A Mechanism for the Activation of the Inﬂuenza Virus Transcriptase Itziar Serna Martin, Narin Hengrung, Max Renner, Jane Sharps, Mónica MartínezAlonso, Simonas Masiulis, Jonathan M. Grimes, and Ervin Fodor

A

B

C

Figure S1. Detailed view of pS5-CTD binding sites on FluPolC. (A) Detailed view of the pS5-CTD peptide inside the omit electron density map (Sigma-A weighted Fo-Fc, 2.5σ, green mesh) for pS5-CTD binding site 1. (B) Same as (A) for pS5-CTD binding site 2. (C) Ribbon representation of the pS5-CTD binding site 2 in the P3 C-ter domain involving a groove formed by P3 β6 and the loop between P3 β11 and β12. The backbone of the 10residue poly-alanine chain built is shown in yellow. Colours of FluPolC subunits are as in Figure 1. Related to Figure 1.

α1

α2

α3

β1

C/Johannesburg/1/66 1

C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68

10

20

30

40

50

α4

α5

β2

80

β3

90

α6

100

110

η1 .

β5

α7

TT 130

140

150

π1

α8

α9

α10

α11

TT 160

170

180

190

200

210

220

230

240

ATAADFLRKENMFNEIFLPDNEETIIE..MKKGKTFLELRDESVPLPFQTYEQMKDYCEKFKGNPRELASKVSQMQSNIKLPIKHYEQNK TELQAELSLKNLWQVLIGEEDVEKGID..FKLGQTISRLRDISVPAGFSNFEGMRSYIDNIDPKG.AIERNLARMSPLVSATPKKLKWED FVLRQELATAGLWDSFRQSEKGEETLEEEFSYPPTFQRLANQSLPPSFKDYHQFKAYVSSFKANG.NIEAKLGAMSEKVNAQIESFDPRT FTIRQEMASRGLWDSFRQSERGEETIEERFEITGTMRRLADQSLPPNFSSLENFRAYVDGFEPNG.CIEGKLSQMSKEVNARIEPFLKTT FTIRQEMANRGLWDSFRQSERGEETIEERFEITGTMRRLADQSLPPNFSCLENFRAYVDGFEPNG.YIEGKLSQMSKEVNAKIEPFLKTT

β6 C/Johannesburg/1/66

α12

α13 .......

T.T 250


β4

120

TAVSLQNELCKSYDLEPLPFLCDIFDREEKQFVEIGITRKADDSYFQSKFGKLG.NSCKIFVFSYDGR.LDKNCEGPM.EEQKLRIFSFL IAWMVQRSLAQEHGIETPKYLADLFDYKTKRFIEVGITKGLADDYFWKKKEKLG.NSMELMIFSYNQD.YSLSNESSLDEEGKGRVLSRL IAWTIVNSICNMTENSKPRFLPDLYDYKTNKFIEIGVTRRKVEDYYYEKASKLKGENVYIHIFSFDGEEMATDDEYILDEESRARIKTRL MAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSEKTHIHIFPFTGEEMATKADYTLDEESRARIKTRL MAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSENTHIHIFSFTGEEMATKADYTLDEESRARIKTRL

C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68

70

MSKTFAEIAEAFLEPEAVRIAKEAVEEYGDH....ERKIIQIGIHFQVCCMFCDEY.LSTNG...............SDRFVLIEGRKRG MDTFI....TRNFQTTIIQKAKNTMAEFSEDPELQPAMLFNICVHLEVCYVISDMNFLDEEGKSYTALEGQGKEQNLRPQYEVIEGMPRT MENFV....RTNFNPMILERAEKTMKEYGENPQNEGNKFAAISTHMEVCFMYSDFHFIDLEGNTIVK.ENDDDNAMLKHRFEIIEGQERN MEDFV....RQCFNPMIVELAEKAMKEYGEDPKIETNKFAAICTHLEVCFMYSDFHFIDERGESIIV.ESGDPNALLKHRFEIIEGRDRT MEDFV....RQCFNPMIVELAEKAMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIVV.ELDDPNALLKHRFEIIEGRDRT


60

260

270

280

η2 TT 290

300

. 310

FRQI.......RLPKGPMAPYTHKFLME.EAWMFTKISDPERSRAGEILI.......DFFKKG...NLSAIRPKDKPLQGKYP.IHYKNL LRPIGPHIYNHELPEV...PY.NAFLLMSDELGL...ANMTEGKSKKPKTLAKECLEKYSTLRDQTDPILIMKSEKA.NENFLWKLWRDC IREL.......ELPEGKFCTQRSKFLLM.DAMKLS.VLNPAHEGEGIPMKDAKACLDTFWGWK...KATIIKKHEKGVNTNYL.MIWEQL PRPL.......RLPDGPPCSQRSKFLLM.DALKLS.IEDPSHEGEGIPLYDAIKCMKTFFGWK...EPNIIKPHEKGINPNYL.LAWKQV PRPI.......RLPDGPPCFQRSKFLLM.DALKLS.IEDPSHEGEGIPLYDAIKCMRTFFGWK...EPYIVKPHEKGINPNYL.LSWKQV

α14

α15

β7

α16

α17



330

340

350

360

370

380

390

400

WNQIKAAIAD....RTMVINENDHSEFLGGIGRASKKIPEISLTQDVITTEGLKQSENKLPEPRSFPRWFNAEWMWAIKDSDLTGWVPMA VNTI...SNE....MSNELQKTNYAKWATGDGLTYQKI....MKEVAIDDETMCQEEPKIPNKCRVAAWVQTEMNLLSTLTS.KRALDLP LESIKEMEG.....KFLNLKKTNHLKWGLGEGQAPEKM....DFEDCKEVPDLFQYKSEPPEKRKLASWIQSEFNKASELTN.SNWIEFD LAELQDIENEEKIPKKKNMKKTSQLKWALGENMAPEKV....DFEDCKDVSDLKQYDSDEPEPRSLASWIQSEFNKACELTD.SSWIELD LAELQDIENEEKIPRTKNMKKTSQLKWALGENMAPEKV....DFDNCRDVSDLKQYDSDEPELRSLSSWIQNEFNKACELTD.STWIELD

α18

α19

β8

β9



420

430

440

450

460

470

480

490

EYPPADNELEDYAEHLNKTMEGVLQGTNCAREMGKCILTVGALMTECRLFPGKIKVVPIYARSKERKSMQEGLPVPSEMDCLFGICVKSK EIGPDVAPVEHVGSERRKYFVNEINYCKASTVMMKYVLFHTSLLNESNASMGKYKVIPITNRVVNEKG........ESFDMLYGLAVKGQ ELGNDVAPIEHIASRRRNFFTAEVSQCRASEYIMKAVYINTALLNSSCTAMEEYQVIPIITKCRDTSG........QRRTNLYGFIIKGR EIGEDVAPIEHIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCRTKEG........RRKTNLYGFIIKGR EIGEDVAPIEYIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCRTKEG........RRKTNLYGFIIKGR

β10

β11

β12

α20

α21



510

530

540

550

560

570

580

SHLNKDDGMYTIITFEFSIREPNLEKH..QKYTVFEAGHTTVRMKKGESVIGREVPLYLYCRTTALSKIKNDWLSKARRCFITTMDTVET SHLRGDTDVVTVVTFEFSSTDPRVDSGKWPKYTVFRIGSLFVS........GREKSVYLYCRVNGTNKIQMKWGMEARRCLLQSMQQMEA SHLRNDTDVVNFISLEFSLTDPRNEIHKWEKYCVLEIGDMEIRTSIS....TIMKPVYLYVRTNGTSKIKMKWGMEMRRCLLQSLQQVES SHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRTAIG....QVSRPMFLYVRTNGTSKIKMKWGMEMRRCLLQSLQQIES SHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIG....QMSRPMFLYVRTNGTSKIKMKWGMEMRRCLLQSLQQIES

α22 .....



520

β13 .

600

β14

α23

α24

β15

TT 610

620

630

640

650

660

ICLRESAKAEENLVEKTL.....NEKQMW.IGKKNGELIAQPLREALRVQLVQQFYFCIYNDSQLEGFCNEQKKILMALEGDKKNKSSFG IVEQESSIQGYDMTKACFKGDRVNSPKTFSIGTQEGKLVKGSFGKALRVIFTKCLMHYVFGNAQLEGFSAESRRLLLLIQALKDRKGPWV MIEAESAVKEKDMTEPFFR....NRENDWPIGESPQGIEKGTIGKVCRVLLAKSVFNSIYASAQLEGFSAESRKLLLLIQAFRDNLDPGT MIEAESSVKEKDMTKEFFE....NKSETWPIGESPKGVEEGSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRRLLLIVQALRDNLEPGT MIEAESSVKEKDMTKEFFE....NKSETWPIGESPKGVEDGSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLVVQALRDNLEPGT

α25

α26



680

690

700

FNPEGLLEKIEECLINNPMCLFMAQRLNELVIEASKRGAKFFKTD...... FDLEGMYSGIEECISNNPWVIQSAYWFNEWLGFE.KEGSKVLESVDEIMDE FDLKGLYEAIEECIINDPWVLLNASWFNSFLKAV.Q..........LSM.. FDLGGLYEAIEECLINDPWVLLNASWFNSFLTHA.L............R.. FDLEGLYEAIEECLINDPWVLLNASWFNSFLTHA.L............R..

Figure S2. Amino acid sequence alignment of FluPol PA/P3 subunit. Amino acid sequence alignment for the PA/P3 subunit from influenza C/Johannesburg/1/66, B/Panama/45/90, A/bat/Guatemala/060/10, A/duck/Fujian/01/02 and A/New Territory/60/68. Highly conserved residues are shown in red, partially conserved residues in yellow. Amino acid numbering and secondary structure correspond to C/Johannesburg/1/66. Residues proximal to FluPolC pS5-CTD binding site 1 are highlighted by green arrows. The residues in FluPolA and FluPolB PA involved in pS5-CTD binding reported by Lukarska et al., 2017 are highlighted. Purple arrows indicate residues at site 1, which are conserved in influenza A and B viruses; blue arrows indicate residues at site 2, conserved only in influenza A viruses. Related to Figure 1 and Figure 2.

β1

η1

α1

η2

α2

C/Johannesburg/1/66

β2

β3

TT 1


10

TTT

20

30

40

50

60

70

80

90

MEINPYLMFLNNDVTSLISTTYPYTGPPPMSHGSSTKYTLETIKRTYDYSRTSVEKTSKVFNIPRRKFCNCLEDKDELVKPTGNVDISSL MNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTGTGHTIDTVIRTHEYSNKGKQYVSDVTGCTMVDPTNGPL..PEDNEPSAYAQLDCV MDVNPMLIFLKVPVQNAISTTFPYTGDPPYSHGTGTGYTMDTVIRTHDYSSRGIWKTNSETGAQQLNPIDGPL..PEDNEPSGYAQTDCV MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPIDGPL..PDNHEPSGYAQTDCV MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPIDGPL..PEDNEPSGYAQTDCV

α3

α4

η3

β4

β5

α5

140

150

α6



110

120

130

160

170

LGLAEMMEKRMGEGFFKHCVMEAETEILKMHFSRLTEGRQTYDWTSERNMPAATALQLTVDAIKETEGPF.KGTTMLEYCNKMIEMLDWK LEALDRMDEE.HPGLFQAASQNAMEALMVTTVDKLTQGRQTFDWTVCRNQPAATALNTTITSFRLNDLNGADKGGLVPFCQDIIDSLDKP LELIEGLDRS.HPGLFETACQETIDAIQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIEVFRKNGYKLNESGRLIDFLKDVLLSFEND LEAMAFLEES.HPGIFENSCLETMEVVQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIEVFRSNDLTANESGRLIDFLKDVMESMDKE LEAMAFLEES.HPGIFENSCLETMEVVQQTRVDRLTQGRQTYDWTLNRNQPAATALANTIEVFRSNGLTANESGRLIDFLKDVMESMDKE

β6

β7

α7

β8

β9

β10

α8

C/Johannesburg/1/66

TTT 180


190

200

210

220

230

240

250

260

EIKFKKVKTVVRREKDKRSGKEIKTKVPVMGIDSIKHDEFLIRALTINTMAKDGERGKLQRRAIATPGMIVRPFSKIVETVAQKICEKLK EMTFFSVKNIKKKLPAKNRKGFLIKRIPMKVKDRITRVEYIKRALSLNTMTKDAERGKLKRRAIATAGIQIRGFVLVVENLAKNICENLE SMEVTTHFQKKKRIRDNHSKKMITQRTIGKKRVKLTKKNYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVELLARNICERLE EMEITAHFQRKRRIRDNMTKKMVTQRTIGKKKQRLNKKSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLE EMEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQRVNKRSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLE

α9

β11

α10

α11

α12

β12

β13



280

290

β14

α13

TT 360

370

η4 .

α14

330

340

350

β15

390

α15

400

410

β16

420

430

TT 440

SNKRKTKEVIIKAEKMGKYK.NLMREEYKNLFEPLEKYIQ.KDVCFLPGGMLMGMFNMLSTVLGVSTLCYMDEELKAKGCFWTGLQSSDD TSKTKRLKAQIPCPDLFSIPLERYNEETRAKLKKLKPFFNEEGTASLSPGMMMGMFNMLSTVLGVAAL..GIKNIGNKEYLWDGLQSSDD ESKSMHLRTQISAENLSDINLRYFNEDTKKKIEKIRHLMV.EGTASLSPGMMMGMFNMLSTVLGVSVLNLGQREILKRTYWWDLLQSSDD ESKSMKLRTQIPAEMLANIDLKYFNESTRKKIEKIRPLLI.DGTASLSPGMMMGMFNMLSTVLGVSILNLGQKRYTKTTYWWDGLQSSDD ESKSMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLI.DGTASLSPGMMMGMFNMLSTVLGVSILNLGQKRYTKTTYWWDGLQSSDD

β17

α16

β18 TTT

450

460

470

β19 TT

480

β20

η5

η6

α17

TT

490

500

510

520

530

FVLFAVASNWSNIHWTIRRFNAVCKLIGINMSLEKSYGSLPELFEFTSMFFDGEFVSNLAMELPAFTTAGVNEGVDFTAAMSIIKTNMIN FALFVNAKDEETCMEGINDFYRTCKLLGINMSKKKSYCNETGMFEFTSMFYRDGFVSNFAMEIPSFGVAGVNESADMAIGMTIIKNNMIN FALIINGHFKEDIQQGVNHFYRTCKLVGINMSQKKSYINKTGTFEFTSFFYRYGFVANFSMELPSFGVAGNNESADMSIGTTVIKTNMIN FALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINRTGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMIN FALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINRTGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMIN

α18

α19

C/Johannesburg/1/66

α20

α21

TT 540


320

TT

380


310

ESGLPVGGNEKKAKLKTTVTSLNARMNSDQFAVNITGDNSKWNECQQPEAYLALLAYITKDSSDLMKDLCSVAPVLFCNKFVKLGQGIRL QSGLPVGGNEKKAKLSNAVAKMLSNCPPGGISMTVTGDNTKWNECLNPRIFLAMTERITRDSPIWFRDFCSIAPVLFSNKIARLGKGFMI QSGLPVGGNEKKAKLANVIKKMMAKSTDEELSYTITGDNTKWNENQNPRIFLAMVLRITAGQPEWFRDLLAVAPIMFSNKVARLGRGYMF QSGLPVGGNEKKAKLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRMFLAMITYITRNQPDWFRNVLSIAPIMFSNKMARLGKGYMF QSGLPVGGNEKKAKLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRMFLAMITYITKNQPEWFRNVLSIAPIMFSNKMARLGKGYMF


300

550

560

570

580

590

600

610

620

NSLSPSTALMALRICLQEFRATYRVHPWDSRVKGGRMKIINEFIKTIENKDGLLIADGGKLMNNISTLHIPEEVLKFEKMDEQYRNRVFN NGMGPATAQTAIQLFIADYRYTYKCHRGDSKVEGKRMKIIKELWENTKGRDGLLVADGGPNIYNLRNLHIPEIVLKYNLMDPEYKGRLLH NDLGPATAQMAIQLFIKDYRYTYRCHRGDTNLETRRTKSIKRLWTETISKAGLLVADGGPNPYNLRNLHIPEVCLKWSLMDPDYRGRLCN NDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCN NDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCN

α22 C/Johannesburg/1/66 630


640

650

660

670

680

690

700

710

PKNPFTNFDK.....TIDIFRAHGPIRVEENEAVVSTHSFRTRANRTLLNTDMRAMMAEEKRYQMVCDMFKSVFESADINPPIGAMSIGE PQNPFVGHLSIEGIKEADITPAHGPVKKMDYDAVSGTHSWRTKRNRSILNTDQRNMILEEQCYAKCCNLFEACFNSASYRKPVGQHSMLE PNNPFVHHMEVESTNLAVVMPAHGPAKSLEYDAVATTHSWTPKRNRSILNTNQRGILEDERIYQKCCQVFEKFFPSSTYRRPIGMASMLD PLNPFVSHKEIESANNAVVMPAHGPAKSMEYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFFPSSSYRRPVGISSMVE PLNPFVSHKEIESVNNAVVMPAHGPAKSMEYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFFPSSSYRRPVGISSMVE

α23

α24



730

740

750

AIEEKLLERAKMKRDIGAIEDSEYEEIKDIIRDAKKARLESR AMAHRLRMDARLDYESGRMSKDDFEKAMAHLGEIGYI..... AMLSRARIDARIDLESGRISSQDFSEITNTCKAIEALKR..Q AMVSRARIDARIDFESGRIKKEEFAEIMKICSTIEELRRQ.K AMVSRARIDARIDFESGRIKKEEFAEIMKICSTIEELRRQ.K

Figure S3. Amino acid sequence alignment of FluPol PB1 subunit. Amino acid sequence alignment for the PB1 subunit from influenza C/Johannesburg/1/66, B/Panama/45/90, A/bat/Guatemala/060/10, A/duck/Fujian/01/02 and A/New Territory/60/68. Highly conserved residues are shown in red, partially conserved residues in yellow. Amino acid numbering and secondary structure correspond to C/Johannesburg/1/66. Residues proximal to FluPolC pS5-CTD binding site 1 are highlighted by green arrows. Related to Figure 1 and Figure 2.

Figure S4. Cryo-EM analysis of FluPolC bound to vRNA promoter. (A) The structure of apo FluPolC in transcriptionally inactive conformation (PDB: 5D98) was fit into the cryo-EM density of FluPol. Two possible fits of the crystal structure are shown: in fit 1, the PA endonuclease domain is placed inside a small peripheral density, which leaves large areas of the density volume unoccupied (red arrow); in fit 2, the PB2 627 domain is placed inside the small density, leaving the endonuclease domain protruding outside the density. (B) Local resolution of the vRNA promoter bound FluPolC cryo-EM density map. Note that the third panel shows a distinct patch of low resolution. (C) Particle orientation distribution in the vRNA promoter bound FluPolC cryo-EM dataset showing two main populations with reduced coverage of other views. Orientation distribution plot generated in cryoSPARC (Punjani et al., 2017). Related to Figure 3.

α1 C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68

α2

α3

α4

α5 .

.. 1

10

20

30

40

50

60

70

80

MS..LLLTIAKEYKRLCQDAKAAQMMTVGTVSNYTTFKKWTTSRKEKNPSLRMRWAMSSKFPIIANKRML.EEAQIP.KEHNNVALWEDT MTLAK....IELLKQLLRDNEAKTVLKQTTVDQYNIIRKFNTSRIEKNPSLRMKWAMCSNFPLALTKGDMANR..IP.LEYKGIQLKTNA ME..R....IKELMEMVKNSRMREILTTTSVDHMAVIKKYTSGRQEKNPALRMKWMMAMKYPISASSRIR.EM..IPEKDEDGNTLWTNT ME..R....IKELRDLMSQSRTREILTKTTVDHIAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRIM.EM..IPERNEQGQTLWSKT ME..R....IKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKRIT.EM..VPERNEQGQTLWSKM

α6

α7

η1

α8

β1

β2

α9

C/Johannesburg/1/66

TT 90

100

110

120

130

140

150

160

170


EDVSKRDHVLASASCINYWNFCGPCVNNSEVIKEVYKSRFGRLERRKEIMWKELRFTLVDRQRRRVDTQPVEQRLRTGEIKDLQMWTLFE EDIGT.KGQMCSIAAVTWWNTYGPIGD.TEGFEKVYESFFLRKMRLDNATWGRITFGPVERVRKRVLLNPLTKEMPPDEASNVIMEILFP KDAGS.NRVLVSPNAVTWWNRAGPVSD.VVHYPRVYKMYFDRLERLTHGTFGPVKFYNQVKVRKRVDINPGHKDLTSREAQEVIMEVVFP NDAGS.DRVMVSSLAVTWWNRNGPTTS.TVHYPKVYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDVNPGHADLSAKEAQDVIMEVVFP SDAGS.DRVMVSPLAVTWWNRNGPMTS.TVHYPKVYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDINPGHADLSAKEAQDVIMEVVFP

C/Johannesburg/1/66

TT

α10 180


α11 190

α12

200

α15

α16

α13 230

α14

β4

240

250

β5

β6

α17

α18

β7

TT 270

280

290

260

β8

β9

TT

300

310

320

330

340

350

RAVCRKVCLAANASIMNAKSKLVEYIKSTSMRIGETERKLEELILETDDVSPEVTLCKSALGGQLGKTLSFGPMLLKKISGSGVKVKDTV IVACRKIIRRSIVAS.NPLELAVEIANKTV..IDTE..PLKSCLTAIDGGDVACDIIRAALGLKIRQRQRFGRLELKRISGRGFKNDEEI IIASRNIVRRSIVAI.DPLASLLSMCHTTS..ISSE..PLVEILRSNPTDEQAVNICKAALGIRINNSFSFGGYNFKRVKGSSQRTEKAV IIAARNIVRRATVST.DPLASLLEMCHSTQ..IGGI..RMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSVKKEEEV IIAARNIVRRAAVSA.DPLASLLEMCHSTQ..IGGT..RMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSIKREEEL 1 1 β10

β11

C/Johannesburg/1/66

β12

β13

TT 360


220

DEAPLASKFILDNYGLVKEMRSKFANKPLNKEVVAHMLEKQFNPESRFLPVFGAIRPERMELIHALGGETWIQEANTAGISNVDQRKNDI KEAGIPRESTWIHRELIKEKREKLKGTMITPIVLAYMLERELVARRRFLPVAGATSAEFIEMLHCLQGENWRQIYHPGGNKLTESRSQSM NEVGARTLSSDAQLTITKEKKEELKNCKISPIMVAYMLERELVRRTRFLPIAGATSSTYVEVLHLTQGTCWEQQYTPGGEAENDDLDQTL NEVGARILTSESQLTITKEKKEELQDCKIAPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSL NEVGARILTSESQLTITKEKKEELQDCKISPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSL


β3

210

370

α19

α20

β14

β15

TT

380

390

400

410

420

430

440

YIQGVRAVQFEYWSEQEEFYGEYKSATALFSRKERSLEWITIGGGINEDRKRLLAMCMIFCRDGDYFKDAPATITMADLSTKLGREIPYQ LIGNGTIQKIGIWDGEEEFHVRCGECRGILKKSKMRMEKLLINSAKKEDMKDLIILCMVFSQDTRMFQGVRGEINFLNRAGQ...LLSPM LTGNLQTLTMTIFEGYEEFNVSGKRASAVLKKGAQRLIQAIIGGRTLEDILNLMITLMVFSQEEKMLKAVRGDLNFVNRANQ...RLNPM LTGNLQTLKIRVHEGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQ...RLNPM LTGNLQTLKIRVHDGYEEFTMVGKRATAILRKATRRLVQLIVSGRDEQSVAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQ...RLNPM

α21

α22

β16

β17

β18

β19

β20



460

470

480

490

500

510

520

530

YVMMNWIQKSEDNLEALLYSRGIVETNPGKMGSSMGI..DGSKRAIKSLRAVTIQSGKIDMPESKEKIHLELSDNLEAFDSSGRIVATIL YQLQRYFLNRS...NDLFDQWGYEESPK.ASE.LHGINEL.MNASDYTLKGVVVTKNVIDDFSSTETEKVSITKNLSLIKRTGEVIMGAN YQLLRHFQKDS...STLLKNWGTEEIDP.IMG.IAGIMPDGTINKTQTLMGVRLSQGGVDEYSFNERIRVNIDKYLRVRNEKGELLISPE HQLLRHFQKDA...KVLFQNWGIEPIDN.VMG.MIGILPDMTPSTEMSLRGVRVSKMGVDEYSSTERVVVSIDRFLRVRDQRGNVLLSPE HQLLRHFQKDA...KVLFQNWGIEHIDN.VMG.MIGVLPDMTPSTEMSMRGIRVSKMGVDEYSSTERVVVSIDRFLRVRDQRGNVLLSPE

α23

α24

α25

α26

η2

η3

α27



550

580

590

600

610

α28

η4

β21

620

β22

TT

TT

630

640

650

660

670

680

690

TT 700

TQV.TTGHYSLHELIKVLPFTYFAPKQGMF..EGRLFF.....SNDSFVEPGVNNNVFSWSKADSSKIYCHGIAIRVPLVV..GDEHMDT QMRDQ.EVMKTDQFIKLLPFCFSPPKLRSNGEPYQFLRLVLKGGGENFIEVRKGSPLFSYNPQTEVL.TICGRMMSLKGKIEDEERNRSM QMRDVEGTFDSIQIIKLLPFSAHPPSLGRT..QFSSFTLNIRGAPLR.LLIRGNSQVFNYNQMENVI.IVLGKSVGSPERSI.LTESSSI QMRDVVGTFDTVQIIKLLPFTAAPPEQSRM..QFSSLTVNVRGSGMR.ILVRGNSPVFNYNKATKRL.TVLGKDAGALTEDP.DEGTAGV QMRDVLGTFDTTQIIKLLPFAAAPPKQSRM..QFSSLTVNVRGSGMR.ILVRGNSPAFNYNKTTKRL.TILGKDAGTLIEDP.DEGTSGV

α29

β23

η5

C/Johannesburg/1/66

β24 TT

710


570

DLPSDKKVTFQDVSFQHPDLAVLRDEKTAITKGYEALIKRLGTGDNDIPSLIAKKDYLSLYNLPEVKLMAPLIRPNRKGVYSRVARKLVS DVSELESQAQLMITYDTPKMWEMGTTKELVQNTYQWVLKNLVTLK.....AQFLLGKEDMFQWDAFEAFESIIPQKMAGQYSGFARAVLK EVSEAQGQEKLPINYNSSLMWEVNGPESILTNTYHWIIKNWELLK.....TQWMTDPTVLYNRIEFEPFQTLIPKGNRAIYSGFTRTLFQ EVSETQGTERLTITYSSSMMWEINGPESVLVNTYQWVIRNWETVK.....IQWSQDPTMLYNKMEFEPFQSLIPKAARGQYSGFVRTLFQ EVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETVK.....IQWSQNPTMLYNKMEFEPFQSLVPKAIRGQYSGFVRTLFQ


560

720

730

β25

α30

TT 740

750

760

770

SLALLEGFSVCE..NDPRAPMVTRQDLIDVGFGQKVRLFVGQGSVRTFKRTASQRAASSDVNKNVKKIKMSN........ GNAVLAGFLVSGKYDPDLGDFKTIEELEKLKPGEKANILLYQGKPVKVVKRKRYSALSNDISQGIKRQRMTVESMGWALS ESAVLRGFLILGKANSKYGPVLTIGELDKLGRGEKANVLIGQGDTVLVMKRKRDSSILTDSQTALKRIRLEESK...... ESAVLRGFLILGKEDKRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAI.N...... ESAVLRGFLILGKEDRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAI.N......

Figure S5. Amino acid sequence alignment of FluPol PB2 subunit. Amino acid sequence alignment for the PB2 subunit from influenza C/Johannesburg/1/66, B/Panama/45/90, A/bat/Guatemala/060/10, A/duck/Fujian/01/02 and A/New Territory/60/68. Highly conserved residues are shown in red, partially conserved residues in yellow. Amino acid numbering and secondary structure correspond to C/Johannesburg/1/66. Residues proximal to FluPolC pS5-CTD binding site 1 are highlighted by green arrows. Related to Figure 4 and Figure 5.

Relative(RNA(levels

1.5

**

1.0

0.5

0.0

** ** BG

'

5 '3 '

5 '3 '*p S 5

5 '3 '*p S 2

5 '3 '*U n P

5 '3 '*S c r

Figure S6. Effect of Pol II CTD peptides on FluPolA transcriptional activity. In vitro cap-dependent transcription initiation assay performed using purified FluPolA in the presence of pS5, pS2, un-phosphorylated (UnP) or scrambled (Scr) Pol II CTD peptides. Assay was carried out in the absence or presence of 5' and 3' vRNA promoter RNAs. The mean of three independent experiments is shown with error bars representing standard deviation. Asterisks indicate a significant difference from the reaction in absence of CTD peptide (**P < 0.01, based on a two-sample t-test). Related to Figure 6.