Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity

Hayward, Joshua A; Tachedjian, Mary; Cui, Jie; Cheng, Adam Z; Johnson, Adam; Baker, Michelle L; Harris, Reuben S; Wang, Lin-Fa; Tachedjian, Gilda

The antiviral activity of bat A3 was assessed by examining the capacity to restrict HIV-1 infectivity. Bat A3 cDNAs were PCR amplified from parental vectors and subcloned into pcDNA3.1 mammalian expression vectors (Thermo Fisher, MA). 293 T cells were cotransfected with different quantities of bat A3 or human A3G (phu-A3GHA; [Mariani et al. 2003 ]) expression plasmids (25-600 ng) and 400 ng of pIIIBDvif (Simon et al. 1995) , which generates HIV-1 virions in the absence of Vif. Untransfected cells and cells transfected only with pIIIBDvif were used as controls. Transfected cell cultures were incubated at 37 C, 5% CO 2 for 48 h, and then virion-containing supernatants were collected and clarified by centrifugation at 200 Â g for 5 min. Virus production was determined by quantifying virion-associated RT activity, as previously described (Wapling et al. 2005) . TZM-bl cells Hayward et al. . doi:10.1093/molbev/msy048 MBE were inoculated with RT normalized supernatant and incubated at 37 C, 5% CO 2 for 48 h. HIV-1 infection of TZM-bl cells was assessed through quantitation of luciferase activity in cell lysates as previously described (Tyssen et al. 2010 ).