Activity of HIV-1, SIVagm and SIVmac Vif against human and Agm APO3G.
(A) HeLa cells were transfected with the Vif-defective proviral clone SIVmac Vif(-) and pCDNA-hAPO3G (left panel) or pCDNA-Agm-Apo3G vector (right panel). In addition, vectors encoding HIV-1 Vif (pNL-A1/43Vif, lanes 1 & 6), Mac Vif (pNL-A1macVif, lanes 2 & 7), or Agm Vif (pNL-A1agmVif, lanes 3 & 8) were co-transfected. Vif-negative samples (lanes 4 & 9) and APO3G-negative samples (lanes 5 & 10) were included as controls. Total amount of DNA was adjusted to 5.5 µg/5×106 cells using pNL-A1vif(-) vector DNA. Whole-cell lysates and concentrated viral extracts were prepared 48 hr after transfection and aliquots from each sample were analyzed by immunoblotting for the presence of APO3G (top panel) or capsid protein (CA, lower panel). (B) Cell-free filtered supernatants from the cultures shown in panel A were normalized for reverse transcriptase activity and used for the infection of LuSIV indicator cells. Infected cells were harvested 24 hr later and analyzed for the expression of Tat-induced luciferase activity. Results are expressed relative to the virus prepared in the absence of APO3G (defined as 100%). Bars reflect the median and error bars indicate the range calculated from two independent infections.