REPORTS which is latently infected with a recombinant MHV68 encoding a hygromycin resistance protein fused to an enhanced green fluorescent protein . We also explored other murine cell lines, such as the MHV68-infected lymphoma cell line, S11, and de novo infected (M12) B cells to examine reactivation from latency . In MHV68, the replication and transcription activator, RTA (encoded by ORF50), is sufficient to disrupt latency and drive lytic replication . TPA (12-O-tetradecanoyl-phorbol-13-acetate) is proposed to upregulate RTA expression in a protein kinase C-dependent manner . Sodium butyrate (NaBut) has also been used to induce reactivation in KSHV cells. In this study, we treated latent cultures with TPA and NF-kB inhibitors, alone or in combination, and examined for reactivation. We have screened Bay11-7082, which prevents IKK-dependent degradation of NF-kB inhibitor, IkBk, and has been shown to increase lytic replication in KSHV- and EBV-positive B lymphocytes . Additional NF-kB inhibitory drugs were utilized, including MG132, a proteasome inhibitor that blocks the degradation of the NF-kB inhibitor IkBk and consequently prevents NF-kB activation. We aim to apply these reactivation conditions to identify viral genes or other aspects of the virus life cycle that are impacted by NF-kB signaling. Further understanding of this pathway will give us better insight into a critical host-virus interaction that influences chronic infection and lymphomagenesis by gammaherpesviruses.
Methods Induction Experiments: A20-HE, M12 and S11 cell lines were grown at 37째C in RPMI media, supplemented with 10% fetal bovine serum, 5% L-glutamine, 5% penicillin/streptomycin and 50 uM betamercaptoethanol. A20-HE cells were maintained using 300 ug/mL hygromycin until the day before induction. All cell lines were subcultured 1:3 on the day before induction. On the day of induction, cells were seeded into 12-well plates at 106 cells/well and the appropriate drug combinations were added. Cells were in-
cubated at 37째C and harvested at the 24or 48-hour time points to extract DNA and virus for quantitative PCR and viral plaque assays, respectively. Drug toxicity was also examined with Trypan Blue staining. 1. S11 cells: Lymphoma B cells from an infected mouse that are latently infected with MHV68 . Final concentrations of TPA and NaBut were 20 ng/mL and 4 mM, respectively, at 24 hours after treatment. 2. MHV68-infected M12 cells: Mature murine B cells newly infected with MHV68 for four days. Before treatment, cells were split 1:4 into 6-well plates and grown in either fresh or conditioned (previously used) media. Final concentration of TPA was 20 ng/ml for 24 hours. 3. A20-HE2 cells: Mature murine B cells latently infected with MHV68. Final concentration of TPA was 20 ng/mL. MG132 concentrations were 1 uM and 5 uM for 24 hours. 4. A20-HE2 cells: As described above. Final concentrations of TPA and BAY-11 were 20 ng/mL and 40 uM, respectively, at 48 hours. De novo Infection of M12 cells (Spinoculation): One day prior to infection, M12 cells were subcultured 1:3. On the day of infection, cells were seeded at 2x106 cells/ mL per well in 12-well plates. Polybrene (8 ug/mL) was added to each milliliter of cells. Concentrated recombinant YFPMHV68 virus (MOI 10) was then applied and the plates were spun at 2500 RPM for 60 minutes. After centrifugation, 10% RPMI was added to bring the final volume to 1 mL. Drug Toxicity. To evaluate drug toxicity in HE and S11 cells, an aliquot of cells was stained with Trypan Blue and counted using a hemacytometer. The number of live cells in treated samples was graphed as the percentage of the number of cells in the untreated sample. Toxicity in M12 cells was measured using flow-cytometry. Quantitative PCR. DNA from untreated and treated cells was purified using the Qiagen DNeasy Kit. Samples were loaded at 200 ng and quantitative PCR was performed using ORF50 and GAPDH primers to determine the concentration of viral and cellular DNA, respectively, for each treatment. The ratios
The Stony Brook Young Investigators Review, Fall 2011
of ORF50/GAPDH were compared to uninduced cells as a measure of fold induction. Plaque Assays: 3T12 cells were previously seeded at 1.8x105 cells/well in 6-well plates. Virus was extracted from uninduced and induced cells through three consecutive cycles of freeze-thawing and bead disruption. Two dilutions of the cell/supernatant mixture were made (101 and 10-2) and 200 uL aliquots of each dilution were added to the 3T12 cells. The plates were swirled every 15 minutes for one hour and methylcellulose was then added. Cells were incubated at 37째C for one week. The number of plaque-forming units per milliliter (viral titer) was calculated for each condition. Western Blot: Cells were lysed with complete RIPA buffer supplemented with 10 uL proteinase inhibitor cocktail (1:100, Sigma) and 100 uM PMSF and quantitated with the Bio-Rad DC protein assay. Samples were heated to 100째C and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were transferred to nitrocellulose and identified using a polyclonal mouse antibody for lytic antigen. Immobilized antibodyantigen was detected using horseradish peroxidase-conjugated secondary antibody and exposed to film. GAPDH was used as a loading control.
Results and Discussion
To determine the role of the NF-kB pathway in the gammaherpesvirus lifecycle, various treatment combinations of lytic activators TPA and NaBut, in combination with NF-kB inhibitors MG132 and Bay11, were used to induce reactivation in three different MHV86-positive murine B cell lines. Following a 24-48 hour post-treatment incubation period, cell viability, increases in viral DNA and infectious virus were considered in determining the optimal conditions for lytic replication. S11 cells can be induced to reactivate. Trypan blue assays suggest that the viability of S11 cells was decreased with TPA and/or NaBut treatment (Figure 2A). At most, the NaBut-treated culture had 60% live cells in contrast to TPA+NaButtreated cells, which had about 30% viabil-