ity. In addition to higher viability, NaBut treated samples also had greater levels of viral (ORF50) DNA compared to the mock (Figure 2B). TPA-treated samples did not differ from the mock in ORF50 content. Cells induced with NaBut were positive for lytic protein in an immunoblot, confirming reactivation took place (data not shown). Although S11 cells can be induced, a high level of spontaneous reactivation occurred, even in untreated samples (3.3x106 PFU/mL, data not shown). Therefore, it would be difficult to distinguish the effect of NF-ÎşB inhibitors in these cells. This stresses the need for a cell line that displays limited gene expression before induction and an increase in viral DNA after exposure to lytic activators. De novo infected M12 cells are induced by TPA. We next examined establishment of latency and reactivation in the newly infected B cell line, M12. Flow-cytometry analysis showed that about 20-25% of cells are capable of being infected, as the expression levels of YFP increases significantly in de novo infected M12 cells for both fresh and conditioned media samples (Figure 3). Following TPA induction, there is no change in YFP expression for fresh media samples, but a slight increase was observed in cells grown in conditioned media. In an immunoblot of M12 lysates, lytic protein is present in all infected samples (Figure 4). However, a stronger positive signal is present in conditioned media TPAtreated infected sample compared to the uninduced. Fresh media infected samples produced less lytic antigen compared to cells grown in conditioned media. These results suggest that reactivation in conditioned media is slightly enhanced. However, the M12 cells do not exhibit a strong induction with TPA, and therefore may not be the best model for evaluating reactivation stimuli. Reactivation of MHV68 by MG132 treatment is dose-dependent. We also explored the latently infected mature B cell line, A20-HE. The HE cells were treated with MG132, a proteasome inhibitor that may inhibit NF-kB by blocking the degradation of its inhibitor, IkBk. Trypan blue assays conducted with MG132treated A20-HE cells indicate that cell viability is largely compromised, with less
Figure 2: Evaluation of TPA and NaBut in S11 cells. A. At 24 hours, treatment of S11 cells with either TPA or NaBut had an adverse effect on cell viability. B. NaBut increased viral copy number by 1.5-fold compared to the mock. TPA alone and in combination with NaBut did not increase ORF50 levels.
Figure 3: Treatment of M12 cells with TPA. De novo infected cells cultured in conditioned media exhibited a significant increase in YFP expression compared to uninfected samples. YFP expression increases slightly after TPA treatment with cells grown in conditioned media.
Figure 4: Immunoblot of TPA-treated M12 lysates. In cells seeded with conditioned media, viral antigen is observed upon de novo infection. This is further enhanced with TPA treatment. In contrast, there is no difference in infected cells with or without TPA stimulation when seeded with fresh media.
The Stony Brook Young Investigators Review, Fall 2011