Table S1. DNA oligonucleo3des used for 3D pol mutagenesis. Fingers Domain Entry Channel. Fidelity

Transcription

1 Table S1. DNA oligonucleo3des used for 3D pol mutagenesis. Fingers Domain Entry Channel Fidelity Charged Residues Interac9ng with Template and Product RNAs Thumb Domain 3D pol Mutations Parental codon Mutant codon Progeny P (1) virus codon Amino Acid in Progeny Virus Forward Primer Reverse Primer Y14A uau gcc gcc ala cct tcg aag gaa gtg gga gcc cca atc ata aat gcc ggc a& tat gat tgg ggc tcc cac &c c& cga agg Y118A uac gcg aug met cc acc agt gct ggc gcg cct tat gta gca atg gg cc cat tgc tac ata agg cgc gcc agc act ggt gg Y118F uac uuu uuu phe cca cca gtg ctg gct <c c& atg tag caa tgg g ccc a& gct aca taa gga aag cca gca ctg gtg g Y148A uau gcc guc val g caa aaa ctg ctc gac aca gcc gga atc aac ctc cca ctg g c cag tgg gag g& gat tcc ggc tgt gtc gagg cag &t &g c Y148F uau uuu uuu phe g caa aaa ctg ctc gac aca <t gga atc aac ctc cca ctg g c cag tgg gag g& gat tcc aaa tgt gtc gagg cag &t &g c Y157A uau gcc uau tyr ctc cca ctg gtg act gcc gta aag gat gaa c& aga tcc gga tct aag &c atc c& tac ggc agt cac cag tgg gag Y157F uau uuu uuu phe ctc cca ctg gtg act <t gta aag gat gaa c& aga tcc gga tct aag &c atc c& tac aaa agt cac cag tgg gag Y118A-Y148A uac - uau gcg - gcc uac - uac tyr - tyr sequenctial mutagenesis with primers above sequenctial mutagenesis with primers above Y118A-Y148F uac - uau gcg - uuu uuu - uuu phe - phe sequenctial mutagenesis with primers above sequenctial mutagenesis with primers above Y118F-Y148A uac - uau uuu - gcc uuu - guc phe - val sequenctial mutagenesis with primers above sequenctial mutagenesis with primers above Y118F-Y148F uac - uau uuu - uuu uuu - uuu phe - phe sequenctial mutagenesis with primers above sequenctial mutagenesis with primers above G64S ggu uca uca ser ggc aat &t ctc caa gta cgt gtc aaa caa aat tac tga agt gg cca c& cag taa &t tgt ttg aca cgt act tgg aga aaa &g cc G64T ggu acc acc* thr* ggc aat &t ctc caa gta cgt gac caa caa aat tac tga agt gg cca c& cag taa &t tgt tgg tca cgt act tgg aga aaa &g cc G64D ggu gau ggu gly ggc aat &t ctc caa gta cgt gga taa caa aat tac tga agt gg cca c& cag taa &t tgt tat cca cgt act tgg aga aaa &g cc K125A aag gcc gcc ala gca atg gga gcc aag aag aga gac atc &g aac aaa caa acc ggt &g &t g& caa gat gtc tct c& c& ggc tcc cat tgc K126A aag gcc gcc ala gca atg gga aag gcc aag aga gac atc &g aac aaa caa acc ggt &g &t g& caa gat gtc tct c& ggc c& tcc cat tgc K125A-K126A aag - aag gcc - gcc mixed mixed gca atg gga gcc gcc aag aga gac atc &g aac aaa caa acc ggt &g &t g& caa gat gtc tct c& ggc ggc tcc cat tgc K127A aag gcc aag lys gca atg gga aag aag gcc aga gac atc &g aac aaa caa acc ggt &g &t g& caa gat gtc tct ggc c& c& tcc cat tgc R128A aga gcc gcc ala gca atg gga aag aag aag gcc gac atc &g aac aaa caa acc ggt &g &t g& caa gat gtc ggc c& c& c& tcc cat tgc K127A-R128A aag - aga gcc - gcc aag - gcc lys - arg gca atg gga aag aag gcc gcc gac atc &g aac aaa caa acc ggt &g &t g& caa gat gtc ggc ggc c& c& tcc cat tgc K133A aaa gcc gcc ala gac atc &g aac gcc caa acc aga gac act aag gaa atg c g cat &c c& agt gtc tct ggt &g ggc g& caa gat gtc R188A aga gcc aga arg gac tca gtg gca atg gcc atg gct &t ggg aac c ggt tcc caa aag cca tgg cca &g cca ctg agt c K276A aag gcc gcc ala c cac ctg tac gcc aat aaa aca tac tgt gtc aag ggc gcc c& gac aca gta tgt &t a& ggc gta cag gtg g N409A aac gcg gcg ala gga cta aag atc cta gg gcg act cag gat cac g c gtg atc ctg agt cgc cc tag gat c& tag tcc D412A gau gcg gcg ala cct agg aac act cag gcg cac g& cgc tct ctg tgc gca cag aga gcg aac gtg cgc ctg agt g& cct agg S416A ucu gcg gcg ala cag gat cac g& cgc gcg ctg tgc c& &a gct tgg c gcc aag c taa aag gca cag cgc gcg aac gtg atc ctg L419A cuu gcc gcc ala cgc tct c& tgc gcc &a gct tgg cac aat ggc gcc a& gtg cca agc taa ggc gca aag aga gcg L420A uua gcc gcc ala cgc tct c& tgc c& gcc gct tgg cac aat ggc gcc a& gtg cca agc ggc aag gca aag aga gcg * G64T mutation was stable based on cdna sequence of P (1) virus populations; however, this mutation causes polyprotein processing defects (Figure S2), decreased titers following transfection as compared to wildtype, and mixed plaque size phenotypes (Table 1). Furthermore, upon additional passage in HeLa cells the G64T mutation reverted to an alanine residue (GCC codon).

2 Table S2. RNA and DNA oligonucleotides. RNA linker 5 phos-cuacgacagu CACCACCGAU ACCCUGUACU ACGCACCACG-3 Linker cdna Primer 5 -CGTGGTGCGT AGTACAG-3 Linker Amplification Primer 5 -CGGTGGTGAC TGTCGT-3 Poliovirus DNA primers Forward Primer GGCAATGACAATTCTACAAGC Forward Primer CTCGGTGGGCGCGAAACTGCTCGTA Forward Primer GAGGATGCCATGTCTGGC Forward Primer GGTATGATGCATCTCTCAGCC Forward Primer GGACTAAAGATCCTAGG Reverse Primer CCCTACAACA GTATGACC Reverse Primer CTCCGAATTA AAGAAAAATT TACCCC EMCV Primers Forward Primer GGTGTTGTCG TACGGAGATG ATCTCC

3 Table S3. Picornavirus 3Dpol residues mutated in study of viral RNA polyadenyla=on. Picornavirus sequence analysis based on bioinforma4cs data from Palmenberg et al. (2009), Knowles et al. (2011) and the Picornaviridae Home Page at hcp:// Palmenberg, A.C., Spiro, D., Kuzmickas, R., Wang, S., Djikeng, A., Rathe, J.A., Fraser- LiggeC, C.M. & LiggeC, S.B. (2009). Sequencing and analyses of all known human rhinovirus genomes reveal structure and evolu4on. Science 324, Knowles, N.J., Hovi, T., Hyypiä, T., King, A.M.Q., Lindberg, M., Pallansch, M.A., Palmenberg, A.C., Simmonds, P., Skern, T., Stanway, G., Yamashita, T. & Zell, R. (2011). Picornaviridae. In Virus Taxonomy: Classifica4on and Nomenclature of Viruses: Ninth Report of the Interna4onal CommiCee on Taxonomy of Viruses (King, A.M.Q., Adams, M.J., Carstens, E.B. & Lefowitz, E.J., eds.), pp Elsevier, San Diego, CA, USA. Virus Strain source nt Group A Enterovirus Nucleotide accession reference protein Accession Template Entry Channel in Fingers Domain G64 64 Lysine and Arginine Residues Contacting RNA templates & Products CV-A2 Fleetwood CG 7398 AY Oberste et al., 2004 AAR38840 R Y Y Y G I K K R P R R N D S L L CV-A3 Olson CG 7395 AY Oberste et al., 2004 AAR38841 R Y Y Y G I K K R P R R N D S L L CV-A4 High Point CG 7434 AY Oberste et al., 2004 AAR38842 R Y Y Y G I K K R P R R N D S L L CV-A5 Swartz CG 7400 AY Oberste et al., 2004 AAR38843 R Y Y Y G I K K R P R R N D S L L CV-A6 Gdula CG 7434 AY Oberste et al., 2004 AAR38844 R Y Y Y G I K K R P R R N D S L L CV-A7 Parker CG 7404 AY Oberste et al., 2004 AAR38845 R Y Y Y G I K K R P R R N D S L L CV-A8 Donovan CG 7396 AY Oberste et al., 2004 AAR38846 R Y Y Y G I K K R P R R N D S L L CV-A10 Kowalik CG 7409 AY Oberste et al., 2004 AAR38847 R Y Y Y G I K K R P R R N D S L L CV-A12 Texas-12 CG 7404 AY Oberste et al., 2004 AAR38848 R Y Y Y G I K K R P R R N D S L L CV-A14 strain G-14 CG 7415 AY Oberste et al., 2004 AAR38849 R Y Y Y G I K K R P R R N D S L L CV-A16 G-10* (South Africa/51) CG 7413 U05876 Pöyry et al., 1994 AAA50478 R Y Y Y G I K K R P R R N D S L L EV-A71 BrCr* (California/70) CG 7408 U22521 Brown and Pallansch, 1995 AAB39968 R Y Y Y G I K K R P R R N D S L L EV-A76 FRA * CG 7438 AY Oberste et al., 2005a AAW30682 N Y Y Y G I K K R P R R N D S L L EV-A89 BAN * CG 7429 AY Oberste et al., 2005a AAW30683 N Y Y Y G I K K R P R R N D S L L EV-A90 BAN * CG 7425 AY Oberste et al., 2005a AAW30684 N Y Y Y G I K K R P R R N D S L L EV-A91 BAN * CG 7427 AY Oberste et al., 2005a AAW30685 N Y Y Y G I K K R P R R N D S L L EV-A92 USA/GA99-RJg-7* CG 7379 EF Oberste et al., 2008 ABV25904 R Y Y Y G I K K R P R R N D S L L Group B Enterovirus CV-B1 Japan CG 7389 M16560 Iizuka et al., 1987 AAC00531 F Y Y Y G I K K R K R K N D S L L CV-B2 Ohio-1/Ohio/US/47* CG 7411 AF Polacek et al., 1999 AAD19874 F Y Y Y G I K K R K R K N D S L L CV-B3 Nancy/Connecticut/US/49* CG 7399 M88483 Consensus of those listed below AAB59927 F Y Y Y G I K K R K R K N D S L L Jenkins et al., 1987; Ramsingh et CV-B4 JVB/New York/US/51 (Benschoten)* CG 7395 X05690 CAA29172 F Y Y Y G I K K R K R K N D S L L al., 1992 CV-B5 Faulkner/Kentucky/US/52* CG 7400 AF Lindberg and Polacek, 2000 AAF21971 F Y Y Y G I K K R K R K N D S L L CV-B6 Schmitt/Philippines/53 ( )* CG 7398 AF Zell et al., 1998 AAD02132 F Y Y Y G I K K R K R K N D S L L CV-A9 Griggs CG 7452 D00627 Chang et al., 1989 BAA00518 F Y Y Y G I K K R K R K N D S L L E-1 Farouk/Egypt/51* CG 7397 AF Filman et al., unpub. AAC63944 F Y Y Y G I K K R K R K N D S L L E-2 Cornelis/Connecticut/US/51* CG 7435 AY Oberste et al., 2004a AAQ73082 F Y Y Y G I K K R K R K N D S L L E-3 Morrisey/Connecticut/US/51* CG 7428 AY Oberste et al., 2004a AAQ73090 F Y Y Y G I K K R K R K N D S L L E-4 Pesacek/Connecticut/US/51* CG 7394 AY Oberste et al., 2004a AAQ73094 F Y Y Y G I K K R K R K N D S L L E-5 Noyce/Maine/54* CG 7433 AF Lindberg et al., 1999 AAD19342 F Y Y Y G I K K R K R K N D S L L E-6 D'Amori* (Rhode Island/55) CG 7418 AY Oberste et al., 2004a AAQ73095 Y Y Y Y G I K K R K R K N D S L L E-7 Wallace* (Ohio) CG 7427 AY Oberste et al., 2004a AAQ73096 F Y Y Y G I K K R K R K N D S L L E-9 Hill/Ohio/US/53* CG 7420 X84981 Zimmermann et al., 1995 CAA59341 F Y Y Y G I K K R K R K N D S L L E-11 Gregory* (Ohio) CG 7438 X80059 Dahllund et al., 1995 CAA56365 F Y Y Y G I K K R R R K N D S L L E-12 Travis* (Philippines/53) (wildtype) CG 7421 X79047 Kraus et al., 1995 CAA55650 F Y Y Y G I K K R K R K N D S L L E-13 Del Carmen* (Philippines/53) CG 7410 AY Oberste et al., 2004a AAQ73076 F Y Y Y G I K K R K R K N D S L L E-14 Tow* (Rhode Island/54) CG 7450 AY Oberste et al., 2004a AAQ73077 F Y Y Y G I K K R K R K N D S L L E-15 Ch (Charleston)* (West Virginia/51) CG 7437 AY Oberste et al., 2004a AAQ73078 F Y Y Y G I K K R K R K N D S L L E-16 Harrington* (Massachusetts/51) CG 7437 AY Oberste et al., 2004a AAQ73079 F F Y Y G I K K R K R K N D S L L E-17 CHHE-29* (Mexico City) CG 7416 AY Oberste et al., 2004a AAQ73080 Y Y Y Y G I K K R K R K N D S L L Andersson, P. and Lindberg, A.M., E-18 Metcalf* (Ohio) CG 7410 AF AAL37163 F Y Y Y G I K K R K R K N D S L L unpub. E-19 Burke* (Ohio) CG 7433 AY Oberste et al., 2004a AAQ73081 F Y Y Y G I K K R K R K N D S L L E-20 JV-1* (Washington DC/55) CG 7394 AY Oberste et al., 2004a AAQ73083 F Y Y Y G I R K R K R K N D S L L E-21 Farina (E26D)* (Massachusetts/50) CG 7426 AY Oberste et al., 2004a AAQ73084 F Y Y Y G I K K R K R K N D S L L E-24 DeCamp* (Ohio/56) CG 7433 AY Oberste et al., 2004a AAQ73085 F Y Y Y G I K K R K R K N D S L L E-25 JV-4* (Washington DC/57) CG 7426 AY Oberste et al., 2004a AAQ73086 F Y Y Y G I K K R K R K N D S L L E-26 Coronel (11-3-6)* (Philippines/53) CG 7424 AY Oberste et al., 2004a AAQ73087 F Y Y Y G I K K R K R K N D S L L E-27 Bacon (1-36-4)* (Philippines/53) CG 7412 AY Oberste et al., 2004a AAQ73088 F Y Y Y G I K K R K R K N D S L L E-29 JV-10* (Washington DC/55) CG 7427 AY Oberste et al., 2004a AAQ73089 F Y Y Y G I K K R K R K N D S L L Lindberg, A.M., Andersson, P. and E-30 Bastianni* (New York/58) CG 7445 AF AAL37155 F Y Y Y G I K K R K R K N D S L L Huseby, L., unpub. E-31 Caldwell* (Kansas/55) CG 7432 AY Oberste et al., 2004a AAQ73091 F Y Y Y G I K K R R R K N D S L L E-32 PR-10 CG 7404 AY Oberste et al., 2004 AAQ73092 F Y Y Y G I K K R K R K N D S L L E-33 Toluca-3* (Mexico/59) CG 7394 AY Oberste et al., 2004a AAQ73093 F Y Y Y G I K K R K R K N D S L L EV-B69 Toluca-1* (Toluca, Mexico/59) CG 7411 AY Oberste et al., 2004a AAQ73097 F Y Y Y G I K K R K R K N D S L L AF241359; EV-B73 CA * CG 7411 Oberste et al., 2001 AAK13071 F Y Y Y G I K K R K R K N D S L L AF EV-B74 USA/CA CG 7410 AY Oberste et al., 2004 AAT65951 F Y Y Y G I K K R K R K N D S L L EV-B75 USA/OK CG 7429 AY Oberste et al., 2004 AAT65964 F Y Y Y G I K K R K R K N D S L L EV-B77 CF CG 7415 AJ Bailly et al., 2004 CAD38168 F Y Y Y G I K K R K R K N D S L L EV-B79 USA/CA * CG 7430 AY Oberste et al., 2007 AAX47033 F Y Y Y G I K K R K R K N D S L L EV-B80 USA/CA * CG 7428 AY Oberste et al., 2007 AAX47034 F Y Y Y G I K K R K R K N D S L L EV-B81 USA/CA * CG 7415 AY Oberste et al., 2007 AAX47035 F Y Y Y G I K K R K R K N D S L L EV-B82 USA/CA * CG 7427 AY Oberste et al., 2007 AAX47036 F Y Y Y G I K K R K R K N D S L L EV-B83 USA/CA * CG 7394 AY Oberste et al., 2007 AAX47037 F Y Y Y G I K K R K R K N D S L L EV-B84 CIV * CG 7423 DQ Oberste et al., 2007 ABK41188 Y Y Y Y G I K K R K R K N D S L L Thumb Alpha Helix in Minor Groove of dsrna Products

4 EV-B85 BAN * CG 7418 AY Oberste et al., 2007 AAX47039 F Y Y Y G I K K R K R K N D S L L EV-B86 BAN * CG 7400 AY Oberste et al., 2007 AAX47040 F Y Y Y G I K K R K R K N D S L L EV-B87 BAN * CG 7422 AY Oberste et al., 2007 AAX47041 F Y Y Y G I K K R R R K N D S L L EV-B88 BAN * CG 7433 AY Oberste et al., 2007 AAX47042 F Y Y Y G I K K R K R K N D S L L EV-B97 BAN * CG 7413 AY Oberste et al., 2007 AAX47043 F Y Y Y G I K K R K R K N D S L L EV-B98 T CG 7422 AB Yamashita et al., unpublished BAG70419 F Y Y Y G I K K R K R K N D S L L EV-B100 BAN * CG 7421 DQ Oberste et al., 2007 ABK41189 F Y Y Y G I K K R K R K N D S L L EV-B101 CIV * CG 7445 AY Oberste et al., 2007 AAX47044 F Y Y Y G I K K R K R K N D S L L EV-B107 TN * CG 7423 AB Yamashita et al., unpublished BAG70420 F Y Y Y G I K K R K R K N D S L L Group C Enterovirus CV-A1 T.T. (Tompkins)* (Coxsackie, NY/47) CG 7397 AF Brown et al., 2003 AAQ02675 Y Y Y Y G K K K R K R R N D S L L CV-A11 Belgium 1* (Belgium/51) CG 7453 AF Brown et al., 2003 AAQ02676 Y Y Y Y G K K K R K R K N D S L L CV-A13 Flores* (Mexico/52) CG 7458 AF Brown et al., 2003 AAQ02677 Y Y Y Y G K K K R K R K N D S L L CV-A17 G-12* (South Africa/51) CG 7457 AF Brown et al., 2003 AAQ02679 Y Y Y Y G K K K R K R K N D S L L CV-A19 NIH-8663 (Dohi)* (Japan/52) CG 7410 AF Brown et al., 2003 AAQ02681 Y Y Y Y G K K K R K R R N D S L L CV-A20 IH-35* (New York/55) CG 7436 AF Brown et al., 2003 AAQ02682 Y Y Y Y G K K K R K R K N D S L L CV-A21 Kuykendall* (California/52) CG 7406 AF Brown et al., 2003 AAQ02684 Y Y Y Y G K K K R K R K N D S L L CV-A22 Chulman* (New York/55 CG 7406 AF Brown et al., 2003 AAQ02683 Y Y Y Y G K K K R K R R N D S L L CV-A24 Joseph CG 7459 EF Brown et al., 2009 ABM54551 Y Y Y Y G K K K R K R K N D S L L EV-C96 BAN * CG 7478 EF Brown et al., 2009 ABM54550 Y Y Y Y G K K K R K R R N D S L L EV-C99 USA-GA CG 7447 EF Brown et al., 2009 ABU41835 Y Y Y Y G K K K R K R K N D S L L EV-C102 BAN * CG 7438 EF Brown et al., 2009 ABU41836 Y Y Y Y G K K K R K R K N D S L L EV-C104 CL FL* CG(p) 7229 EU Tapparel et al., 2009 ACU27241 Y L D Y G K K K R K R K N D S L L EV-C105 34S/DRC/2010 CG 7299 JX Lukashev et al., 2012 AFQ62100 Y Y Y Y G K R K R K R K N D S L L EV-C109 NICA * CG 7354 GQ Yozwiak et al., 2010 ADK22846 Y Y Y Y G K K K R K R K N D S L L EV-C /Russia/2010* CG 7395 JX Lukashev et al., 2012 AFQ62099 Y Y Y Y G K K K R K R R N D S L L EV-C117 LIT22 CG 7363 JX Daleno et a., 2012 AFR46521 Y Y Y Y G K K K R K R K N D S L L Kitamura et al., 1981; Racaniello & PV-1 Mahoney (Ohio/41) CG 7440 V01149 CAA24461 Y Y Y Y G K K K R K R K N D S L L Baltimore, 1981 PV-1 Sabin (LSc, 2ab) CG 7441 V01150 Nomoto et al., 1982 CAA24465 Y Y Y Y G K K K R K R K N D S L L PV-2 Lansing* (Michagan/37) CG 7437 M12197 La Monica et al., 1986 AAA46912 Y Y Y Y G K K K R K R K N D S L L PV-2 Sabin (P712, Ch, 2ab) CG 7439 X00595 Toyoda et al., 1984 CAA25246 Y Y Y Y G K K K R K R K N D S L L PV-3 Leon* (California/37) CG 7431 K01392 Stanway et al., 1984 AAA46914 Y Y Y Y G K K K R K R K N D S L L PV-3 Sabin (Leon 12a-1-b) CG 7432 K00043 Stanway et al., 1983 CAA25444 Y Y Y Y G K K K R K R K N D S L L Group D Enterovirus EV-D68 Fermon* (California/62) CG 7367 AY Oberste et al., 2004b AAR Y Y F G K K K R R R K N D S L L EV-D70 J670/71* (Japan/71) CG 7391 DQ Kim and Racaniello, 2007 ABA Y Y F G K K K R R R K N D S L L EV-D94 E210* (Egypt) CG 7364 DQ Smura et al., 2007 ABK F Y F G K K K R R R K N D S Y L Group A Rhinovirus HRV-A1A 2060 (Ohio) (ATCC VR-1559) CG 7137 FJ Palmenberg et al., 2009 ACK37367 L F Y F G I K K R N R K Q E S H L HRV-A1B B632 CG 7133 D00239 Hughes et al., 1988 BAA00168 L F Y F G I K K R N R K Q E S H L HRV-A2 HGP CG 7102 X02316 Skern et al., 1985 CAA26181 L F Y F G V K K R N R K Q E S H L HRV-A7 68-CV11 CG 7146 DQ Kistler et al., 2007 ABF51197 L F Y F G I K K K K R K C E S H L HRV-A8 MRH-CV12 (ATCC VR-1118) CG 7108 FJ Palmenberg et al., 2009 ACK37369 Y F Y Y G I K K K K R K C E S H L HRV-A9 211-CV13 (ATCC VR-489) CG 7128 FJ Palmenberg et al., 2009 ACK37433 Y F Y F G I R K R N R R Q E S H L HRV-A CV14 CG 7137 DQ Kistler et al., 2007 ABF51192 L F Y F G I K K K K R K Q E S H L HRV-A11 1-CV15 CG 7125 EF Tapparel et al., 2007 ABO69370 L F Y F G I K K K N R K Q E S H L HRV-A CV16 CG 7124 EF Tapparel et al., 2007 ABO69371 M Y Y F G I K K R R R R C E S Y L HRV-A (South Carolina/59) CG 7140 FJ Palmenberg et al., 2009 ACK37372 L Y Y F G I K K R N R R Q E S H L HRV-A (South Carolina/60) CG 7134 DQ Kistler et al., 2007 ABF51187 Y F Y F G I K K R N R K Q E S H L HRV-A (Washington DC/60) CG 7124 L24917 Lee et al., 1994 AAA69862 L F Y F G I K K K N R K Q E S H L HRV-A CV17 CG 7119 FJ Palmenberg et al., 2009 ACK37374 Y Y Y F G I R K R K R K Q E S H L HRV-A19 ATCC VR-1129 CG 7135 FJ Palmenberg et al., 2009 ACK37375 L F Y F G I K K R N R R Q E S H L HRV-A20 ATCC VR-1130 CG 7163 FJ Palmenberg et al., 2009 ACK37376 L F Y F G I K K K K R K N E S H L HRV-A21 ATCC VR-1131 CG 7134 FJ Palmenberg et al., 2009 ACK37377 L Y Y F G I K K R K R K Q E S H L HRV-A22 ATCC VR-1132 CG 7129 FJ Palmenberg et al., 2009 ACK37378 L F Y F G I K K R N R R Q E S H L HRV-A CV24 CG 7025 DQ Kistler et al., 2007 ABF51191 L F Y F G I K K R N R K Q E S H L HRV-A CV25 CG 7132 EF Tapparel et al., 2007 ABO69372 L F Y F G I K K R K R K Q E S H L HRV-A25 ATCC VR-1135 CG 7126 FJ Palmenberg et al., 2009 ACK37379 L F Y F G I K K R K R K Q E S H L HRV-A CV29 CG 7148 DQ Kistler et al., 2007 ABF51202 L F Y F G I K K K K R K N E S H L HRV-A29 ATCC VR-1139 CG 7123 FJ Palmenberg et al., 2009 ACK37381 L Y Y F G I K K R K R K Q E S H L HRV-A30 106F CG 7099 DQ Kistler et al., 2007 ABF51206 L F Y F G I K K R N R K Q E S H L HRV-A31 ATCC VR-506 CG 7131 FJ Palmenberg et al., 2009 ACK37382 L F Y F G I K K R K R K Q E S H L HRV-A32 ATCC VR-1142 CG 7133 FJ Palmenberg et al., 2009 ACK37383 Y F Y F G I K K R N R K Q E S H L HRV-A33 ATCC VR-330 CG 7133 FJ Palmenberg et al., 2009 ACK37384 L F Y F G I K K K N R K Q E S H L HRV-A CG 7119 DQ Kistler et al., 2007 ABF51195 L Y Y F G I K K R K R K Q E S H L HRV-A36 342H CG 7141 DQ Kistler et al., 2007 ABF51199 L F Y F G I K K K K R K N E S H L HRV-A38 CH79 CG 7136 DQ Kistler et al., 2007 ABF51189 L F Y F G V K K R K R K Q E S H L HRV-A (Maryland/62) CG 7137 AY Harris and Racaniello, 2005 AAV27300 L F Y F G I K K R N R R Q E S H L HRV-A40 ATCC VR-341 CG 7138 FJ Palmenberg et al., 2009 ACK37385 L F Y F G I K K R K R K Q E S H L HRV-A (North Carolina/61) CG 7145 DQ Kistler et al., 2007 ABF51185 L F Y F G I K K R N R R Q E S H L HRV-A43 ATCC VR-1153 CG 7129 FJ Palmenberg et al., 2009 ACK37387 F F Y F G I K K R K R R Q E S H L HRV-A (North Carolina/61) CG 7123 DQ Kistler et al., 2007 ABF51193 V F Y F G I K K R K R K Q E S H L HRV-A45 ATCC VR-1155 CG 7114 FJ Palmenberg et al., 2009 ACK37388 Y F Y Y G I K K K R R K C E S H L HRV-A46 Crell (Baylor 2) (Texas/64) CG 7149 DQ Kistler et al., 2007 ABF51200 M Y Y F G I K K K K R K N E S H L HRV-A47 N13 (China 2007) CG 7136 GQ Huang et al., 2009 ACU00187 L F Y L G I K K R K R K Q E S H L HRV-A CG 7106 DQ Kistler et al., 2007 ABF51190 L F Y F G I K K R N R R Q E S H L HRV-A50 ATCC VR-517 CG 7118 FJ Palmenberg et al., 2009 ACK37391 L F Y F G I K K R K R K Q E S R L

5 HRV-A51 ATCC VR-1161 CG 7152 FJ Palmenberg et al., 2009 ACK37392 L Y Y F G I K K K K R K N E S H L HRV-A53 F CG 7143 DQ Kistler et al., 2007 ABF51201 L Y Y F G I K K K K R K N E S H L HRV-A54 ATCC VR-1164 CG 7134 FJ Palmenberg et al., 2009 ACK37394 L F Y F G I K K R K R K Q E S H L HRV-A55 Wis315E (Wisconsin/64) CG 7036 DQ Kistler et al., 2007 ABF51205 L F Y F G I K K R K R K Q E S H L HRV-A56 CH82 [V ] CG 7136 FJ Palmenberg et al., 2009 ACK37396 L F Y F G I K K K K R K Q E S R L HRV-A57 fs ship#1-hrv-57 CG 7134 FJ Palmenberg et al., 2009 ACK37397 L F Y F G I K K R K R K Q E S H L HRV-A58 ATCC VR-1168 CG 7140 FJ Palmenberg et al., 2009 ACK37398 L F Y F G I K K K K R K C E S H L HRV-A CV35 CG 7135 DQ Kistler et al., 2007 ABF51194 L F Y F G I K K K N R K Q E S H L HRV-A60 ATCC VR-1473 CG 7139 FJ Palmenberg et al., 2009 ACK37399 L F Y F G I K K R K R K Q E S H L HRV-A61 ATCC VR-1171 CG 7139 FJ Palmenberg et al., 2009 ACK37400 Y F Y F G I R K R N R K Q E S H L HRV-A62 ATCC VR-1172 CG 7131 FJ Palmenberg et al., 2009 ACK37401 L F Y F G I K K R K R K Q E S H L HRV-A63 ATCC VR-1173 CG 7141 FJ Palmenberg et al., 2009 ACK37402 L F Y F G I K K R K R K Q E S H L HRV-A CV44 CG 7129 EF Tapparel et al., 2007 ABO69373 L F Y F G I K K R N R R Q D S H L HRV-A65 ATCC VR-1175 CG 7162 FJ Palmenberg et al., 2009 ACK37403 L Y Y F G I K K K K R K N E S H L HRV-A66 ATCC VR-1176 CG 7139 FJ Palmenberg et al., 2009 ACK37404 L F Y F G I K K R K R K Q E S H L HRV-A67 ATCC VR-1177 CG 7135 FJ Palmenberg et al., 2009 ACK37405 Y F Y F G V K K R N R K Q E S H L HRV-A68 ATCC VR-1178 CG 7164 FJ Palmenberg et al., 2009 ACK37406 L Y Y F G I K K K K R K N E S H L HRV-A71 ATCC VR-1181 CG 7161 FJ Palmenberg et al., 2009 ACK37408 L Y Y F G I K K K K R K N E S H L HRV-A73 107E CG 7140 DQ Kistler et al., 2007 ABF51186 L F Y F G I R K K N R R Q E S H L HRV-A74 328A CG 7120 DQ Kistler et al., 2007 ABF51188 Y F Y F G I K K R N R R Q E S H L HRV-A75 328F CG 7137 DQ Kistler et al., 2007 ABF51204 Y F Y F G I K K R N R K Q E S H L HRV-A76 H00062 CG 7129 DQ Kistler et al., 2007 ABF51196 L Y Y F G I K K K N R K Q E S H L HRV-A77 ATCC VR-1187 CG 7136 FJ Palmenberg et al., 2009 ACK37410 L F Y F G I K K R K R K Q E S H L HRV-A CG 7145 EF Tapparel et al., 2007 ABO69374 F Y Y F G I K K K T R S N E S Q L HRV-A80 ATCC VR-1190 CG 7138 FJ Palmenberg et al., 2009 ACK37412 I Y Y F G I K K K K R K N E S H L HRV-A81 ATCC VR-1191 CG 7116 FJ Palmenberg et al., 2009 ACK37413 L Y Y F G I K K K N R K Q E S H L HRV-A82 Santa Cruz, CA CG 7129 DQ Kistler et al., 2007 ABF51203 F F Y F G I K K R K R K Q E S H L HRV-A85 ATCC VR-1195 CG 7140 FJ Palmenberg et al., 2009 ACK37419 Y F Y F G I R K R K R K Q E S H L HRV-A88 CVD Dambrauskas CG 7143 DQ Kistler et al., 2007 ABF51198 L F Y F G I K K K K R K C E S H L HRV-A Gallo CG 7152 M16248 Duechler et al., 1987 AAA45762 L F Y F G I K K K K R K C E S H L HRV-A90 ATCC VR-1291 CG 7124 FJ Palmenberg et al., 2009 ACK37423 L F Y F G I K K R K R K Q E S H L HRV-A94 SF-1803 CG 7132 EF Tapparel et al., 2007 ABO69375 L F Y F G I K K R N R R Q E S H L HRV-A95 ATCC VR-1301 CG 7110 FJ Palmenberg et al., 2009 ACK37426 Y F Y Y G I K K K K R K C E S H L HRV-A96 ATCC VR-1296 CG 7134 FJ Palmenberg et al., 2009 ACK37427 Y F Y F G I R K R N R K Q E S H L HRV-A98 ATCC VR-1298 CG 7133 FJ Palmenberg et al., 2009 ACK37429 L F Y F G I K K R K R K Q E S H L HRV-A100 ATCC VR-1300 CG 7140 FJ Palmenberg et al., 2009 ACK37431 L F Y F G I K K R K R K Q E S H L HRV-A101 hrv-a101 CG 7130 GQ Rathe et al., 2010 ACT89355 L Y Y F G I K K K K R K N E S H L HRV-A102 AMS323 (Netherlands) CG 7160 EF de Vries et al., 2008 ABO69521 L Y Y F G I K K K K R K N E S H L HRV-A103 WA327E/09 CG 7168 JF Chidlow et al., unpub. AEG75268 L Y Y F G V K K K K R K N E S H L HRV-B3 Group B Rhinovirus FEB CG 7211 EF Tapparel et al., 2007 ABO69378 L Y Y Y G I K K R K R R N D S M L HRV-B4 16/60 CG 7212 DQ Kistler et al., 2007 ABF51184 I Y Y Y G I N K K R R R N D S M L HRV-B5 Norman (ATCC VR-485) CG 7212 FJ Palmenberg et al., 2009 ACK37368 I Y Y Y G I K K R K R K N D S M M HRV-B6 Thompson CG 7216 DQ Kistler et al., 2007 ABF51180 M Y Y Y G I K K R R R K N D S M L HRV-B (South Carolina/59) CG 7212 L05355 Lee et al., 1993 AAA45758 I F Y Y G I K K R K R R N D S M L HRV-B (North Carolina/59) CG 7219 EF Tapparel et al., 2007 ABO69376 L Y Y Y G I K K R R R R N D S M L HRV-B CV27 (ATCC VR-1136) CG 7211 FJ Palmenberg et al., 2009 ACK37380 I Y Y Y G I K K R K R K N D S M M HRV-B27 (ATCC VR-1137) CG 7217 FJ Palmenberg et al., 2009 ACK37442 L Y Y Y G I K K R K R K N D S M L HRV-B (V ) (ATCC 1145) CG 7224 FJ Palmenberg et al., 2009 ACK37443 L Y Y Y G I K K R R R K N D S M L HRV-B CG 7216 EF Tapparel et al., 2007 ABO69379 I Y Y Y G I K K R R R K N D S M L HRV-B (North Carolina/61) (ATCC VR-338) CG 7223 FJ Palmenberg et al., 2009 ACK37386 I Y Y Y G I K K R K R K N D S M M HRV-B CG 7214 DQ Kistler et al., 2007 ABF51182 L Y Y Y G I R K R K R R S D S M L HRV-B52 F (ATCC VR-1162) CG 7216 FJ Palmenberg et al., 2009 ACK37444 L Y Y Y G I K K K R R R N D S M L HRV-B69 F Mitchinson (ATCC VR-1179) CG 7211 FJ Palmenberg et al., 2009 ACK37407 L Y Y Y G I K K K R R R N D S M L HRV-B70 F Treganza CG 7223 DQ Kistler et al., 2007 ABF51183 L Y Y Y G I K K R R R R N D S M L HRV-B72 K2207 (ATCC VR-1182) CG 7216 FJ Palmenberg et al., 2009 ACK37409 M Y Y Y G I K K R K R R N D S M L HRV-B (ATCC VR-1189) CG 7224 FJ Palmenberg et al., 2009 ACK37411 L Y Y Y G I K K R K R K N D S M L HRV-B83 Baylor 7 (ATCC VR-1193) CG 7230 FJ Palmenberg et al., 2009 ACK37417 L Y Y Y G I K K R K R K N D S M L HRV-B84 432D (ATCC VR-1194) CG 7201 FJ Palmenberg et al., 2009 ACK37418 I Y Y Y G I K K R K R R N D S M L HRV-B Johnson (ATCC VR-1196) CG 7213 FJ Palmenberg et al., 2009 ACK37420 L Y Y Y G I K K R K R R N D S M L HRV-B91 JM1 (ATCC VR-1292) CG 7221 FJ Palmenberg et al., 2009 ACK37424 L Y Y Y G I K K R K R R N D S M L HRV-B92 SF-1662 (ATCC VR-1293) CG 7233 FJ Palmenberg et al., 2009 ACK37425 L Y Y Y G I K K R K R R N D S M L HRV-B93 SF-1492 CG 7215 EF Tapparel et al., 2007 ABO69381 M Y Y Y G I K K R K R K N D S M L HRV-B97 SF-1372 (ATCC VR-1297) CG 7207 FJ Palmenberg et al., 2009 ACK37428 L Y Y Y G I K K R K R K N D S M L HRV-B (ATCC VR-1299) CG 7208 FJ Palmenberg et al., 2009 ACK37430 L Y Y Y G I K K R K R R N D S M M Group C Rhinovirus HRV-C1 NAT001* CG 6944 EF Kistler et al., 2007 ABQ16587 L F Y Y G V K K K R R K N D S L L HRV-C2 NAT045* CG 7015 EF Kistler et al., 2007 ABQ51392 Y F Y Y G I K K R R R K N D S T L HRV-C3 QPM* CG 6948 EF McErlean et al., 2007 ABO76708 L F Y Y G V K K K R R K N D S L L HRV-C4 024* CG 7099 EF Lau et al., 2007 ABU62848 W F Y F G V K K R N R R N D S L L HRV-C5 025* CG 7114 EF Lau et al., 2007 ABU62849 M Y Y Y G V K K R R R K N D S L L HRV-C6 026* CG 7086 EF Lau et al., 2007 ABU62850 L F Y Y G I K K R R R K N D S L L HRV-C7 NY-074 (1078)* CG 7072 DQ Lamson et al., 2006 ABK29455 L F Y Y G V K K R R R K N D S L L HRV-C8 N4* CG 7107 GQ Huang et al., 2009 ACU00185 Y Y Y Y G I K K R K R K N D S Q L HRV-C9 N10* CG 7111 GQ Huang et al., 2009 ACU00186 I F F Y G I K K R K R K N D S Q L HRV-C10 QCE* CG 7010 GQ Arden et al., unpub. ACV51798 L F Y Y G V K K R R R K N D S L L HRV-C11 CL * CG 7108 EU Tapparel et al., 2009a,b ACR14890 I Y Y Y G I K K K R R K N D S L L

6 HRV-C15 W10 CG 7119 GU Rathe et al., unpub. ACZ67658 L F Y Y G I K K K K R K N D S L L Aphthovirus FMDV strain C, isolate c-s8c1 CG 8115 AJ Toja et al., 1999 CAB L - F G K R R G F R E T E S G L

7 A. 5 T7 PV A (32) 3 NTR GGAG 7440 AAAAAAAAAA 10 AAAAAAAAAA 20 AAAAAAAAAA 30 AA CGCG 3 5 T7 PV A (51) 3 NTR GGAG 7440 polya( 51 bases long) CGCG 3 5 T7 PV A (84) 3 NTR GGAG 7440 polya( 84 bases long) CGCG 3 B. T7 PV A (32) T7 PV A (51) T7 PV A (84) Clone GGAG 7440 Poly(A) 1 Yes 28 2 Yes 28 3 Yes 29 4 Yes 31 5 Yes 31 6 Yes 31 7 Yes 32 8 Yes 32 9 Yes Yes 33 Range A (28-33) GGAG 7440 Poly(A) Yes 45 Yes 46 Yes 48 Yes 49 Yes 49 Yes 49 Yes 50 Yes 52 Yes 52 Yes 53 A (45-53) GGAG 7440 Poly(A) Yes 73 Yes 73 Yes 79 Yes 80 Yes 80 Yes 81 Yes 82 Yes 84 Yes 85 Yes 85 A (73-85) Figure S1. Validating TOPO-TA cloning and sequencing methods to analyze 3 -terminal GGAG-poly(A) tails in viral RNA. Poliovirus cdna clones encoding 3 poly(a) tails 32, 51 and 84 bases in length were transcribed by T7 DNA-dependent RNA polymerase into viral RNAs as described in Materials and Methods (Steil et al., 2010). An RNA linker was added to the 3 end of the viral RNA transcripts and a cdna primer complementary to the RNA linker was used in conjunction with Superscript III reverse transcriptase to make cdna. cdnas were amplified by PCR and TOPO-TA cloned into plasmids in E. coli. DNA from ten random TOPO-TA cdna clones was sequenced. A. Diagram of T7 transcripts of poliovirus RNA with 32, 51 and 84 base poly(a) tails. B. Sequence of 3 -terminal GGAG-poly(A) tail from 10 random TOPO-TA cdna clones from each transcript. Yes indicates that the GGAG sequence is invarient from that in the original cdna clone used to transcribe the viral RNA. The size of poly(a) in each cdna clone is indicated. Results: The GGAG sequence 3 proximal to the poly(a) tail was invarient in 30 out-of 30 cdna clones. The size of poly(a) tails in the cdna clones reflected the size of poly(a) tail in the respective T7 transcripts (as shown in Figures 3-5 of the manuscript).

8 A." WT! G64S! G64T! G64D! Mock! P1" P3" 3CD" VP0-VP3" B." WT! Y14A! Y118A! Y118F! Y148A! Y148F! Y157A! Y157F! Y118A-Y148A! Y118A-Y148F! Y118F-Y148A! Y118F-Y148F! Mock! P1" P3" 3CD" VP0-VP3" 2BC" VP0" 2C" VP1" 2BC" VP0" 2C" VP1" VP3" VP3" 2A" 3AB" 2A" 3AB" C." WT! ! K125A! K126A! K125A-K126A! K127A! R128A! K127A-R128A! K133A! R188A! K276A! Mock! P1" P3" 3CD" VP0-VP3" D." ! WT! N409A! D412A! S416A! L419A! L420A! Mock! P1" P3" 3CD" VP0-VP3" 2BC" VP0" 2C" VP1" VP3" 2BC" VP0" 2C" VP1" VP3" 2A" 3AB" 2A" 3AB" ! ! Figure S2. Impact of 3D pol mutations on viral RNA translation and polyprotein processing. Poliovirus RNAs with the indicated 3D pol mutations were incubated in cytoplasmic extracts from uninfected HeLa cells as previously described (7-10). SDS-PAGE and phosphorimaging were used to detect [ 35 S]-methionine labeled viral proteins. A. G64 mutations in 3D pol. B. Mutation of tyrosines in the fingers domain of 3D pol. C. Alanine substitution mutations of charged residues in 3D pol. D. Alanine substitution mutations of thumb residues N409, D412, S416, L419, and L420 in 3D pol. The mobilities of individual viral proteins are indicated.

9 A. 5 NTR 3 NTR VPg VP4 VP2 VP3 VP1 2A Pro 2B 2C ATPase 3A 3B VPg 3C Pro 3D Pol A (20-120) open-reading frame 3 NTR GGAG 7440 AAAAAAAAAA (N) B. Virus GGAG GGAGG GGAA GGGG Poliovirus Type 1 V01149, AJ416942, AJ430385, AF405666, AF405669, AF405682, AF405690, AY278553, AF111961, AF111966, AF111981, AF111982, AF V01148, V01150, AF462418, AF462419, AF AJ132960, AJ Poliovirus Type 2 AF448782, AY278550, AY278551, AY278552, AJ M12197, D00625, AY X00595, AF AY Poliovirus Type 3 K01392, X04468, AJ K00043 AJ Coxsackievirus A1 Coxsackievirus A11 Coxsackievirus A13 Coxsackievirus A17 Coxsackievirus A19 Coxsackievirus A20 Coxsackievirus A21 Coxsackievirus A22 Coxsackievirus A24 AF499636, AF499638, AF AF499637, AF465511, AF499640, DQ AF AF499642, AF465514, EF D00538, AF546702, AF466515, EF AF499643, DQ AF AF D90457 Coxsackievirus A96 FJ751915, EF FJ Coxsackievirus A99 Coxsackievirus A102 Coxsackievirus A104 Coxsackievirus A109 Coxsackievirus A116 Coxsackievirus A117 EF EF AB GQ JX JX AF Figure S3. Polymorphisms in the GGAG sequence adjacent to the poly(a) tail of group C enteroviruses. A. Diagram highlighting the GGAG sequence adjacent to the 3 poly(a) tail of poliovirus and other group C enteroviruses. B. Polymorphisms in the sequences adjacent to the poly(a) tail of group C enteroviruses. There are frequent polymorphisms in the reported sequence adjacent to the poly(a) tail of group C enteroviruses. NCBI accession numbers are in the column of the corresponding polymorphic sequence for each virus.