FIG. 1.

Transiently expressed A3F wt and A3F mt are packaged into Vif-deficient HIV-1 virions and have antiviral activity. (A) HeLa cells were transfected with vif-defective pNL4-3 (4 μg) together with 1 μg of a human A3G wt (lane 2)-, A3F wt (lane 3)-, or A3F mt (lane 4)-expressing vector. Lane 1 is a control consisting of vif-defective pNL4-3 without APOBEC. Virus-containing supernatants were collected at 48 h posttransfection and analyzed by immunoblotting for the presence of APOBEC proteins using a myc-specific antibody (A3G/A3F). The same blot was subsequently reprobed with an HIV-positive patient serum to identify the CA protein. (B) Virus-associated deaminase activity was determined on viruses from panel A using a 5′-end ³²P-labeled single-stranded DNA template with signature motifs for A3G and A3F. Oligonucleotides were separated by 15% acrylamide-7 M urea gel electrophoresis. The positions of the cleaved products and the uncleaved probe are indicated on the right. (C) HeLa cells were cotransfected with 4 μg of pNL4-3Vif(−) (lanes 3 to 15) and increasing amounts of A3G wt (lanes 4 to 7), A3F wt (lanes 8 to 11), or A3F mt (lanes 12 to 15). The amounts of transfected APOBEC DNA used were 30 ng (lanes 4, 8, and 12), 100 ng (lanes 5, 9, and 13), 300 ng (lanes 6, 10, and 14), and 1 μg (lanes 7, 11, and 15). Lane 3 is a control consisting of HeLa cells transfected with 4 μg of pNL4-3Vif(−) in the absence of APOBEC. Lane 2 shows HeLa cells transfected with 4 μg of pNL4-3 wt in the absence of APOBEC. The total DNA amount in all samples was adjusted to 5 μg with the appropriate amount of empty vector DNA. Lane 1 is a mock-treated control consisting of HeLa cells transfected with 5 μg of empty vector DNA. Cells and virus-containing supernatants were harvested at 24 h following transfection. Whole-cell lysates were subjected to immunoblot analysis using a myc-specific antibody (A3G/A3F) or an antibody to Vif or HIV-1 Gag (CA). (D) The infectivity of viruses produced in panel G was determined by infecting TZM-bl indicator cells. Virus input was normalized by reverse transcriptase activity. Virus-induced luciferase activity was determined at 24 h after infection in a standard luciferase assay. The infectivity of virus produced in the absence of APOBEC was defined as 100% (lane 3). The infectivity of the other viruses was expressed as a percentage of that of the control virus. Error bars reflect standard deviations from three independent infections.