Article Figures & Data
Figures
- Fig. 1.
The proteasome inhibitor MG132 increases HIV infection. (A) P4 indicator cells (HeLa CD4+LTR-lacZ +) were incubated with HIV-1 (left panels) or with HIVΔenv(VSV), a defective HIV-1 strain with a deletion in the env gene and pseudotyped with the VSV-G envelope, in the absence and in the presence of MG132 (25 μM). Five hours later, virus and MG132 were removed. After 24 h, HIV-infected cells were revealed by in situ staining for β-galactosidase (β-gal) activity. (B) The effect of MG132 is independent of the viral input. HIV infection was quantified in P4 cells in a β-galactosidase based colorimetric assay. P4 cells were infected with the indicated amounts of HIV-1 (left panel) or HIVΔenv(VSV) (right panel) with or without MG132 (50 μM) for 1 h. After 24 h, cells were lysed, and β-galactosidase activity (optical density [O.D.] units) in cell extracts was measured. Values are means of triplicates. Variation between each point of triplicate determinations was below 10%. Data are representative of three independent experiments.
- Fig. 2.
Analysis of HIV-1 infection in the presence of MG132 on various cell lines. HeLa-CD4 cells and the Jurkat, CEM, and HUT78 T-lymphoid cell lines were infected with the HIV-HSA reporter virus pseudotyped with the VSV-G envelope, which contains, in the place ofnef, the gene coding for HSA (CD24). Following integration and proviral expression, cells synthesize CD24, which can be detected at the cell surface. Cell lines were infected with HIV-HSA(VSV) (50 ng of p24Gag) for 1 h, with or without MG132 (50 μM). Infection was revealed 24 h later by staining with anti-CD24 monoclonal antibodies and flow cytometry analysis of gated living cells. Data are representative of three independent experiments.
- Fig. 3.
Analysis of the effect of various protease inhibitors on HIV infection and on LTR activity. (A) Effect of MG132 on HIV-1 LTR activity. P4 cells were transfected with 1 μg of pLTRX-Luc and the indicated amounts of pCMV-Tat DNA. After 24 h, cells were pulse-incubated for 1 h with or without MG132 (50 μM), and 5 h later, luciferase activities in cell extracts were measured. Results are expressed as RLU per microgram of cellular protein. Values are means of triplicate determinations, and variation between each point was below 10%. (B) P4 cells were infected with HIV-1 (left panel) or HIVΔenv(VSV) (right panel) in the presence of the proteasome inhibitors MG132 (50 μM) or lactacystin (40 μM) or with calpain inhibitor I (50 μM) or calpain inhibitor II (50 μM) for 1 h and washed. Infections were revealed 24 h later by measuring β-galactosidase activity (optical density [O.D.] units). Data are representative of three independent experiments.
- Fig. 4.
MG132 induces an accumulation of proviral DNA and of cytosolic p24Gag 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 p24Gag 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 cytochromeb (cyt. b) (lower panel). (B) Accumulation of cytosolic p24Gag 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. p24Gag contents (in picograms) were measured in the cytosolic fraction. Time zero corresponds to the beginning of infection. Data are representative of three independent experiments.
- Fig. 5.
In vitro degradation of HIV-1 Gag proteins by purified 20S proteasome. HIV-1 virions were pelleted by ultracentrifugation and resuspended in a buffer containing 0.4% Nonidet P-40 to remove the viral membrane. HIV-1 virions (10 ng of p24Gag) were then incubated with purified 20S proteasome (0.4 μg) for 30 min at 37°C, with or without MG132 (20 μM) or the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (50 μM). Viral proteins were then revealed in a Western blot assay with an anti-HIV-1 serum. Molecular mass markers are indicated (in kilodaltons) on the right. Data are representative of three independent experiments.
