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.

CHO-K1 cells were plated on 96-well plates and grown to 70%-90% confluence. The following day, CMV gB plasmid (pCCMVgB) from the Tandon lab, was transfected into cells using Lipofectamine 2000 (ThermoFisher, Waltham, MA, USA) and incubated overnight. Transfection media was replaced with complete media to allow cell recovery. Following, transfected cells were incubated with dilutions of SPGG for 1 h in SFM under standard conditions. The cells were then washed in TBS, fixed with methanol for five minutes and washed again. Cell-ELISA protocol described previously was then followed using 0.5 µg/mL monoclonal antibody to HCMV gB (Acris, Rockville, MD, USA, cat. no. BM3261) and goat anti-mouse peroxidase conjugated secondary antibody diluted 1:10,000 (ThermoFisher, Waltham, MA, USA). Final washing of the plate was done three times in TBST before 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate (Pierce Biotechnology, Rockford, IL, USA) was added and the reaction was stopped. The enzymatic activity was measured at OD 450 nm by microplate photometer (Thermo Scientific Multiskan FC).