2.1 Cells and viruses
Akata cells were kindly provided by John Sixbey (Louisiana State University, Baton Rouge, LA). Cells were maintained in RPMI 1640, (Mediatech, Inc, Herndon, Va.) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah), L-glutamine, penicillin and gentamicin at 37°C in a humidified 5% CO2 atmosphere. Cells were passaged 2–3 days prior to performing the assay, enumerated on a hemacytometer and diluted to a final concentration of 4×105 cells/ml. Latently infected cells were induced to undergo a lytic infection by adding a F(ab′)2 fragment of goat anti-human IgG antibody (MP Biomedicals, Aurora OH).
2.2 DNA hybridization assay
Growth media containing 50 μg/ml goat anti-human IgG antibody was added to all wells except the cell control wells of a round bottom 96-well plate. Experimental compounds were serially diluted in duplicate at 1:5 with concentrations ranging from 100 μg/ml to 0.032 μg/ml. Latently infected Akata cells were diluted to a final concentration of 4×105
cells/ml and 100 μl was added to each well. The plates were incubated for 72 hours after which antiviral activity was assessed using the blot hybridization assay. After incubation 100 μl of denaturation buffer (1.2 M NaOH, 4.5 M NaCl) was added to each well and 50 μl was added to the wells of a Biodot apparatus (Bio-Rad, Hercules, CA) containing an Immobilon nylon membrane (Millipore, Bedford, A). A 50 μl aliquot of denatured DNA from the cells that were induced to undergo a lytic infection was aspirated through the membrane and then 50 μl of denaturation buffer was added. The membrane was allowed to dry before beginning the hybridization process. The membrane was then incubated in DIG Easy Hyb (Roche Diagnostics, Indianapolis, IN.) at 56°C for 30 minutes. A specific digoxigenin (DIG)-labeled probe was prepared according to the manufacturer’s protocol (Roche Diagnostics) using PCR primers, EBNA probe forward 5′ CCC AGG AGT CCC AGT AGT CA 3′ and EBNA probe reverse 5′ CAG TTC CTC GCC TTA GGT TG 3′. The resulting probe corresponds to coordinates 96802–97234 in EBV genome (AJ507799) and was hybridized to the blots overnight at 56°C. Blots were then washed at 56°C with 0.2X SSC with 0.1% SDS and 0.1X SSC with 0.1% SDS. Detection of specifically bound DIG probe was performed with anti-DIG antibody using the manufacturer’s protocol (Roche Diagnostics). An image of the film was captured and quantified with QuantityOne software (Bio-Rad). Drug concentrations sufficient to reduce the accumulation of viral DNA by 50% (EC50
), were interpolated from the dose response using standard methods (Prichard and Shipman, 1990
2.3 EBV Real-Time PCR
EBV DNA was purified according to the manufacturer’s instructions using the Wizard SV 96 Genomic DNA Purification System (Promega, Madison, WI). The purified DNA was then subjected to Real-Time PCR to quantify viral DNA. The primers 5′-CGG AAG CCC TCT GGA CTT C-3′ and 5′-CCC TGT TTA TCC GAT GGA ATG-3′ were used with the fluorescent probe, 6FAM-TGT ACA CGC ACG AGA AAT GCG CC-TAMRA corresponding to coordinates 155959–155981 in the EBV genome (Applied Biosystems). A plasmid containing the amplified region was diluted to produce the standards used to calculate genome equivalents and ddH2O as a negative control. The PCR was performed in an optical 96 well plate using an ABI 7300 Real-Time PCR system. The PCR reaction contained 900nM primers, 200nM probe, 12.5 μL Taqman Universal Master Mix (Applied Biosystems, Foster City, CA), and 5 μL target DNA in a final volume of 25 μL. Amplification conditions were: 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each sample was run in duplicate and the copy number was calculated using the 7300 system software.
2.4 Cytotoxicity assays
Cytotoxicity was evaluated by both MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazolium) and CellTiter-Glo (Promega). Assay plates for toxicity were prepared in the same manner as those for the antiviral assay, except that the cells were not induced to undergo a lytic infection and a set of control wells were added that contained only growth media. Cytotoxicity was assessed at three days, the same time that viral DNA was harvested. In the MTS assay, 20 μl of MTS was added to each well and the plate was wrapped in aluminum foil and incubated at 37°C for 4 hours. The quantity of formazan product was measured at 490 nm in a microplate reader and the CC50
values were calculated by standard methods (Prichard & Shipman, 1990
). Cell viability was also assessed with the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Briefly, assay plates were incubated at ambient temperature for 30 minutes, 50 μl of CellTiter-Glo reagent was added to each well and the plates were mixed for 2 minutes on an orbital shaker to induce cell lysis. The plates were then incubated for an additional 10 minutes at room temperature and the luminescence was quantified on a luminometer. Standard methods were used to calculate drug concentrations that inhibited the proliferation of uninduced Akata cells by 50% (CC50
Proteins from cells induced to undergo a lytic infection in 96-well plates from hybridization assays described above, were solubilized in Laemli buffer, separated using 10% Tris HCl gels, and transferred to PVDF membranes. Viral proteins were specifically detected with monoclonal antibodies to EA-D and VCA (Chemicon, Temecula, CA). Bound antibodies were detected with alkaline phosphatase conjugated goat anti-mouse antisera (Southern Biotech, Birmingham, AL) and visualized with CDP* (Roche, Indianapolis IN) and Biomax film (Kodak, Rochester, NY).
2.6 Antiviral Drugs
Cidofovir (CDV) was a gift of Mick Hitchcock (Gilead Sciences, Foster City, CA). Acyclovir (ACV), ganciclovir (GCV), and thymidine (THD) were obtained from a commercial source (Sigma-Aldrich, St. Louis, MO). Maribavir (MBV), cyclopropavir (CPV) and (N)-methanocarbathymidine (MCT) were described previously (Gershburg & Pagano, 2002
, Kushner et al., 2003
, Prichard et al., 2006
), and were obtained through the NIAID, NIH, Bethesda, MD.