FIG. 4.

MG132 induces an accumulation of proviral DNA and of cytosolic p24^Gag proteins in target cells. (A) Synthesis of proviral DNA in newly infected cells. P4 cells were infected by a 5-h incubation period with HIV at the indicated amounts of p24^Gag in the absence or in the presence of MG132 (25 μM). Seventeen hours later, low-molecular-weight DNA was extracted and analyzed by Southern blotting with an HIV probe (upper panel). Digestion with EcoRI produced diagnostic fragments with sizes of 5.7 and 9.1 kb from linear DNA (L) and DNA containing one LTR circle (C), respectively. Since variations in the yield of cellular low-molecular-weight DNA were observed, samples were normalized by hybridization with the mitochondrial gene coding for cytochrome b (cyt. b) (lower panel). (B) Accumulation of cytosolic p24^Gag proteins. P4 cells were incubated with HIV-1 (1 μg of p24) in the absence (untreated columns) and in the presence of MG132 (50 μM) or lactacystin (40 μM) for 1 h at 37°C. Cells were then washed to remove unbound virus and further incubated at 37°C in medium containing the indicated inhibitors. At each time point, cells were treated with Pronase to eliminate virus adsorbed at the cell surface and lysed. Postnuclear supernatants were separated in cytosol and pellet fractions. The pellet fraction corresponds to cellular membranes and vesicles. p24^Gag contents (in picograms) were measured in the cytosolic fraction. Time zero corresponds to the beginning of infection. Data are representative of three independent experiments.