Figure 5: Evaluation of MG132 A. Treatment of HE cells with MG132 is extremely toxic at 24 hours post-treatment, with a survival rate of less than 20%. B. MG132 causes a dose-dependent increase in viral copy-number
than 20% live cells (Figure 5A). However, application of TPA and increasing concentrations of MG132 produced about 4 to 7-fold greater levels of ORF50 DNA compared to the mock, a much greater increase in viral copy number in comparison to the previous two cell lines. MG132 in combination with 5 uM of MG132 in particular showed the greatest increase in viral copy number (Figure 5B). TPA and 1 uM MG132 contained the lowest levels of ORF50 DNA, with no corresponding differences in viability. No plaques were observed and therefore viral titer could not be determined. The cell toxicity of MG132 may be linked to the drugâ€™s nonspecific degradation of proteins within the cell, leading to the disruption of multiple signaling pathways that lead to cell death. Therefore, MG132 would not be
an ideal reagent to test NF-kB inhibition in the context of reactivation. Additional induction experiments were conducted in which MG132 was removed after 2 hours (data not shown). These experiments revealed increased cell viability but no notable changes in viral DNA (0.94-fold increase in ORF50 DNA). Treatment with TPA and Bay11 increases viral DNA 4-fold. Treatment of HE cells with TPA, Bay11 and in combination was conducted in triplicate to determine statistical significance. Based on Trypan blue viability staining, cells treated with TPA have decreased cell viability by about 50%, while application of Bay11 does not impact viability (Figure 6A). Quantitative PCR analysis indicates that treatment with TPA+Bay11 yields
a significant 4-fold increase in ORF50 content compared to TPA alone (Figure 6B). By itself, TPA increases reactivation in comparison to the mock, while Bay11 samples have no changes in viral DNA levels. In samples treated with TPA and Bay11 together, higher levels of infectious virus as well as a positive result for lytic antigen complement this increase in viral DNA (Figure 6C, 7). Unfortunately, the plaque assay results varied slightly between replicates and conditions are still being optimized in order to confirm the significance of this enhancement. The immunoblot of treated cell lysates reveals the importance of the 48 hours post-treatment timepoint. While no lytic antigen is detected for any treatment at 24 hours, there is a faint positive signal for TPA and a strong positive signal for TPA+Bay11 at 48 hours post-treatment compared to the mock (Figure 7). This data suggests that more lytic protein is produced with TPA and Bay11 together and reactivation has been successfully initiated. As a NF-kB specific inhibitor, Bay11 appears to work synergistically with TPA by inducing the transcription activator RTA and removing the signal for IKK-dependent degradation of IkBk. Thus, Bay11 may be a better approach for observing NF-kBâ€™s role in the switch from latency into lytic replication. In comparison to S11 and M12 cells, induced A20-HE cells show the greatest fold-increase in viral DNA ver-
Figure 6: Evaluation of Bay11. A. At 48 hours post-treatment, there is no significant change in HE cell viability in samples treated with Bay11 and TPA in combination compared to the mock. B. The combination of TPA and Bay11 increases viral copy number as compared to TPA alone (p=0.05). C. There appears to be a greater level of infectious virus with the combination of treatments in contrast to the treatments alone.
The Stony Brook Young Investigators Review, Fall 2011