The expression vectors for EGFP-fused APOBEC3G and Vif were previously described [21, 22]. The expression vector for HA-tagged APOBEC3G (pcDNA3/HA-A3G) was previously described . The expression vector for luciferase-fused APOBEC3G was generated by inserting coding sequence of luciferase gene amplified with primers NNN NGC TAG CGC CAC CAT GGA AGA CGC CAA AAA CAT and NNN NCT CGA GCA CGG CGA TCT TTC CGC CCT at Nhe I and Xho I sites of pcDNA3/HA-A3G. pNL4-3/ΔEnv-Luc and pNL4-3/ΔenvΔVif-Luc vectors were previously described .
Cell culture and transfection
293 T cells were maintained in DMEM (Nacalai) containing 10% FBS and 1% penicillin–streptomycin and glutamine (PSG, Invitrogen). CEM and CEM-SS cells were maintained in RPMI1640 containing 10% FBS and 1% PSG. 293 T cells on 6-well plates were transfected with about 1 μg of plasmid DNA in total using X-tremegene HP DNA transfection reagent (Roche) according to manufacturer’s instruction.
Screening for small molecules that inhibit Vif-mediated degradation of APOBEC3G
A library of small compounds was purchased from Enamine. For the primary screening, 293 T cells on 24-well plates were transfected with expression vectors for EGFP-APOBEC3G and Vif and treated with individual molecules from a library of small compounds for 24 hours, then cells were lysed with M-PER (Pierce), and fluorescence intensity was measured by plate reader (2030 Arvo X, Perkin Elmer). DMSO was used for control for the compounds and cells transfected with only EGFP-APOBEC3G were used for positive control for fluorescence intensity. Experiments were performed in duplicate and repeated once more, and the compounds repeatedly recovered fluorescence intensity to more than 50% were selected to secondary screening. For the secondary screening, 293 T cells on 24-well plates were transfected with expression vectors for Luc-APOBEC3G and Vif and treated with compounds for 24 hours, Passive lysis buffer (Promega) and luciferase activity was determined by luminometer (2030 Arvo X, Perkin Elmer) using Luciferase Assay System (Promega). DMSO was used for control for the compounds and cells transfected with only Luc-APOBEC3G were used for positive control. Experiments were performed in duplicate and the compounds which recovered chemiluminescence intensity to more than 50% were selected for tertiary screening. For the tertiary screening, CEM and CEM-SS cells were challenged with NL4-3 at 0.01 MOI and treated with the compounds for 10 days. The compounds which rescued virus-inducing cell death of CEM cells, but not that of CEM-SS cells were selected.
Cells were lysed with triton-based buffer (20 mM HEPES-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 0.1% triton X-100) supplemented with protease inhibitor cocktail (Nacalai). After centrifugation at 20, 000 × g for 10 minutes, supernatant was mixed with sample buffer (Biorad), boiled for 5 minutes, resolved on 10% (w/v) polyacrylamide gel and transferred to PVDF membrane (Immobilon, Millipore). The membrane was blocked for non-specific binding of antibodies with 5% BSA containing buffer for 1 hour and analyzed by standard immunoblotting procedure. Primary antibodies for immunoblotting against Vif, A3G and p24Gag were obtained from the NIH AIDS Research and Reference Reagent Program. Mouse anti-tubulin antibody was purchased from Covance. Rabbit anti-GFP serum was purchased from Invitrogen. Mouse anti-ubiquitin monoclonal antibody was purchased form Santa Cruz. HRP-conjugated secondary antibodies against mouse and rabbit were purchased from GE Healthcare.
Luciferase encoding HIV-1 particles were produced by transiently transfecting 293 T cells at 50% confluency using 0.6 μg pNL43/ΔEnv-Luc or pNL43/ΔEnvΔVif-Luc, 0.15 μg pVSV-G and 0.25 μg pcDNA3/HA-A3G, or an empty vector. After 48 hours, virus-containing supernatants were harvested through PVDF filter with 0.45 μm pores (Millipore), and challenged to fresh 293 T cells. Medium were changed after 12 hours, cells were incubated for additional 36 hours, lysed with Passive lysis buffer (Promega) and luciferase activity was determined by luminometer (2030 Arvo X, Perkin Elmer) using Luciferase Assay System (Promega). Sample preparation of producer cells and virus for immunoblotting was performed as described .
For co-immunoprecipitation, 293 T cells were transfected with the expression vectors for Vif, with co-transfection of the expression vector for APOBEC3G-myc or empty vector. Cells were treated with 10 μM of MM-1, MM-2 or DMSO only for 24 hours and 2.5 μM MG132 for 16 hours, then washed with PBS, lysed with co-IP buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% triton X-100, 1 mM EDTA, 1 mM MgCl2, and 10% glycerol) supplemented with protease inhibitor cocktail and 10 μM MG132, centrifuged for 10 minutes at 20,000 × g. Supernatant was incubated with anti-myc mouse monoclonal antibody (clone 9E11) for 1 hour, and then mixed with 20 μl protein A sepharose (Pharmacia) for 1 hour. Beads were washed with co-IP buffer three times, and bound protein was eluted with 1 × SDS sample buffer.
For testing ubiquitination of APOBEC3G, 293 T cells were transfected with expression vectors for HA-tagged ubiquitin and EGFP-fused APOBEC3G, with or without co-transfection of the expression vector for Vif. Cells were treated with 2.5 μM MG132 for 16 hours before harvest, then washed with PBS, lysed with RIPA buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% triton X-100, 1 mM EDTA) supplemented with protease inhibitor cocktail and 10 μM MG132, centrifuged for 10 minutes at 20,000 × g. Supernatant was incubated with anti-GFP rabbit serum (Invitrogen) for 1 hour, and then mixed with 20 μl protein A sepharose (Pharmacia) for 1 hour. Beads were washed with RIPA buffer three times, and bound protein was eluted with 1 × SDS sample buffer. Samples were analyzed by immunoblotting as described above.
Proteasomal activity studies
Proteasome 20S assay kit (Enzo) was used according to manufacturer’s instruction. 10 μM of MM-1 or MM-2 was added to the reaction, and DMSO and epoxomicin are used as controls.
293 T cells were incubated with various concentration of MM-1 or MM-2 for 48 hours, and cell viability was measured by MTS assays using a kit (Promega). Cell viability was normalized to that of cells incubated with the same concentration of DMSO.