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. 2007 May 23;81(15):8080–8090. doi: 10.1128/JVI.02727-06

FIG. 2.

FIG. 2.

SIVagm Vif inhibits the packaging of CypA in SIVagm virions in Jurkat cells. (A) Virus-containing supernatants from SIVagm (lanes 1 to 3) and HIV-1-infected Jurkat cells (lanes 4 to 6) shown in Fig. 1A were harvested at peak virus production. Virus-containing supernatants were normalized for equal RT activities and concentrated by pelleting through 20% sucrose. Pelleted viruses were then analyzed by immunoblotting using antibodies specific to CypA or SIVagm and HIV-1 CA protein. Proteins are identified on the left. (B) Vif-deficient virions from infected Jurkat cells (as shown in Fig. 1A) were subjected to step gradient analysis in the absence (untreated) or presence (Triton X-100) of detergent. Three fractions (a to c) were collected from each gradient and analyzed by immunoblotting for the presence of SIV or HIV-1 CA protein or CypA. (C) Jurkat cells were nucleofected with pSIVagmVif(−) in the absence of Vif (lane 2) or together with increasing amounts of pNL-A1 (lanes 3 to 4), pNL-A1/agmVif (lanes 5 to 6) or pNL-A1/macVif (lanes 7 to 8). The plasmid ratios (provirus:Vif) were 4:1 (lanes 3, 5, and 7) and 1:1 (lanes 4, 6, and 8). A mock-transfected sample was included as a control (lane 1). Total amounts of transfected plasmid DNA were kept constant by adjusting with appropriate amounts of vif-defective pNL-A1vif(−) DNA. Virus-containing supernatants were harvested 3 days after transfection and processed for immunoblotting as shown in panel A (upper panels). CypA-specific protein bands were quantified by densitometric scanning of the gel and were plotted as a percentage of the Vif-negative control (lane 2) which was defined as 100% (lower panel). Lane numbers correspond to lanes on the immunoblot. (D) Virus-containing supernatants were normalized for equal reverse transcriptase activity and used for the infection of LuSIV indicator cells. Infection was determined 24 h later as described in the legend to Fig. 1B. Error bars reflect the standard deviations calculated from triplicate infections. Lane numbers correspond to lanes on the immunoblot in panel C (top).