Supplementary Material - Materials and Methods

1 SUPPLEMENTARY MATERIAL - Materials and Methods

2 Cells and media. HEp-2c cells (derived from a human laryngeal carcinoma cell line expressing the

3 human poliovirus receptor CD155 and the ICAM-1 receptor for some non-polio enteroviruses, and

4 L20B cells (mouse L cells expressing the CD155 gene) were grown as monolayers in DMEM

5 (GIBCO) supplemented with 10% new-born calf serum (GIBCO). For the multiplication of viruses

6 and for cotransfection experiments, HEp-2c and L20B cell monolayers were grown in DMEM

7 supplemented with 3% fetal calf serum (FCS).

8 Construction of full-length and deleted cDNA-derived RNAs. The molecular cloning of the S2

9 cDNA, MAD4(2A)/S2 cDNA and CA17.67591 cDNA in a modified pBR-322 vector, generating

10 plasmids pBR-S2, pBR-MAD4(2A)/S2 and pBR-CA17, respectively, has been described

11 elsewhere (22, 23). We first deleted nt 7301 to 7455 (155 nt) of the S2 genome from pBR-S2 and

12 pBR-MAD4(2A)/S2, using the MfeI and NotI restriction sites (Fig. 1A). This resulted in the

13 deletion of the 23 C-terminal codons of the 3D polymerase and the entire 3’UTR from the viral

14 cDNAs. We deleted nt 291 to 480 of the CA17 genome from pBR-CA17, using the BlpI and SacII

15 restriction sites. This 190 nt deletion concerns domains IV and V of the 5’UTR. After ligation and

16 the transformation of JM109 chemocompetent bacteria (PROMEGA), we obtained the cloned

17 deleted viral genomes pBR-ΔS2, pBR-∆MAD4(2A)/S2 and pBR-ΔCA17, respectively. We also

18 introduced larger deletions into the S2 and CA17 genomes (Fig.1A). We deleted nucleotides 6251

19 to 7455 (1205 nt) from the S2-derived cDNA, using the XbaI and NotI restriction sites. We deleted

20 nt 33 to 1766 (1734 nt) from the CA17-derived cDNA, using the AscI and StuI restriction sites.

21 After ligation and the transformation of JM109 chemocompetent bacteria, we obtained pBR-ΔLS2

22 and pBR-ΔLCA17, in which large deletions had eliminated almost all the 3D polymerase coding

23 region and the entire 3’UTR (pBR-ΔLS2), and most of the 5’UTR and all of the regions encoding

24 the viral proteins VP4 and VP2 (pBR-ΔLCA17).

25 The T7 RNApol promoter upstream from the cloned viral cDNA was used to transcribe infectious

26 RNA and RNA with deletions from plasmids linearized with the T7 RiboMAXTM, polymerase kit

1 27 (PROMEGA). DNA templates were eliminated by treatment with the RQ1 RNase-free DNase

28 provided with the T7 RiboMAX kit. RNA was then purified with the RNeasy Mini Kit

29 (QIAGEN), quantified on an ND-1000 (NanoDrop) spectrophotometer and used for cotransfection

30 experiments.

31 RNase/DNase treatment. Additional RNase or DNase treatments of genomic RNA were

32 occasionally performed before the cotransfection assay. RNA (1 µg) was treated with 0.5 µg of

33 DNase-free RNase (ROCHE) or 10 U of recombinant RNase-free DNase I (ROCHE) and

34 incubated at 37.0°C for 1 h. The RNA was then used for the cotransfection assays, as described in

35 the main text (Materials and Methods).

36 Isolation of recombinant viruses. For the isolation of recombinant viruses, we cotransfected L20B

37 cell monolayers in semi-solid medium, as described in the main text (Materials and Methods),

38 except that the Avicel solution was replaced with a mixture containing 1 volume of 1.8% agarose

39 in water and 1 volume of 2 x DMEM supplemented with 4% FCS. Plates were incubated at

40 34.0°C, under an atmosphere containing 5% CO2. Three days after transfection, we added 1 ml of

41 a similar agarose/DMEM mixture containing 0.01% neutral red to the plates, which were then

42 incubated at 34.0°C for one day. Plaques formed by recombinant viruses were then picked and

43 transferred to 50 µl of DMEM for storage at -25.0°C until use. Recombinant viruses were then

44 amplified to obtain a viral stock at passage P1. This stock was used for the analysis of recombinant

45 genomes.

46 Some recombinant viruses were plaque-purified again (passage P2), on L20B cells, to prevent the

47 production of mixed viral stocks. Picked plaques were amplified in L20B cells to obtain small

48 viral stock suspensions (1.5 ml) at passage P3. Large viral stocks of each virus were also obtained

49 at passage P3, by infecting L20B cells in 25 ml of DMEM medium supplemented with 3% FCS

50 and incubating at 37.0°C under an atmosphere containing 5% CO2. These viral stocks were called

51 P4 and were used for studies of replication kinetics and assays of stability in vitro and in vivo. The

2 52 titration of viral stocks and phenotypic analyses of viruses were performed in HEp-2c cells, which

53 are the most permissive for enteroviruses.

54 Analysis of recombinant genomes: RT-PCR and sequencing. The oligonucleotides used for RT-

55 PCR and sequencing are presented in Table S3; most of the PV- and enterovirus-specific primers

56 have been described elsewhere (12, 22). Viral recombinant RNAs were extracted from viral

57 supernatants and from the spinal cords of infected mice, with the QIAamp® Viral RNA Mini Kit

58 (QIAGEN), according to the manufacturer’s instructions. Reverse transcription was performed as

59 described by Bessaud et al. (50), with the heptaN primer. PCR was carried out in a final volume of

60 50 µl, including 5 µl of 10 x Taq Buffer with 1.5 mM MgCl2, 200 µM of each dNTP, 10 pmoles of

61 each primer, 2 µl of cDNA and 2.5 U of Taq DNA polymerase (Taq CORE kit 10, Q-BIOGEN).

62 The cDNA was denatured by heating for 3 minutes at 95°C, amplified by 40 cycles of 30 s at

63 95°C, 30 s at 45°C, 1 min at 72°C, and subjected to a final elongation step consisting of 10

64 minutes at 72°C. PCR products were analyzed by electrophoresis in ethidium bromide-stained

65 agarose gels and purified by ultrafiltration (MILLIPORE). The sequences of the resulting

66 amplicons were determined with the BigDye Terminator v3.1 kit (Applied Biosystems) and an

67 ABI Prism 3730XL DNA Analyzer automated sequencer (Applied Biosystems), mostly with the

68 primers used for PCR (Table S4). The RT-PCR screening process was designed to facilitate the

69 localization of recombination sites. Several direct primers specific for S2 and reverse primers

70 specific for CA17 (Table S4) were used to generate RT-PCR products including the recombination

71 site. RNA was extracted directly from viruses at passage P1, with the High Pure Viral RNA kit

72 (Roche Diagnostics, Meylan, France), according to the manufacturer’s instructions. Reverse

73 transcription was performed in a single step, with the SuperScript™ One-Step RT-PCR with

74 Platinum Taq Polymerase System (Invitrogen). Reactions were carried out in a final volume of 25

75 µl, including 12.5 µl of reaction mixture (a buffer containing 0.4 mM of each dNTP and 2.4 mM

76 MgSO4), primers at a final concentration of 0.2 μM, 5 µl of viral RNA and 0.5 µl of RT/Platinum

77 Taq Mix. The thermocycler incubation conditions were as follows: 55°C for 30 minutes and 94°C

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

79 extension step of 5 minutes at 72°C. The RT-PCT products were subjected to agarose gel

80 electrophoresis and sequencing, as described above. The sequences of the resulting amplicons

81 were determined with the primers used for PCR.

82 Replication kinetics of recombinant viruses. We compared the replication kinetics of parental and

83 recombinant viruses at a low multiplicity of infection (MOI). HEp-2c cells in DMEM medium

84 supplemented with 3% FCS were added to plates and subsequent cell monolayers were infected at

85 a MOI of 0.1. Viruses were allowed to adsorb onto the cells for 30 minutes and infected cells were

86 then washed twice and incubated at 37.0°C under an atmosphere containing 5% CO2. Infected

87 cultures were frozen at various time points after infection. Viral titers were determined for each

88 time point (TCID50/ml). The results are expressed as means ± standard errors for two independent

89 experiments.