Inhibition of Human Cytomegalovirus Entry into Host Cells through A Pleiotropic Small Molecule

Elste, James; Kaltenbach, Dominik; Patel, Vraj R.; Nguyen, Max T.; Sharthiya, Harsh; Tandon, Ritesh; Mehta, Satish K.; Volin, Michael V.; Fornaro, Michele; Tiwari, Vaibhav; Desai, Umesh R.

We monitored IE gene expression using AD169, a widely characterized HCMV strain [65] , using a cell-ELISA assay (Figure 4 ). Control mock-treatment (PBS) experiments with HCMV strain AD169 showed robust expression of both IE 1 and 2 genes within one hour of incubation with host cells. Pretreatment of AD169 strain with SPGG followed by incubation with either HFF-1 or SK-N-MC cells showed significant reductions in expression of both IE1 and IE2. A clear dose-dependent decrease in IE 1 and 2 expression was evident, which gave IC50s of 3.1 µM (HFF-1) and 5.6 µM (SK-N-MC) ( Figure 4A ,B). As with puncta formation, the best results were observed at 100 µM SPGG. Thus, the results show that SPGG-mediated interference in viral entry negatively impacts the expression of viral genes, which probably contribute to reducing the viral infectivity. High-resolution confocal microscopy. HFF-1 and SK-N-MC cells were challenged with the HCMV strain BAD32GFP which was pre-incubated with or without SPGG 100µM for 1 h. The double immunolabeling in confocal microscopy shows an increased number of GFP puncta (green highlighted with white arrows) in HFF-1 cells (A) and SK-N-MC cell (D) infected with HCMV and mock-treated compared to HCMV treated with SPGG (B,E). Cells were counterstained with phalloidin (red) and DAPI (blue) and images were processed and enumerated using ImageJ. The number of GFP-positive puncta detected in HFF-1 (C) and SK-N-MC (F) cells infected with HCMV exposed to different concentrations of SPGG was quantified in ImageJ using 3 random views per slide. The Graphs show a clear dose-dependent reduction in number of GFP-positive for both cell lines. Data are shown as mean value and SD for a triplicate experiment. Scale bar (A-E) = 50 µm.