Figure 3

The same major RT isoforms are present in virus producer cells, newly infected cells and HIV RTCs. H3B and Hut-78 cells were co-cultured for the indicated time period then lysed. For panels A and B, lysates were immunoprecipitated using heat-inactivated AIDS patient sera cross-linked to protein A sepharose beads and washed. In panels A, B and D, E samples were subjected to 2D gel electrophoresis on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel along with 3 μg of GAPDH protein. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. RT was detected by Western blot using an anti-RT antibody and RT isoforms are designated by a black arrow (n = 2 for each panel). Minor differences in the p66 and p51 profiles were observed between experiments and spots not routinely observed are indicated by a white arrow. (A) H3B virus producer cells. H3B and Hut-78 cells were co-cultured and lysed immediately. (B) Infected cell lysates. H3B and Hut-78 cells were co-cultured and lysed at 40 min post-cell mixing. (C-E) HIV RTC's. Lysates were subjected to 15–30% sucrose velocity gradient sedimentation. Fractions (1 ml) were collected from the top of the gradient and viral -ssDNA analysed by real time PCR (C). The remainder of two selected fractions; (D) from the top of the gradient (fraction 1) and (E) co-incident with the known sedimentation of RTCs (fraction 5), were TCA precipitated and subjected to 2D gel electrophoresis, as for panels A and B, above. Experiments were replicated, at least n = 2, for each presented biological situation.