Figure 2

Delayed growth kinetics displayed by HIV-YRHHY > A5 in non-permissive cells but not in permissive cells. (A) Expression levels of A3G in CEM and CEM-SS cells. To confirm that the non-permissive CEM cells expressed A3G and the permissive CEM-SS cells did not, cell lysates were analyzed by western blotting for expression of both A3G and A3F. Expression of α-tubulin in the cell lysates was also analyzed to control for the amount of cell lysate examined. As positive controls 293T cell lysates transfected with FLAG-tagged A3G and A3F were also analyzed. (B) Schematic representation of the virus-passage protocol used. The different steps carried out at each round of infection are shown. (C) Virus growth in permissive CEM-SS cells. To determine the growth kinetics of HIV-YRHHY > A5 in permissive CEM-SS cells, 1000 RT units were added to 1 × 10⁶ CEM-SS cells and the virus and cells were cultured at 37°C. At various time points virus-containing supernatant was removed and the RT levels were determined. As controls, HIV WT and NL4-3ΔVif were also included. The results are plotted as the scintillation counts/minute measured at each time point for 3 independent infections of HIV-YRHHY > A5 and two independent infections of HIV WT and NL4-3ΔVif. (D) Virus growth in Round 1 infection of non-permissive CEM cells. The experiment was carried out as described in FIG. 1C legend except that 10 independent infections were used for HIV-YRHHY > A5. (E) Virus growth in Round 2 infections of non-permissive CEM cells. Virus from the peak of infection of HIV-YRHHY > A5 Round 1 samples YA, YB, and YC and HIV WT was added to fresh CEM cells and passaged as described in Fig. 2C legend. (F) Virus growth in Round 3 infections of non-permissive CEM cells. Virus from the peak of infection of HIV-YRHHY > A5 Round 2 samples YA, YB and YC and HIV WT A was added to fresh CEM cells and passaged as described in FIG. 2C legend.