Cells, plasmids, and viruses.
S3 HeLa, A549, and L20B (31
) cells were grown in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, Calif.), 10% bovine calf serum (HyClone, Logan, Utah), and 1% penicillin-streptomycin (Invitrogen). Neuro-2A and SH-SY5Y cells were grown in the same medium except that 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.) was used. For plaque assays HeLa cells were grown in Dulbecco's modified Eagle medium (Specialty Media, Philipsburg, N.J.), 0.2% NaHCO3
, 5% bovine calf serum, 1% penicillin-streptomycin, and 0.9% Bacto-agar (Difco, Franklin Lakes, N.J.).
Plasmid pP1/HRV2, an infectious poliovirus DNA clone in which nucleotides 108 to 745 are replaced with HRV2 sequence, was created as follows. Nucleotides 108 to 610 of the HRV2 genome were amplified by PCR from an infectious HRV2 DNA clone (12
) using primers SK95 (5′-CGC GAA TTC TAG AAG TTT TTC ACA AAG-3′) and SK96 (5′-GCG GAG CTC GGT GCC AAT ATA TAT ATT-3′), cleaved with EcoRI/SacI, and used to replace the 640-bp EcoRI/SacI fragment from pP1/CVB3 (20
A plasmid encoding a bicistronic mRNA with the HRV2 IRES flanked by firefly and Renilla
luciferase coding regions (Fig. ) was created as follows. The HRV2 IRES was amplified by PCR from pP1/HRV2 using primers SK1 (5′-CGC GTCGACTTAAAACAGCTCTGGGGT-3′) and SK96 (5′-GCGGAGCTCGGT GCCAATATATATATT-3′), cleaved with SalI, blunt-ended with T4 DNA polymerase, cleaved with SacI, and ligated to an EcoRV/SacI-cleaved pDC516 (Microbix, Toronto, Ontario, Canada)-based bicistronic expression plasmid that has been described previously (20
). The creation of a plasmid encoding a bicistronic mRNA with the poliovirus IRES flanked by the firefly and Renilla
luciferase coding regions has also been described previously (20
FIG. 1. IRES-mediated translation in cultured cells. (A) Schematic of bicistronic reporter DNA encoded by reporter plasmids and recombinant adenoviruses. The arrow indicates the transcription initiation site of the murine cytomegalovirus immediate early promoter. (more ...)
Recombinant human adenoviruses encoding bicistronic reporter mRNA were produced using the Admax system (Microbix, Toronto, Ontario, Canada). Viruses encoding bicistronic mRNAs were created by recombination in 293 cells between the calcium phosphate-transfected adenovirus genome plasmid pBHGfrtΔE1,3FLP and the pDC516-based plasmids described above that encode bicistronic mRNA. Recovered virus was subjected to two rounds of plaque purification as described by the manufacturer (Microbix, Toronto, Ontario, Canada). Virus stocks were established by infection of 293 cells, and virus was purified from cells by centrifugation onto a cushion of cesium chloride as described by the manufacturer (Microbix, Toronto, Ontario, Canada).
To produce poliovirus strains P1/M and P1/HRV2, viral DNA clones pT7M and pP1HRV2 were linearized by restriction enzyme cleavage and used as templates for runoff transcription by T7 RNA polymerase (Promega, Madison, Wis.). RNA was transfected into HeLa cells using DEAE-Dextran, and after 3 days intracellular virus was released by three freeze-thaw cycles and passed once in HeLa cells. Virus was subjected to two rounds of plaque purification, and virus stocks were produced in HeLa cells.
Poliovirus replication in cultured cells.
Monolayers of adherent cells were infected at a multiplicity of 10 PFU per cell. At different times after infection, cells were scraped into tubes and subjected to three freeze-thaw cycles to release intracellular virus. The virus titer in each sample was determined by plaque assay of HeLa cell monolayers.
Assay for IRES-dependent translation in continuous cell lines.
Cells at 70% confluence in 35-mm dishes were transformed with 3 μg of plasmids encoding bicistronic mRNAs using Lipofectamine Plus (Invitrogen, Carlsbad, Calif.). Growth medium was replaced after 3 h. After 24 h, cells were washed with 1 ml of phosphate-buffered saline (PBS) and lysed in 1 ml of passive lysis buffer (Promega, Madison, Wis.). A dual luciferase assay (Promega, Madison, Wis.) and a Lumat LB9507 luminometer (EG&G Bertold, Oak Ridge, Tenn.) were used to determine firefly luciferase and Renilla luciferase activity levels in lysates. To control for variations in transformation or transcription, the ratio of firefly luciferase activity to Renilla luciferase activity was determined and defined as IRES activity. To control for IRES-independent Renilla luciferase translation, the activity of each IRES was normalized to the ratio determined in lysates from cells transfected with a plasmid lacking an IRES. The concentration of luciferase protein was calculated with reference to a standard curve generated by using known concentrations of recombinant firefly luciferase (Fisher Scientific, Springfield, N.J.) and Renilla luciferase (Chemicon, Temecula, Calif.).
Assay for IRES-dependent translation in murine organs.
C57BL/6J mice (The Jackson Laboratory, Bar Harbor, Maine) were injected (at 4 weeks or 1 to 2 days old) as follows: intraperitoneally with 109 PFU of recombinant human adenovirus for assays of heart, lung, liver, kidney, and ileum; intramuscularly with 5 × 108 PFU for assays of muscle; and intracerebrally with 5 × 108 PFU for assays of brain and spinal cord. The volume of the inoculation was 50 μl for 4-week-old mice or 15 μl for 1- to 2-day-old mice. Sixteen to 24 h after infection, mice were sacrificed, and organs were removed and homogenized with a PowerGen 125 homogenizer (Fisher Scientific, Springfield, N.J.) in 0.5 ml of passive lysis buffer. Crude protein extracts were prepared, and the dual luciferase assay and the Lumat LB9507 luminometer were used to determine firefly luciferase and Renilla luciferase activities in the extracts. To control for variation in adenovirus infection or transcription, the ratio of firefly luciferase activity to Renilla luciferase activity was determined and defined as IRES activity. To control for IRES-independent Renilla luciferase translation, the activity of each IRES was normalized to the ratio determined in organs from mice infected with an adenovirus lacking an IRES. All experimental mouse protocols adhered to Institutional Animal Care and Use Committee guidelines and were approved by the Institutional Animal Care and Use Committee of Columbia University Medical Center, New York, N.Y.
Infection of TgPVR mice with poliovirus.
TgPVR mice transgenic for the human poliovirus receptor (38
) were genotyped to ensure that they carried the human poliovirus receptor gene. Mice lacking the CD155
gene were used as negative controls. Tail fragments were incubated overnight at 55°C in 0.2 ml of 50 mM KCl, 10 mM Tris, pH 8.3, 2.5 mM MgCl2
, 0.1 mg/ml gelatin, 0.45%NP-40, 0.45% Tween 20, and 60 μg of proteinase K (The Jackson Laboratory, Bar Harbor, Maine). Two microliters of the digestion product were used as a template for PCR under standard conditions using primers 20A1C (5′-CTCACC ACTGTACTCTAGTCTG-3′) and 20A1W (5′-AGAAGGACTCACTAGACT CAGG-3′). A 350-bp PCR product indicated the presence of the human poliovirus receptor gene.
To assay viral replication, the following inoculations were performed. TgPVR mice (either 4 weeks old or 1 to 2 days old) were inoculated intraperitoneally with 105 PFU or 103 PFU of poliovirus type 1 Mahoney strain in a volume of 50 μl (4-week-old mice) or 15 μl (1- to 2-day-old mice). TgPVR mice (either 4 weeks old or 1 to 2 days old) were inoculated intraperitoneally with 2 × 107 PFU or 5 × 106 PFU of poliovirus strain P1/HRV2 in a volume of 50 μl (4-week-old mice) or 15 μl (1- to 2-day-old mice). At different times after infection, mice were sacrificed, and organs were removed and homogenized in PBS with 0.2% bovine calf serum with a PowerGen 125 homogenizer. Intracellular virus was released by three freeze-thaw cycles, and cellular debris was removed by centrifugation at 16,100 × g for 15 min at 4°C. The titer of infectious virus in the supernatant of each sample was determined by plaque assay of HeLa cell monolayers.
Generation of virulent P1/HRV2.
TgPVR mice were genotyped as described above. Neonatal TgPVR mice were inoculated intracerebrally with 5 × 106 PFU of P1/HRV2. Mice were sacrificed after 3 days, and brains were homogenized in 1 ml of PBS with 0.2% bovine calf serum. Intracellular virus was released by three freeze-thaw cycles, and cellular debris was removed by centrifugation at 16,100 × g for 15 min at 4°C. The recovered virus was amplified by infection of HeLa cells in 35-mm dishes with 100 μl of supernatant. This process was repeated three times. The titer of infectious virus in the supernatant of each brain lysate was determined by plaque assay of HeLa cell monolayers.
To assay P1/HRV2 virulence, TgPVR mice (1 to 2 days old) were inoculated intracerebrally with 5 × 106 PFU of virus in a volume of 15 μl. Eleven TgPVR mice were inoculated with P1/HRV2, 16 were inoculated in the second round of infection, and 14 were inoculated in the third round of infection. Eight TgPVR mice (4 weeks old) were inoculated with 2 × 107 PFU of P1/HRV2 recovered from the second round of infection in a volume of 50 μl. Mice were observed daily for paralysis or death, and paralyzed mice were sacrificed immediately.
To determine the nucleotide sequence of viral genomes recovered from paralyzed mice, total RNA was extracted from 0.2 ml of virus stock grown in HeLa cells using Trizol (Invitrogen, Carlsbad, Calif.). First-strand cDNA was produced using 1/20 of the RNA preparation with the Improm II reverse transcription system (Promega, Madison, Wis.) and oligonucleotide primers SCK7, SCK14, SCK20, or oligo(dT) anchor primer (5′/3′ RACE Kit; Roche Diagnostics GmbH, Mannheim, Germany) (Table ). These four cDNAs were amplified in a PCR using 5 μl of each reverse transcription reaction and oligonucleotide pairs SK103/SCK7, SCK6/SCK14, SCK13/SCK20, or SCK18/oligo(dT) anchor primer (Table ), respectively. Nucleotide sequencing reactions used oligonucleotides SKC1, SCK2, SCK3, SCK4, SCK5, SCK8, SCK9, SCK10, SCK11, SCK12, SCK15, SCK16, SCK17, SCK19, SK104, and SK105 (Table ). The nucleotide sequences of PCR products were determined by the Herbert Irving Comprehensive Cancer Center DNA Facility, Columbia University Medical Center, New York, N.Y.
Oligonucleotides used for sequence analysis of the P1/HRV2 genome