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.

HFF-1 and or SK-N-MC cells were seeded on cover glass in a 24-well plate and grown to 40% confluency overnight. Virus strain BAD32GFP was preincubated with dilutions of SPGG for 1 h with agitation. Cells were infected with 5.0 MOI of treated or untreated virus for 2 h at RT. The infection media was replaced with SFM and incubated for an additional 2 h under standard conditions. Samples were washed three times in PBS, fixed for 30 min, and washed an additional three times. Samples were then incubated overnight at 4 • C with anti-GFP mouse IgG2a secondary antibody (Thermo Fisher, Waltham, MA, USA) diluted 1:500 in blocking buffer. Samples were washed three times in PBS and incubated in normal goat serum diluted 1:100 in wash buffer overnight at 4 • C. Samples were then washed 3 times in PBS and then incubated with goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA) diluted 1:500 in wash buffer for 1 h at RT. Samples were washed three times in PBS and incubated in phalloidin (Thermo Fisher, Waltham, MA, USA) diluted 1:40 in blocking buffer for 30 min in a humidity chamber. Samples were washed two times in PBS 0.2% Triton X-100, and once in dH 2 O before mounting to slides with hardset mounting media with DAPI (Vector Laboratories, Burlingame, CA, USA). Slides were imaged using the Nikon A1R confocal microscope and images were processed using ImageJ (version 1.52p, National Institutes of Health, USA) [78] and the GFP-positive punctae were enumerated in 3 random views per slide.