HeLa S3 (human cervical carcinoma), LLC-MK2 (rhesus monkey kidney), 15C4 (human Chang conjunctiva), L (L2929, murine fibroblast), NIH 3T3 (murine fibroblast), T98-G (human glioblastoma multiforme), U373-MG (human glioblastoma), SY5Y (human neuroblastoma), and RK13 (rabbit kidney) cell lines were propagated in Dulbecco modified Eagle growth medium (DMEM) containing 10% bovine calf serum and 1% penicillin-streptomycin (Gibco). These cell lines were obtained from American Type Culture Collection (Manassas, VA) with the exception of the U373-MG and T98-G, which were obtained from the laboratory of C. S. Young at Columbia University. The HCE cell line (for human cornea fibroblast, obtained from Peter Reinach, SUNY State College of Optometry) was propagated in 50% DMEM-50% F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 250 ng of mouse epidermal growth factor (Sigma-Aldrich, St. Louis, MO)/ml, and 2.5 μg of bovine insulin (Sigma-Aldrich, St. Louis, MO)/ml.
The stable L cell line L-hDAF was isolated after cotransformation of cells with a plasmid carrying DNA of the GPI-linked form of DAF (pDAFBOS, provided by D. Lublin, Washington University, St. Louis, MO) with a plasmid encoding the puromycin resistance gene (pRSVPR puro-R). Transformants were selected in 10 μg of puromycin/ml for 2 weeks. DAF production was determined by flow cytometry using anti-human CD55 (DAF) SCR1 domain antibody (BD Biosciences Pharmingen, San Diego, CA) and anti-human CD55 (DAF) SCR3 (clone BRIC 216, MCA914; Serotec, Raleigh, NC). The cell line used in these experiments, L-DAF8H4, was obtained after four cycles of sorting for high DAF SCR1 domain expression. This cell line is maintained in DMEM, 10% bovine calf serum, 1% penicillin-streptomycin, and 10 μg of puromycin/ml.
EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C. The virus was plaque purified three times in LLC-MK2 cells and designated EV70-Rmk. This virus was passaged nine times in HeLa cells, after which cytopathic effects became evident 8 h postinfection. The virus was plaque purified three times in HeLa cells and designated EV70-Dne. Other virus stocks were obtained by transfection of cell monolayers with RNA produced by in vitro transcription of cloned genomic DNA, including EV70-Rmk (from plasmid pRMK14), EV70-Dne (from plasmid pDNE9), and poliovirus P2/Lansing (from plasmid PT7L) (32
). RNA transcripts were produced by in vitro transcription of a linearized template using T7 RNA polymerase (Invitrogen) as previously described (26
). Virus titers were determined by plaque assay in HeLa cells or LLC-MK2 cells as described previously (26
Isolation of viral RNA.
Virion RNA for the production of DNA clones was isolated by ultracentrifugation of infected cell supernatants in an SW28 rotor at 28,000 rpm for 3 h at 4°C. Supernatants were discarded, and pellets were pooled and suspended in 500 μl of DMEM. An equal volume of TRIzol reagent (Invitrogen) was added to the suspended pellet in 1.5-ml Eppendorf tubes. This mixture was centrifuged at 14,000 rpm for 10 min to induce phase separation. The top, aqueous phase was harvested, an equal volume of isopropanol was added, and the mixture was centrifuged for 10 min at 14,000 rpm. The supernatant was aspirated, leaving the RNA pellet to air dry. The RNA was resuspended in H2O and incubated for 10 min at 65°C, and then 10 mM dithiothreitol and 5% (vol/vol) RNasin (Promega, Madison, WI) were added.
Production of infectious viral DNA clones.
DNA copies of the EV70-Rmk and EV70-Dne viral genomes were cloned into the plasmid pATEVL. Viral RNA was reverse transcribed at 42°C for 1 h by using Superscript II reverse transcriptase (Invitrogen) and a 3′ primer which encoded (5′ to 3′) a BamHI restriction site, 20 polyadenine bases, and the last 15 bases at the 3′ end of the EV70 genome. After 1 h, 1 U of RNase H was added, and the sample was incubated for 30 min at 37°C. Viral DNA was amplified by PCR using the 3′ primer described above, and a 5′ end primer comprising from 5′ to 3′ an AscI restriction site, a T7 promoter, and the first 12 bases of the viral RNA. EV70 primers were derived from the EV70 reference sequence D00820 (National Center for Biotechnology Information). PCR products were cloned into plasmid pATEVL. This vector was produced by introducing a polylinker containing cleavage sites for AscI, SpeI, ApaI, BsteII, and PacI into the EcoRI and HindIII sites of plasmid pAT153. RNA transcripts were produced by in vitro transcription of a BamHI-linearized template using T7 RNA polymerase (Promega) and introduced into LLC-MK2 cells using DEAE-dextran (40
). EV70-Rmk virus produced after transfection with RNA from clone 14 was selected for further studies and named EV70-Rmk14.
Nucleotide sequence analysis of subgenomic DNA clones produced from EV70-Dne RNA revealed that it differed from the genome of EV70-Rmk by five coding changes. A full-length infectious DNA copy of the EV70-Dne genome was produced from the Rmk14 full-length DNA clone by using PCR mutagenesis to change five amino acids in the RMK polyprotein: E14K, I557M, F694L, R719P, and N787D. Mutagenic primers were annealed to both strands of the parental supercoiled plasmid, and then gaps between sites of hybridization were filled in during the elongation cycle of the PCR. After thermocycling, 1 μl of DpnI restriction enzyme (New England Biolabs, Beverly, MA) was added to each reaction to digest parent double-stranded plasmids. After heat inactivation at 80°C for 20 min, samples were introduced into XL10Gold bacteria (Stratagene, La Jolla, CA). The sequence of the entire EV70-Dne DNA was determined to ensure that no other mutations were present. RNA transcripts were produced by in vitro transcription of a BamHI-linearized template using T7 RNA polymerase and introduced into HeLa cells using DEAE-dextran. EV70-Dne virus produced after transfection with RNA from clone 9 was selected for further studies and named EV70-Dne.
Single amino acid coding changes were introduced into the genome of EV70-Dne and EV70-Rmk by PCR mutagenesis of cloned DNA as described in the preceding paragraph. The presence of the desired nucleotide was confirmed by nucleotide sequencing of full-length viral cDNA derived from plasmid DNA isolated from individual bacterial colonies. (The nucleotide sequence of EV70-Rmk has been deposited in GenBank under accession number 76496395.)
Quantitation of viral RNA.
Slot blot hybridization analysis was performed by extracting total cytoplasmic RNA from infected cells and vacuum blotting samples onto a GeneScreen membrane (New England Nuclear, Boston, MA). Samples were cross-linked to the membrane by using UV irradiation in a Stratalinker (Stratagene, La Jolla, CA). Membranes were prehybridized for 1 h prior to the addition of the probe overnight. Membranes were washed six times under increasingly stringent conditions and then quantitated using a PhosphorImager instrument and ImageQuant software (Amersham Radiochemicals, Piscataway, NJ). Positive-strand amplification was detected using a negative-strand RNA produced by in vitro-transcribed RNA probe complementary to nucleotides 5832 to 7221 of the EV70 genome. The hybridization probe was radiolabeled with [α-32P]UTP (Amersham Biosciences, Piscataway, NJ) during an in vitro transcription reaction using T7 RNA polymerase.
EV70 plaque assay.
Titers of EV70-Dne were determined by plaque assay on HeLa cell monolayers as described previously (26
). This procedure was modified to determine titers of EV70-Rmk by plaque assay on LLC-MK2 cells. After virus adsorption, 2 ml of an overlay composed of 0.9% agar noble and 1× DMEM supplemented with 10% bovine calf serum (BCS), 2% penicillin-streptomycin, and 0.5% sodium bicarbonate was added. After solidification of the overlay, 2 ml of DMEM (supplemented with 10% BCS, 1% penicillin-streptomycin) was added to further nourish the cell monolayer. The plates were incubated for 72 h at 37°C and then stained as described previously (26
One-step growth analysis.
Cells (5 × 105 cells in a 3.5-cm plate) were infected with virus at a multiplicity of infection (MOI) of 5. After adsorption for 30 min at 37°C, the inoculum was aspirated, and 1 ml of fresh growth medium was added. At each time point, monolayers were scraped into the medium and frozen and thawed three times, and cell debris was removed by centrifugation at 2,000 rpm for 10 min. Viral supernatants were titrated on HeLa monolayers for Dne virus and LLC-MK2 monolayers for Rmk virus as described in the previous section. Each one-step growth analysis was repeated three times, and one representative graph is presented.
Measurement of cytopathic effect.
Virus-induced cell killing was quantitated by staining with trypan blue. HeLa cells were infected at an MOI of 3, the inoculum was aspirated, and the monolayer washed twice, and then 0.5 ml of growth medium was added. At different times after infection, cells and medium were collected and the cells were pelleted by low-speed centrifugation. Pellets were resuspended in phosphate-buffered saline, trypan blue dye was added, and the cells were examined by light microscopy. Approximately 150 to 200 cells were counted for each sample, and the percent viability was determined by dividing the number of cells that excluded dye by the number of cells examined.
Treatment of cells with antibodies or enzymes.
Monoclonal antibodies (0.5 μg) were added to monolayers of 106 HeLa cells, followed by incubation for 20 min at room temperature. The monoclonal antibodies used were mouse immunoglobulin G2a (IgG2a) anti-human SCR1 DAF (CD55; clone IA10; BD Biosciences Pharmingen), mouse IgG1 anti-human SCR3 DAF (CD55; clone BRIC 216, MCA914; Serotec), and mouse IgG2a anti-trinitrophenol hapten (BD Biosciences Pharmingen). Antibodies were aspirated from the wells prior to infection at an MOI of 3. At 24 h postinfection, the monolayers and their supernatants were harvested and subjected to RNA slot blot analysis. Positive-strand RNA replication was determined using a negative-strand RNA hybridization probe as described above. The replication signal for each sample was normalized to the signal for mock-infected cells.
Enzymatic blocking of viral replication was performed as described above, except that cells were incubated with enzymes diluted in DMEM without serum at 37°C for 30 min. The quantity of enzyme used per 106 HeLa cells was: 0.1 U of phosphoinositol-specific phospho-lipase C (PI-PLC from Bacillus cereus; Sigma-Aldrich, St. Louis, MO), and 0.01 U of neuraminidase (from Vibrio cholerae; Biochemika, St. Louis, MO).
Structural location of amino acid changes in the EV70 capsid.
EV70 strain-specific amino acid changes were mapped onto the capsid of bovine enterovirus (BEV) type 1 (GenBank accession number NC_001859) as follows. Amino acid alignments of BEV and EV70 were performed independently for each of the four structural proteins using the maximum matching tool of MacVector (Accelerys, Inc.), and the BEV residue that corresponds to each changed amino acid in EV70 was identified. The BEV capsid structure was visualized by using MolScript. Protomer coordinates were used to produce pentamers using Virus Particle Explorer (VIPER) (36