Figure 1.
Productive replication of HIV-1ΔVif in CEM-T4 cells.
(A) Cell surface expression of HIV-1 receptors. HUT 78, H9, PM1, CEM-SS, CEM-T4, and A3.01 cells were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CXCR4 antibodies (BD Biosciences). Expression of these surface molecules was determined by flow cytometry. (B) HIV-1 replication. Wild-type or vif-defective HIV-1 was produced from 293T cells after transfection with pNL4-3 or pNL4-3ΔVif. HUT 78, H9, PM1, CEM-SS, CEM-T4, and A3.01 cells were then infected with these viruses, and viral production was monitored daily using a p24Gag ELISA for 8 d. Results shown are from 1 of 3 independent experiments. (C) A3G and A3F expression. Cell lysates were prepared from indicated human T cell lines, and the expression of A3G or A3F was determined by Western blotting.
Figure 2.
Characterization of A3G in CEM-T4 cells.
(A) Isolation of A3G protein complexes. Cytosolic fractions were prepared from CEM-T4, A3.01, and 293T cells transfected with human A3G expression vector. These lysates were treated or untreated with RNase A and then loaded on the top of a 4% to 40% sucrose gradient. After centrifugation at 200,000g for 4 h, 9 fractions were collected, and A3G expression was determined by Western blotting. (B) A3G deaminase activity. Cell lysates were prepared from indicated cell lines with or without RNase A treatment and then subjected to scintillation proximity-based cytidine deaminase assay. Error bars represent standard deviations in 3 independent experiments. (C) Production of HIV-1 from human T cell lines. H9, A3.01, CEM-SS, and CEM-T4 cells were infected with wild-type or vif-defective HIV-1 carrying a neomycin-resistant gene, and stable cell lines were generated by G418 selection. Viruses were then collected from these cultures, and virion-associated proteins were determined by Western blotting. (D) Viral infectivity assay. Viruses produced from the stable cell lines were collected and used to infect TZM-bI cells. Viral infectivity was determined and normalized to viral input and expressed as relative values to the wild-type virus infectivity from CEM-SS cells. The values for wild-type and vif-deficient virus infectivity are 100 and 90 from CEM-SS, 105 and 23 from A3.01 cells, and 72 and 89 from CEM-T4 cells. Error bars represent standard deviations in 3 independent experiments.
Figure 3.
CEM-T4 cells lack an A3G/A3F cofactor.
(A) A schematic description for transient trans-complementation assay. T cells were infected with env-defective HIV-1 virus pseudotyped with VSV-G, and 293T cells were transfected with the Env-expressing vector pNLΔGag. These two types of cells were cocultured for heterokaryon formation. Infectious particles were then detected by infection of TZM-bI cells. (B) Infectivity of HIV-1 produced from heterokaryons. 293T cells were transfected with either pNLΔGag or pNLΔGagΔVif in the presence or absence of a human A3G or A3F expression vector and then cocultured with A2.01, A3.01, HUT 78, H9, CEM-SS, or CEM-T4 T cells that were infected with VSV-G–pseudotyped env-defective HIV-1 expressing or not expressing Vif protein. Infectious particles were collected 48 h later and used to infect TZM-bI cells. Viral infectivity was finally determined by measuring firefly luciferase activity in TZM-bI cell lysates. Results shown here were from 1 of 3 independent experiments.
Figure 4.
Anti-HIV activity of A3F/A3G proteins expressed in cis from an HIV-based vector.
(A) Expression of A3G/A3F from an HIV-based vector and the antiviral infectivity in a single round replication assay. An A3F, A3G, or A3GE259Q gene with an HA tag was inserted into the Nef open reading frame in pNL4-3 or pNL4-3ΔVif. These constructs were then cotransfected into 293T cells with either pNL-A1 or its control pNL-A1ΔVif as indicated. Expressions of Vif and A3 proteins in these transfections were determined by Western blotting, and viral infectivity was determined in TZM-bI cells. (B) Replication kinetics of HIV-1 viruses expressing A3G or A3F. CEM-SS and CEM-T4 cells were infected with the same amount of A3G- or A3F-expressing HIV-1 viruses used as in (A); viral growth curves were determined by measuring p24Gag in the supernatants. pNLA3FΔVif + Vif, pNLA3GΔVif + Vif, and pNLA3GE259QΔVif + Vif are viruses produced in the presence of pNL-A1 during transfection of 293T cells.
Figure 5.
Anti-HIV activity of A3F/A3G proteins expressed in trans from a MuLV-based vector.
(A) Stable transduction of CEM-T4 and CEM-SS cells by MuLV-based vector. An A3F, A3G, A3GE259Q, or GFP gene with an HA tag was inserted into the pMSCVneo vector, and recombinant MuLV was produced. CEM-T4 and CEM-SS cells were then infected with these viruses, and stably infected cells were selected by G418 treatment. The expression of A3F/A3G proteins in each individual cell line was determined by Western blotting. (B) A3G subcellular localization. CEM-T4 and CEM-SS cells stably transduced with A3G were fixed with formaldehyde and then stained with a mouse anti-A3G monoclonal antibody and a rabbit anti-MOV10 polyclonal antibody. A3G was visualized using Alexa Fluor 488–conjugated secondary antibody (green), and MOV10 was visualized by Alexa Fluor 594–conjugated antibody (red). The cells were also stained with Hoechst 33342 to visualize nuclei (blue). Areas of overlap between A3G and MOV10 appear as yellow. (C) Replication kinetics of HIV-1 in stably transduced cell lines. CEM-T4 and CEM-SS cells stably transduced with A3G, A3GE259Q, A3F, or GFP were infected with the same amount of wild-type or vif-deficient HIV-1. Viral growth curves were determined by measuring p24Gag in the supernatants using ELISA.