CEMx174 cells were infected with HIV-1 primary isolate 89.6 (From infectious clone p89.6, NIH AIDS Reagent Program) at approximately at 10% of cell population determined by FACS analyses. To determine antiviral activity during virus production, STP0404 was added at concentration in the range of 0.1 nM—10 μM during media exchange at 4 hrs post-infection. DMSO was used as a negative control. Cell-free supernatants were measured for p24 antigen production, 5 days post-infection. The anti-HIV-1 efficacy of the two enantiomers of STP0404 were also determined by using the same protocol. All assays were conducted in triplicates. The IC50 values were computed using GraphPad Prism (Version 9) and presented as means ± S.D. of the triplicates. For SIVmac239, CEMx174 cells were infected with viral supernatant containing SIVmac293 at 50,000 TCID50/mL (kind gift from Dr. G. Silvestri, Emory Yerkes National Primate Research Center) with 100 μL/106 cells at varying concentrations of STP0404, BI224436, and Raltegravir, and the same above protocol was followed except for that p27 capsid antigen content was measured daily for 5 days post infection.

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