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 cells were grown to semi-confluency overnight in a 12-well plate. The following day, RC256 was pretreated with 100 µM SPGG in serum free media (SFM) for 1 h at room temperature (RT) with agitation. Cells were infected with treated or untreated virus at a multiplicity of infection (MOI) of 1.0 in SFM for 2 h with rocking. After viral absorption, samples were washed three times in phosphate buffered saline (PBS) before the addition of overlay media (DMEM containing 1% heat-inactivated FBS and 0.5% methyl cellulose). Once a cytopathic effect was observed (four-seven days post infection), overlay media was removed and wells were washed three times with PBS. Cells were then fixed with fixative buffer (4% paraformaldehyde 0.2% glutaraldehyde in PBS) for 30 min, washed three times in PBS and incubated in permeabilization buffer (2 mM MgCl 2 , 0.01% Deoxycholate, 0.02% Nonidet P40 in PBS) for 30 min. Cells were then incubated in a substrate solution containing 500 µg/mL 5-bromo-4-chloro-3-indolyl-β-galactoside (X-gal, Thermo Fisher, Waltham, MA, USA). Blue-stained infected cells were counted under 10× magnification on an inverted microscope.