Cell lines and chemicals.
Cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA; http://www.atcc.org/
). The Vero cell line (AGM kidney cells; ATCC catalog no. CCL-81, lot no. 3645301; passage 120) was grown in Eagle minimum essential medium (EMEM) (modified), with Earle's salts and without l
-glutamine (Mediatech, Manassas, VA; catalog no. 15-010-CV), supplemented with 5% fetal bovine serum (FBS), per ATCC instructions (heat inactivated at 56°C for 30 min; HyClone, Logan, UT; catalog no. SH30071.03), 2 mM l
-glutamine, 250 U of penicillin per ml, 250 μg of streptomycin per ml, 1× nonessential amino acids (MEM-NEAA 100×; Quality Biological Inc., Gaithersburg, MD, catalog no. 116-078-061), and 1 mM sodium pyruvate (Quality Biological, Inc.), designated complete medium. To maximize reproducibility of results in the induction assays, a cell bank was established at passage 123. A new vial was used in each experiment to maintain similar passage numbers in the induction studies.
For virus-induction studies, Vero cells were treated with different concentrations of IUdR (stock solution, 75 mg per ml in 1 N NH4OH; Sigma, St. Louis, MO; catalog no. 17125), AzaC (1 mg/ml in complete Vero cell medium; Sigma catalog no. A1287), and sodium butyrate (NaBut; 0.9 M in sterile H2O; Sigma catalog no. B5887).
Target cell lines used in infectivity studies were A-204 (human rhabdomyosarcoma; ATCC catalog no. HTB-82), Raji (human B cell lymphoma; ATCC catalog no. CCL-86), and Cf2Th (dog thymus; ATCC catalog no. CRL-1430). Cf2Th cells were grown in Dulbecco's modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA; catalog no. 119955) supplemented with 20% FBS; A-204 and Raji cells were grown in RPMI 1640 (Quality Biological catalog no. 112-024-101) supplemented with 10% FBS and 1× MEM-NEAA. Additionally, both media contained 2 mM l-glutamine, 250 U of penicillin per ml, and 250 μg of streptomycin per ml.
Growth curve and population doubling time (PDT).
Cells were counted using an automated Guava PCA flow cytometer according to the manufacturer's protocol (Guava ViaCount assay; Hayward, CA). Cells were diluted, and the numbers of viable cells, dead cells, and apoptotic cells were counted in triplicate. The average count of the viable cell numbers was used in the experiments. For cell cycle analysis, 0.5 × 106 cells were processed and stained according to the manufacturer's protocol (Guava cell cycle assay).
To determine the optimum number of cells for obtaining a sigmoidal growth curve, Vero cells (0.5 × 106
, 0.75 × 106
, and 1.0 × 106
) were planted in 5 ml complete medium in 25-cm2
flasks, and viable cells were counted at various times using a Guava PCA cytometer. Cell cycle analysis was done to determine the cell phases. PDT was calculated as 1/k
= PDT) from the linear curve in the log phase from the formula N
= total viable cell number at end time t
= total viable cell number at initial time t0
= hours from N0
, and k
= regression constant) (60
). Results were confirmed in three independent assays.
Drug dose evaluation.
Vero cells (1 × 106; passages 125 to 131) were planted for 16 h in 25-cm2 flasks before replacing medium with fresh medium containing different concentrations of AzaC (0.3125 to 40 μg/ml), IUdR (50 to 3,200 μg/ml), or NaBut (1 to 6 mM). After 48 h of drug treatment, cells were washed with medium three times (designated day 0), trypsinized, and counted by using a Guava PCA cytometer. Another set of flasks were further incubated after the medium change, and the cells were trypsinized and counted at day 3. Untreated cells, at confluence, were used as a control to evaluate cell toxicity and cell recovery based upon the cell-confluence ratio at day 0 and at day 3, which was calculated by dividing the number of viable cells in the drug-treated flask by the number of viable cells at confluence in the untreated control flask (2.3 × 106 cells) and expressed as a percentage. Furthermore, in the case of IUdR-treated cells, additional controls were included to evaluate toxicity due to NH4OH used for dissolving the drug. In case the number of cells in the NH4OH-treated flask was less than that of the untreated control flask, the number of viable cells in the IUdR-treated flask was divided by the number of viable cells in the NH4OH-treated flask before determining the cell-confluence ratio.
Chemical treatment and evaluation for induced retroviruses by the STF-PERT assay.
Vero cells were drug treated under optimized induction conditions: cells (1 × 106
) were planted for 16 h and then treated with drug for 48 h (AzaC, 1.25 μg/ml; IUdR, 200 μg/ml; and NaBut, 3 mM); untreated cells were included as a control. For kinetics of virus induction, medium was replaced daily and filtered supernatant collected for detection of reverse transcriptase (RT) activity by the single-tube fluorescent PCR-enhanced reverse transcriptase (STF-PERT) assay (53
). Supernatants were collected and filtered (Costar Spin-X centrifuge tube filters, 0.45-μm-pore-size CA membrane; Corning, Corning, NY, catalog no. 8162) on the day of drug removal (day 0), prior to washing the cells, and then daily, at each medium change. Filtered supernatants were stored at −80°C in single-use, 10-μl aliquots for STF-PERT analysis and in 0.5-ml aliquots for additional use. Each sample was tested at a 1:10 dilution (per the assay protocol), and results were obtained from triplicate samples. The PERT assays for testing supernatant from drug-treated cells met the acceptability criteria (53
): IUdR, slope = −3.96, y
intercept = 48.06, r2
= 0.999; AzaC, slope = −3.14, y
intercept = 42.59, r2
= 0.996; NaBut, slope = −3.97, y
intercept = 48.05, r2
= 0.996. Negative controls were cells without drug (or with NH4
OH in the case of IUdR cell toxicity studies) and were set up in parallel.
Total cellular RNAs were extracted by using the RNeasy Plus minikit (Qiagen, Valencia, CA; catalog no. 74134) in combination with the RNase-free DNase set (Qiagen; catalog no. 79254) according to the manufacturer's instructions. Concentration and purity were determined by using UV absorbance.
A low-concentrated (10×) supernatant sample was prepared from normal and drug-treated cells by ultracentrifugation of filtered supernatant (1.5 ml) at 45,000 rpm (Beckman TLA 45 rotor) for 90 min at 4°C. RNA was prepared from the pellet by resuspending it in 130 μl of Promega DNase buffer and adding 10 μl DNase (1 U per μl; RNase-free DNase; Promega, Madison, WI, catalog no. M6101) for incubation at 37°C for 30 min. RNA was extracted from the entire sample by using the QIAamp viral RNA minikit (Qiagen catalog no. 52904).
A high-concentrated (1,000×) supernatant sample was prepared from normal and from AzaC-treated Vero cells (1.25 μg/ml for 48 h) on day 4 after drug treatment (medium was changed on day 1) by ultracentrifugation of pooled (180 ml), filtered supernatant (tube top vacuum filters, 0.45-μm-pore-size CA membrane, Corning catalog no 430314) on a 20% sucrose cushion (25,000 rpm in a Beckman SW-28 rotor for 4 h at 4°C). Pellets were pooled, resuspended in 4 ml phosphate-buffered saline (PBS) (pH 7.4), and ultracentrifuged immediately at 35,000 rpm (Beckman SW-41 rotor) for 90 min at 4°C. The pellet was resuspended in 180 μl in PBS (pH 7.4) and stored in aliquots at −80°C to minimize freeze-thaw of test samples. RNA was extracted from 50 μl using the QIAamp viral RNA minikit after DNase I digestion (1 U per μl), as described above.
One-half of the RNA sample was used for cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA; catalog no. 170-8890) according to the manufacturer's instructions. The other half of the RNA was used for control without RT. Additionally, PCR amplification using human β-actin primers was performed to demonstrate absence of cellular DNA according to the manufacturer's protocol (Clontech, Mountain View, CA; catalog no. 639008).
Consensus PCR primers (SRV/SERV) were designed based upon GenBank sequences of SRV-1 type D retrovirus (M11841), SRV-2 complete genome (AF126467), simian Mason-Pfizer D-type retrovirus or SRV-3 (M12349), and simian type D virus 1, complete proviral genome (U85505; designated SERVbab in this paper). The location of the SRV/SERV primers is given in : a long terminal repeat (LTR) gag fragment (553 bp) was amplified using forward primer F04, 5′-CTGTCTTGTCTCCATTTCT-3′, and reverse primer R10, 5′-ACSGCAGCCATKACTTGYGG-3′; a pol fragment (610 bp) was amplified using forward primer F41, 5′-TACAAGAYCCMTAYACCTA-3′, and reverse primer R46, 5′-TTDGGTGGRTAATGGTTRTC-3′; and an env fragment (548 bp) was amplified using forward primer F65, 5′-CAYATNTCYGATGGAGGAGG-3′, and reverse primer R70, 5′-CCYGTCCARTTTGTRGGTA-3′. PCR conditions were 95°C for 3 min, followed by 35 amplification cycles of 95°C for 30 s, 56°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min.
Comparative nucleotide sequence analysis of cloned SERVagm-Vero DNAa
Primers for amplification of BaEV sequences and PCR cycle conditions were as described previously (82
): RT1 and RT2 in the pol
region, and ENV1/ENV4 with nested primers ENV2/ENV3 in the env
region. Additional primers were made for PCR amplification in the gag
region: outer primer pairs were GAG1 (5′-GAGTGGCCCACCCTTCATGT-3′) and GAG2 (5′-CAGTACTGGATCGTGCGGTT-3′), at nucleotide positions 1108 to 1127 and 1697 to 1678, respectively, and inner primer pairs were GAG3 (5′-CCCCGGGACGGAACTTTTGA-3′) and GAG4 (5′-GATGAGGTAGAGGGTCTTGGAAG-3′) at nucleotide positions 1135 to 1154 and 1420 to 1398, respectively. The nucleotide positions are based upon the sequence of the BaEV by Kato et al. (35
PCRs were done in 25 μl using 2 μl cDNA template, 10× PCR buffer containing 15 mM MgCl2, and 1.5 U Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN; catalog no. 11647687001). The final concentration of deoxynucleotide triphosphates were 200 μM each, and primers were 1 μM each.
Nucleotide sequence analysis.
PCR-amplified DNA fragments were isolated from agarose gels by using the Zymoclean gel DNA recovery kit (Zymo Research Corporation, Orange, CA; catalog no. D4001) and cloned into the pGEM-T Easy vector (Promega catalog no. A1360). Nucleotide sequences were determined with T7 and SP6 primers by using an ABI 3130xl genetic analyzer according to the manufacturer's standard protocol (Applied Biosystems, Foster City, CA). Sequence analysis and alignment of the sequences were done using nucleotide BLAST (National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD).
Supernatant (160 ml) was collected from Vero cells without drug treatment and from Vero cells on day 4 after drug treatment for 48 h with IUdR (200 μg/ml) and AzaC (1.25 μg/ml) for 48 h. Ultracentrifuged pellets were obtained through a 20% sucrose cushion by using the procedure described above for preparation of the 1,000× supernatant sample. The pellets were fixed overnight in McDowell and Trumps fixative and sent to Charles River Pathology Associates (Durham, NC) for evaluation of viruslike particles. The volume of the pellet was measured by comparison to known standards and processed for transmission electron microscopy (TEM) analysis. Thin sections were cut, stained with methanolic uranyl acetate and Reynolds lead citrate, and examined by TEM. Ten grid spaces were examined and evaluated for numbers of particles with retrovirus-like morphology. The number of viruslike particles in the entire pellet was calculated by multiplying the number of particles tabulated in the examined section by the number of potential sections in the pellet (calculated by dividing the volume of the entire pellet by the volume of the section examined). The limit of sensitivity of the assay was calculated as the smallest detectable amount of virus in the samples or one particle in the section examined by TEM. Thus, to obtain the limit of sensitivity, the number of potential sections in the pellet is multiplied by 1.
Cell pellets (2 × 106
to 4 × 106
) for TEM analysis were prepared by trypsinizing cells from normal or AzaC-treated Vero cells, as previously described (41
Combined infectivity and coculture studies were set up with cells and supernatant from drug-treated cells that were prepared by planting Vero cells (1 × 106; passage 131) in 25-cm2 flasks for 16 h and then treating the cells with drug for 48 h (1.25 μg/ml AzaC or 200 μg/ml IUdR). Cells were washed three times with plain medium to remove the drug, and fresh complete medium was added (day 0). Medium was replaced the next day (day 1); on day 4, unfiltered supernatant and cells from eight 25-cm2 flasks were pooled and used for infection/coculture with target cells at predetermined cell ratios for equivalent growth of test and target cells. Target control cells were set up without coculture, and control cocultures were set up with target cells and uninduced Vero cells.
In the case of the A-204 and Cf2Th adherent target cells, 2.7 × 106 and 1.5 × 106 cells, respectively, were set up in 10 ml medium for preincubation with 5 ml unfiltered supernatant from AzaC-treated and from IUdR-treated Vero cells (passage 131) at 37°C for 45 min in 75-cm2 flasks, after which trypsinized, AzaC- or IUdR-treated Vero cells (about 3.0 × 106 cells per flask) were added into the corresponding flasks. The coculture ratio of Vero cells to target cells was 1:1 for A-204 cells and 2:1 for Cf2Th cells. In the case of the Raji suspension target cells, 8 × 106 cells in 10 ml per flask were incubated at 37°C for 45 min with unfiltered supernatant from AzaC- or IUdR-treated Vero cells (5 ml), and then all of the cells and supernatant (total volume of 15 ml) were added by replacing the medium in flasks containing drug-treated Vero cells (3 × 106), which had been preincubated for 45 min in 75-cm2 flasks. The target cells were demonstrated to be susceptible to SRV (A-204 and Raji) and to squirrel monkey retrovirus (SMRV) (Cf2Th). Medium was replaced with 13 ml of target cell medium following overnight incubation. In the case of Raji cells, the supernatant was collected and spun at 1,200 rpm (GS-6KR centrifuge with a GH-3.8 rotor; Beckman Instruments, Columbia, MD) for 10 min at 4°C, and the Raji cells were resuspended in 10 ml medium and then added back to the flask containing Vero cells in 10 ml fresh medium. Upon reaching 95% confluence, all of the cells were passaged the next day (day 2) into 162-cm2 flasks. Cultures were propagated in 162-cm2 flasks with passage every 2 to 3 days until termination on day 32.
Extended cell culture was done on AzaC-induced Vero cells (passage 135; 1.0 × 106 cells in 25-cm2 flasks; 1.25 μg/ml in 5 ml). For extended culture, cells were washed 48 h after drug treatment (day 0, the day of drug removal) and then cultured in fresh complete medium. Upon reaching confluence, cells were passaged into 75-cm2 and then into 162-cm2 flasks at the same time as the cocultures and continued in 162-cm2 flasks until termination on day 34. Uninduced Vero cells were included as a control.
The cultures were regularly monitored for cytopathic effect (CPE). Filtered supernatants were collected starting at the first passage at day 4 after coculture or after drug treatment, until termination, and stored at −80°C for the STF-PERT assay.