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 or SK-N-MC cells were grown to semi-confluency overnight in black opaque 96-well plates. The following day, AD169 was preincubated with different dilutions of SPGG for 1 h with agitation and cells were infected with 0.3 MOI of treated or untreated virus for 2 h at RT. The infection media was replaced with SFM and incubated for an additional 5 h under standard conditions. The samples were fixed for 20 min with methanol, washed three times in Tris-buffered saline (TBS), and blocked for 2 h with protein-free blocking buffer (Thermo Fisher, Waltham, MA, USA). Samples were incubated overnight at 4 • C with a mouse monoclonal antibody against immediate early genes 1 and 2 (Virusys, Taneytown, MD, USA, cat. no. P1215) diluted 1:5000 in blocking buffer. Next, samples were washed three times with wash buffer (0.05% tween 20 in TBS) and then incubated for 1 h at RT with goat anti-mouse IgG (H+L) peroxidase-conjugated secondary antibody (Thermo Fisher, Waltham, MA, USA) diluted 1:20,000 in wash buffer. Samples were washed 3 times in wash buffer, Super Signal ELISA Femto Substrate (Thermo Fisher, Waltham, MA, USA) was added, and total luminescence was analyzed using a microplate luminometer (Beckman DTX 880).