Figure 4: F49A-FTP kills experimentally latently infected monocytes, reducing HCMV reactivation events.

CD14+ peripheral blood monocytes were isolated and experimentally infected at an MOI of 5 with HCMV SV40-GFP-TB40E wild type that led to a mean average of 10.9% latently infected cells. After 24 h, F49A-FTP was added to monocyte cultures, and incubated for 72 h. Changes in the number of GFP-positive cells was observed by fluorescence microscopy and compared with a mock-treated latently infected cell controls that was set at 100% (a). These monocytes were then differentiated using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) stimulation followed by LPS treatment to mature dendritic cells (mDCs). Similarly, changes in the percentage of GFP-positive mDCs were then measured (b). Finally, the cells shown in b were co-cultured with fibroblasts for 2 weeks and the numbers of IE foci were counted (c). Means and error bars (showing s.d.) were generated from five independent experiments. (d) Monocytes were isolated and experimentally infected with either HCMV Titan wild-type or HCMV Titan with a US28 deletion at an MOI of 5. Cultures were then either mock-treated with PBS or treated with F49A-FTP for 3 days and then reactivated by differentiation into mDCs as above. Using the UL32-GFP tag on these viral isolates, reactivated dendritic cells were then counted and compared with levels of reactivation of monocytes infected with Titan wild-type virus in the absence of drug that was set to 100%. (e) After co-culture with reporter fibroblast cells, incubation for 2 weeks and staining for IE, reactivation events were quantified. Means and error bars (showing s.d.) were generated from three independent experiments. Statistical analyses were carried out using a paired two-tailed t-test and P values expressed as *P=0.05, **P=0.01 or ***P=0.001.