Viral stocks. HIV-1 particles or pseudoparticles containing BlaM-Vpr were produced by cotransfection of HEK293T cells with proviral plasmids (pHIV-1ADA or pNL4.3ΔEnv and pEnvVSVG), pCMV-BlaM-Vpr encoding β-lactamase fused to the viral protein Vpr, and pAdvantage, as described elsewhere (22). After 48 hours of culture at 37°C, the virus-containing supernatant was filtered and stored at −80°C. Pseudoparticles containing BlaM-Vpr were ultracentrifuged at 60,000g for 90 min at 4°C on a sucrose cushion (20%). The virion-enriched pellet was resuspended in PBS and aliquoted for storage at −80°C. The amount of p24 antigen in the supernatants was quantified using an ELISA kit (PerkinElmer). HIV-1 infectious titers were also determined in HeLa TZM-bl cells (LTRlacZ, NIH reagent program) by scoring β-lactamase–positive cells 24 hours after infection, as described previously (43).

BlaM-Vpr viral fusion assay. After 18 hours of incubation with compounds, 1.5 × 105 primary macrophages were inoculated with the BlaM-Vpr–containing viruses (15 ng of p24 Gag) by 1-hour spinoculation at 4°C and incubated for 2.5 hours at 37°C. Cells were then loaded with CCF2/AM, the BlaM-Vpr substrate (2 hours at room temperature), and fixed. Enzymatic cleavage of CCF2/AM by β-lactamase (22) was measured by flow cytometry (LSR II, BD), and data were analyzed with FACSDiva software. The percentage of fusion corresponds to the percentage of cells displaying increased cleaved CCF2/AM fluorescence (447 nm).

HIV-1 infections. After treatment for 18 hours, human primary macrophages were infected with HIV-1ADA in six-well trays with a multiplicity of infection of 0.2, incubated for 24 hours at 37°C, and washed with culture medium without FCS. Cells were cultured for 24 hours at 37°C in complete culture medium supplemented with library compounds. After 24 hours, supernatant was harvested, and cells were lysed for 15 min at 4°C in lysis buffer [20 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 50 mM NaF, and 1 mM sodium orthovanadate, supplemented with complete protease inhibitor cocktail; Roche Diagnostic]. Lysates were centrifuged at 10,000g for 10 min at 4°C, and the quantity of HIV-1 p24 in the postnuclear supernatants was determined by ELISA.

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