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 proteins was determined by assessing the capacity of representatives of each bat A3 subtype to restrict HIV-1 infectivity in target cells. HIV-1 encodes the anti-A3 counter-measure, Vif, a protein that causes the proteolytic degradation of A3 through the recruitment of the cellular E3 ubiquitin ligase (Marin et al. 2003; Sheehy et al. 2003; Kane et al. 2015) . HIV-1 stocks were generated by cotransfection of 293 T producer cells with bat A3 and an HIV-1 variant that does not express Vif. HIV-1 particles were harvested from the cell culture supernatant, and virion production was determined by quantifying virion-associated reverse transcriptase (RT) activity. HIV-1 infectivity was assessed by inoculating human TZM-bl target cells, which express luciferase in the presence of HIV-1 infection, with virus normalized to equivalent levels of virion-associated RT activity. Four bat A3 proteins demonstrated a dose-dependent inhibition of HIV-1 infectivity ( fig. 4) . The sole double Z-domain bat A3 protein, A3Z2Bd-Z3, produced the largest reduction in HIV-1 infectivity, which was comparable to inhibition by the human A3 protein, A3G. These data indicate that bat A3 proteins are functional restriction factors.