Characterization of inhibitory activity of CEM.NKR. A) CEM.NKR cells do not secrete a soluble HIV inhibitory factor. Either 2 × 105 H9 or CEM.NKR cells were infected with 150 ng p24Gag of HIV-1 (NL4-3) for 3 hours at 37°C individually. After extensive washing, they were either cultured in separated wells, co-cultured by mixing them in the same well (CEM.NKR+H9) at 1:1 ratio, or co-cultured in the same well at 1:1 ratio separated by a insert with a 1.0 μm transparent PET membrane (H9/CEM.NKR) in 24-well plates (BD Biosciences). Viral production was determined by p24Gag ELISA over an 11-day period. B) CEM.NKR cells express lower levels of A3G and A3F than H9. Cytosolic fractions were prepared from equal numbers of CEM.NKR and H9 cells and the cytosol from H9 was subjected to a two-fold serial dilution. The levels of A3G and A3F expression were analyzed by Western blotting. The levels of actin contained lysate from 5 × 106 cells (undiluted lanes) served as an internal control. C) A3G R14Q and A3F Q275E mutants retain anti-HIV-1 activity. The cDNAs of A3G R14Q and A3F Q275E were amplified from CEM.NKR by RT-PCR and cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen). Wild-type or vif-defective HIV-1 (NL4-3) were produced from 293T cells in the presence of these wild-type or mutant A3G or A3F proteins and viral infectivity was determined in GHOST cells. Ctrl, control vector (pcDNA3.1). D) Vif effectively inactivates A3G/A3F in CEM.NKR cells. H9 and CEM.NKR cells were infected with HIV-1 or vif-deficient HIV-1 (NL4-3) expressing a neomycin-resistant gene in the nef gene locus and infected cells were selected by G418 treatment. HIV genes from nucleotides 5693 to 5912 corresponding to pNL4-3 sequence were amplified from these infected cells by PCR. After cloning into TA-cloning vector, multiple clones were collected for nucleotide sequencing. The type of mutation is summarized in tabular form, where the original HIV-1 sequence is given at left, and the new sequence is given across the top. N at the lower right of each box indicates the total numbers of bases sequenced.