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.

Preliminary studies with BAD32GFP were performed to assess infectivity of HFF-1 and SK-N-MC cells. Following a 2-h incubation at RT at an MOI of 5.0, we observed excellent infectivity of the viral strain for both cell-types as monitored by the presence of multiple GFP punctae (aggregates of virions; Figure 3 , panels A and D). Addition of SPGG resulted in reduced number of GFP-puncta for both cell types (Supplementary Figure S1 ). Using ImageJ, the immunolabeled punctae with SPGG treatment were quantified as compared to the mock-treated cell lines. The relative decrease in GFP-puncta as a function of SPGG dose followed the traditional semi-log relationship with an IC 50 of 2.0 ± 0.3 µM for HFF-1 and 1.8 ± 0.2 µM for SK-N-MC cells (Figure 3 panels C, and F). In fact, at the maximal dose studied (100 µM), SPGG treatment almost completely abolished BAD32GFP internalization ( Figure 3B ,E). This implied that SPGG exhibit excellent 'therapeutic' window of more than 50 and 55-fold (ratio of LC 50 /IC 50 ), respectively for the two cell lines. . 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.