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. 2009 Sep 30;83(24):13032–13036. doi: 10.1128/JVI.01786-09

FIG. 1.

FIG. 1.

Effect of CAML overexpression on HIV-1 particle release from permissive Cos-7 cells. (A and B) Cos-7 cells were seeded onto six-well plates and transfected using Lipofectamine 2000 according to the recommendations of the manufacturer (Invitrogen). Triplicate wells received fixed amounts of HxBH10vpu_wt (wt) or HxBH10vpu− (−) proviral DNA (2 μg) and pCMV-HA-hCAML (1 μg), along with the pCMV-HA empty vector to obtain the same final amounts of total DNA. (A) Western blot for Cos-7 cells transfected with the proviral DNA plasmid HxBH10vpu− (lanes 2, 4, and 7) or HxBH10vpu_wt (lanes 3, 5, and 8) or mock transfected (m; lanes 1 and 6). Samples from lanes 4 and 5 were treated with 10,000 U/ml of IFN-α, while samples from lanes 6 to 8 were cotransfected with the HA-tagged hCAML-expressing plasmid. Cells and supernatants containing viral particles were harvested 48 h posttransfection, and lysates were analyzed by Western blotting to detect steady-state levels of target proteins. Cell lysates were analyzed to detect HA-tagged CAML (by using anti-HA antibodies), as well as Gag products, Vpu, and cellular actin by using specific antibodies. Virus lysates were analyzed for the presence of p24 by using specific antibodies. (B) Quantification of virus particle release efficiency. (Top) Densitometric quantification of HIV-1 particle release efficiency in the presence of hCAML or upon IFN-α-treatment was performed by determining the ratio between the virion-associated Gag signal (corresponding to mature p24) and all cell-associated Gag signals (corresponding to p24/p25 and precursors p55 and p41) by Western blotting. Bands corresponding to all Gag products in cells and virus particles were scanned by laser densitometry and quantified using ImageQuant 5.0. (Bottom) Levels of released infectious virus were determined using HeLa-TZM indicator cells. HeLa-TZM indicator cells (AIDS Research and Reference Program, NIH) were inoculated with an aliquot of virus-containing supernatant. After 48 h, cells were lysed and luciferase activity was evaluated using the Promega luciferase assay system. For both top and bottom panels, the release efficiency of HxBH10vpu_wt was arbitrarily set at 100%. Error bars indicate the standard deviations of the means of results from two independent experiments. (C) Effect of increasing amounts of HA-hCAML on HIV-1 particle release. Cos-7 cells were seeded onto six-well plates and transfected as described in the legend to panel A. The cells received fixed amounts of HxBH10vpu_wt or HxBH10vpu− proviral DNA (2 μg) and increasing amounts of pCMV-HA-hCAML (from 2 to 8 μg), along with the pCMV-HA empty vector to obtain the same final amounts of total DNA. Cells and virus-containing supernatants were harvested 48 h posttransfection, and lysates were analyzed by Western blotting to detect Gag products and Vpu by using specific antibodies as indicated. Shown is a Western blot for Cos-7 cells transfected with the proviral DNA plasmid HxBH10vpu− (lanes 2, 4, 6, 8, and 10) or HxBH10vpu_wt (lanes 1, 3, 5, 7, and 9). Samples from lanes 3 to 10 were cotransfected with the HA-tagged hCAML-expressing plasmid.

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