<p> 1 SUPPLEMENTARY MATERIAL - Materials and Methods </p><p>2 Cells and media. HEp-2c cells (derived from a human laryngeal carcinoma cell line expressing the</p><p>3 human poliovirus receptor CD155 and the ICAM-1 receptor for some non-polio enteroviruses, and</p><p>4 L20B cells (mouse L cells expressing the CD155 gene) were grown as monolayers in DMEM</p><p>5 (GIBCO) supplemented with 10% new-born calf serum (GIBCO). For the multiplication of viruses</p><p>6 and for cotransfection experiments, HEp-2c and L20B cell monolayers were grown in DMEM</p><p>7 supplemented with 3% fetal calf serum (FCS).</p><p>8 Construction of full-length and deleted cDNA-derived RNAs. The molecular cloning of the S2</p><p>9 cDNA, MAD4(2A)/S2 cDNA and CA17.67591 cDNA in a modified pBR-322 vector, generating</p><p>10 plasmids pBR-S2, pBR-MAD4(2A)/S2 and pBR-CA17, respectively, has been described</p><p>11 elsewhere (22, 23). We first deleted nt 7301 to 7455 (155 nt) of the S2 genome from pBR-S2 and</p><p>12 pBR-MAD4(2A)/S2, using the MfeI and NotI restriction sites (Fig. 1A). This resulted in the</p><p>13 deletion of the 23 C-terminal codons of the 3D polymerase and the entire 3’UTR from the viral</p><p>14 cDNAs. We deleted nt 291 to 480 of the CA17 genome from pBR-CA17, using the BlpI and SacII</p><p>15 restriction sites. This 190 nt deletion concerns domains IV and V of the 5’UTR. After ligation and</p><p>16 the transformation of JM109 chemocompetent bacteria (PROMEGA), we obtained the cloned</p><p>17 deleted viral genomes pBR-ΔS2, pBR-∆MAD4(2A)/S2 and pBR-ΔCA17, respectively. We also</p><p>18 introduced larger deletions into the S2 and CA17 genomes (Fig.1A). We deleted nucleotides 6251</p><p>19 to 7455 (1205 nt) from the S2-derived cDNA, using the XbaI and NotI restriction sites. We deleted</p><p>20 nt 33 to 1766 (1734 nt) from the CA17-derived cDNA, using the AscI and StuI restriction sites.</p><p>21 After ligation and the transformation of JM109 chemocompetent bacteria, we obtained pBR-ΔLS2</p><p>22 and pBR-ΔLCA17, in which large deletions had eliminated almost all the 3D polymerase coding</p><p>23 region and the entire 3’UTR (pBR-ΔLS2), and most of the 5’UTR and all of the regions encoding</p><p>24 the viral proteins VP4 and VP2 (pBR-ΔLCA17).</p><p>25 The T7 RNApol promoter upstream from the cloned viral cDNA was used to transcribe infectious</p><p>26 RNA and RNA with deletions from plasmids linearized with the T7 RiboMAXTM, polymerase kit</p><p>1 27 (PROMEGA). DNA templates were eliminated by treatment with the RQ1 RNase-free DNase</p><p>28 provided with the T7 RiboMAX kit. RNA was then purified with the RNeasy Mini Kit</p><p>29 (QIAGEN), quantified on an ND-1000 (NanoDrop) spectrophotometer and used for cotransfection</p><p>30 experiments.</p><p>31 RNase/DNase treatment. Additional RNase or DNase treatments of genomic RNA were</p><p>32 occasionally performed before the cotransfection assay. RNA (1 µg) was treated with 0.5 µg of</p><p>33 DNase-free RNase (ROCHE) or 10 U of recombinant RNase-free DNase I (ROCHE) and</p><p>34 incubated at 37.0°C for 1 h. The RNA was then used for the cotransfection assays, as described in</p><p>35 the main text (Materials and Methods).</p><p>36 Isolation of recombinant viruses. For the isolation of recombinant viruses, we cotransfected L20B</p><p>37 cell monolayers in semi-solid medium, as described in the main text (Materials and Methods),</p><p>38 except that the Avicel solution was replaced with a mixture containing 1 volume of 1.8% agarose</p><p>39 in water and 1 volume of 2 x DMEM supplemented with 4% FCS. Plates were incubated at</p><p>40 34.0°C, under an atmosphere containing 5% CO2. Three days after transfection, we added 1 ml of</p><p>41 a similar agarose/DMEM mixture containing 0.01% neutral red to the plates, which were then</p><p>42 incubated at 34.0°C for one day. Plaques formed by recombinant viruses were then picked and</p><p>43 transferred to 50 µl of DMEM for storage at -25.0°C until use. Recombinant viruses were then</p><p>44 amplified to obtain a viral stock at passage P1. This stock was used for the analysis of recombinant</p><p>45 genomes. </p><p>46 Some recombinant viruses were plaque-purified again (passage P2), on L20B cells, to prevent the</p><p>47 production of mixed viral stocks. Picked plaques were amplified in L20B cells to obtain small</p><p>48 viral stock suspensions (1.5 ml) at passage P3. Large viral stocks of each virus were also obtained</p><p>49 at passage P3, by infecting L20B cells in 25 ml of DMEM medium supplemented with 3% FCS</p><p>50 and incubating at 37.0°C under an atmosphere containing 5% CO2. These viral stocks were called</p><p>51 P4 and were used for studies of replication kinetics and assays of stability in vitro and in vivo. The</p><p>2 52 titration of viral stocks and phenotypic analyses of viruses were performed in HEp-2c cells, which</p><p>53 are the most permissive for enteroviruses.</p><p>54 Analysis of recombinant genomes: RT-PCR and sequencing. The oligonucleotides used for RT-</p><p>55 PCR and sequencing are presented in Table S3; most of the PV- and enterovirus-specific primers</p><p>56 have been described elsewhere (12, 22). Viral recombinant RNAs were extracted from viral</p><p>57 supernatants and from the spinal cords of infected mice, with the QIAamp® Viral RNA Mini Kit</p><p>58 (QIAGEN), according to the manufacturer’s instructions. Reverse transcription was performed as</p><p>59 described by Bessaud et al. (50), with the heptaN primer. PCR was carried out in a final volume of</p><p>60 50 µl, including 5 µl of 10 x Taq Buffer with 1.5 mM MgCl2, 200 µM of each dNTP, 10 pmoles of</p><p>61 each primer, 2 µl of cDNA and 2.5 U of Taq DNA polymerase (Taq CORE kit 10, Q-BIOGEN).</p><p>62 The cDNA was denatured by heating for 3 minutes at 95°C, amplified by 40 cycles of 30 s at</p><p>63 95°C, 30 s at 45°C, 1 min at 72°C, and subjected to a final elongation step consisting of 10</p><p>64 minutes at 72°C. PCR products were analyzed by electrophoresis in ethidium bromide-stained</p><p>65 agarose gels and purified by ultrafiltration (MILLIPORE). The sequences of the resulting</p><p>66 amplicons were determined with the BigDye Terminator v3.1 kit (Applied Biosystems) and an</p><p>67 ABI Prism 3730XL DNA Analyzer automated sequencer (Applied Biosystems), mostly with the</p><p>68 primers used for PCR (Table S4). The RT-PCR screening process was designed to facilitate the</p><p>69 localization of recombination sites. Several direct primers specific for S2 and reverse primers</p><p>70 specific for CA17 (Table S4) were used to generate RT-PCR products including the recombination</p><p>71 site. RNA was extracted directly from viruses at passage P1, with the High Pure Viral RNA kit</p><p>72 (Roche Diagnostics, Meylan, France), according to the manufacturer’s instructions. Reverse</p><p>73 transcription was performed in a single step, with the SuperScript™ One-Step RT-PCR with</p><p>74 Platinum Taq Polymerase System (Invitrogen). Reactions were carried out in a final volume of 25</p><p>75 µl, including 12.5 µl of reaction mixture (a buffer containing 0.4 mM of each dNTP and 2.4 mM</p><p>76 MgSO4), primers at a final concentration of 0.2 μM, 5 µl of viral RNA and 0.5 µl of RT/Platinum</p><p>77 Taq Mix. The thermocycler incubation conditions were as follows: 55°C for 30 minutes and 94°C</p><p>3 78 for 2 minutes, followed by 35 cycles of 15 s at 94°C, 30 s at 55°C, 1 minute/kb at 72°C, and a final</p><p>79 extension step of 5 minutes at 72°C. The RT-PCT products were subjected to agarose gel</p><p>80 electrophoresis and sequencing, as described above. The sequences of the resulting amplicons</p><p>81 were determined with the primers used for PCR.</p><p>82 Replication kinetics of recombinant viruses. We compared the replication kinetics of parental and</p><p>83 recombinant viruses at a low multiplicity of infection (MOI). HEp-2c cells in DMEM medium</p><p>84 supplemented with 3% FCS were added to plates and subsequent cell monolayers were infected at</p><p>85 a MOI of 0.1. Viruses were allowed to adsorb onto the cells for 30 minutes and infected cells were</p><p>86 then washed twice and incubated at 37.0°C under an atmosphere containing 5% CO2. Infected</p><p>87 cultures were frozen at various time points after infection. Viral titers were determined for each</p><p>88 time point (TCID50/ml). The results are expressed as means ± standard errors for two independent</p><p>89 experiments.</p><p>90</p><p>91</p><p>4</p>