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.

HCMV strain AD169 was pre-incubated with dilutions of SPGG for 1 h with agitation at RT. Following incubation, 10 µL spots containing 5 × 10 5 PFU AD169 and SPGG were applied to a 0.2 µm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Spots were dried and the membrane was blocked in protein-free blocking buffer (ThermoFisher, Waltham, MA, USA) for 1 h. Standard ELISA protocol previously described was then followed using 1 µg/mL monoclonal antibody to HCMV gB (Acris) and goat anti-mouse peroxidase conjugated secondary antibody diluted 1:10,000 (ThermoFisher). After the final wash, the blot was incubated with 3-amino-9-ethylcarbazole (AEC) staining solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 min and rinsed in water before imaging. The intensity of the signal was quantified using ImageJ gel analysis function to calculate and plot the area under each peak.