The HIV-1 proviral constructs pNL4-3 (T-tropic), pNL-AD8 (M-tropic), pWT/Bal (M-tropic), pNL4-3.Luc.R-E-, and pSG3Δenv were kindly provided by NIH AIDS Research and Reference Reagent Program [70–76]. The construct pNL4-3.Luc.R-E- is defective for env and nef where as pSG3Δenv has intact nef, but a deletion in env. An expression plasmid for vesicular stomatitis virus envelope G protein (pCI-VSV) was kindly provided by Jiing-Kuan Yee of City of Hope National Medical Center, Duarte, California. A Cav-1 expressing plasmid, pCZ-cav-1, was generated as described previously . pCZ-vector is the same as pCZ-cav-1 except it lacks the coding sequence of cav-1. The Nef expression plasmid pcDNA3.1SF2Nef was provided by NIH AIDS Research and Reference Reagent Program [77, 78]. A construct expressing Nef tagged with HA (pCI NL4-3 Nef-HA-WT) was purchased from Addgene Inc (Cambridge, MA). The NefG2A mutation plasmid was generated using a site-directed mutagenesis kit according to the manufacturer’s protocol (Strategene). Briefly, the mutation was generated by PCR amplification using pCI NL4-3 Nef-HA-WT as template and the following pair of primers: 5′-ggattttgctataagatggctggcaagtggtcaaaaagt-3′ and 5′-actttttgaccacttgccagccatcttatagcaaaatcc-3′. The PCR products were digested with the restriction enzyme DpnI to destroy template plasmids and were then transformed into DH5α competent cells. Introduction of the mutation (pCI NL4-3 NefG2A-HA) was confirmed by sequence analysis. Wild type AD8 and a replication competent nef defective AD8 derived HIV provirus DNA construct ADnefmut  were provided by Dr. Maureen Goodenow of the University of Florida. Adenovirus particles (Ad) for expressing Cav-1 (Ad-Cav-1) and GFP (Ad-GFP) were obtained from Vector Biolabs (Philadelphia, PA).
Human U87MG-CD4 cells stably transfected with CXCR4 (U87-CD4-CXCR4) or CCR5 (U87-CD4-CCR5), human acute monocytic leukemia (THP-1),and an indicator cell line for tittering HIV (TZM-bl) was kindly provided by the NIH AIDS Research and Reference Reagent Program. U87-CD4-CXCR4 were maintained in DMEM containing 15% FBS, penicillin-streptomycin (100 μg/mL), glutamine, puromycin (1μg/ml; Sigma Chemical), and neomycin (G418; 300μg/ml; Sigma). THP-1 cells were grown in RPMI-1640 containing 10% FBS, 1.0mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol. For differentiation into macrophages, THP-1 cells were treated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma Chemical) for 5 days until the cells adhered and exhibited macrophage-like morphology. TZM-bl and 293T cells were grown in DMEM medium supplemented with 10% FBS and penicillin-streptomycin (100μg/ml). All cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats prepared from healthy donors by centrifugation through a Ficoll gradient (Sigma-Aldrich, St. Louis, MO). Monocytes were isolated by negative selection with a human monocyte enrichment kit according to the manufacturer’s instructions (EasySep® Human Monocyte Enrichment Kit, Stemcell Technologies). The monocyte preparations contained 97% CD14+ cells, as determined by flow cytometry. For differentiation of monocytes into macrophages (MDMs), monocytes were seeded into Biocoat poly-D-lysine plates (B.D. Bioscience), and cultured in DMEM, supplemented with 10% heat-inactivated human serum, gentamicin (50μg/ml), ciprofloxacin (10μg/ml), and M-CSF (1000U/ml) for 7 days. MDM culture medium was half-exchanged every 2 to 3 days.
Transfection of siRNA
Small interfering RNA (siRNA) targeting Cav-1 and control siRNA were purchased from Santa Cruz Biotechnology, Inc. Transfection of siRNA was performed using OligofectaminTM Reagent (Invitrogen Corp., Carlsbad, Calif.) according to the manufacturer’s protocol. Briefly, the day before transfection, U87cells were seeded into a 24 well plate and cultured with antibiotics free medium to 30% confluency. Cells were washed and resuspended in 200ul serum free medium. Transfection mixture was prepared by incubating 50pmol of siRNA duplexes with 3ul of Oligofectamin in a final volume of 50ul Opti-MEM I Medium. After a 5 hour incubation, 125ul of growth medium with 3 times the normal concentration of serum was added to cells. Transfection was repeated once the next day. For THP1 macrophages, cells were first transfected with siRNA followed by HIV infection, and cells were then transfected again with siRNA the day after infection. The efficiency of Cav-1 knock-down by the siRNA transfection was monitored using Western blot analysis.
Virus production and concentration
Infectious virus HIV-1 AD8, ADnefmut, Bal, and NL4-3 were generated by calcium phosphate transfection of monolayers of 293T cells in 75-cm2 flasks with 25μg provirus DNA. Supernatants containing virus were harvested 4 days after transfection and quantified using the TZM-bl indicator cells as well as by measuring reverse transcriptase and a p24 ELISA method as described previously . When required, virus was produced from U87-CD4-CXCR4 cells transfected with 18μg proviral HIV NL4-3 along with 9μg pCZ-Cav-1 or pCZ-vector. To generate pseudotyped HIV particles 20μg pSG3Δenv or pNL4-3.Luc.R-E- was co-transfected with 3μg pCI-VSV into monolayers of 293T cells in 75-cm2 culture flasks by the calcium phosphate method. Pseudotyped viral supernatants were harvested 4 days post-transfection and were clarified by centrifugation at 3,000 rpm for 20 min and then by filtering through a 0.45 μm-pore size filter. Virus particles were concentrated using virus precipitation reagent Retro-ConcentinTM (System Biosciences) according to the manufacturer’s protocol.
Oil red O staining
To determine the influence of Cav-1 on the level of lipid accumulation in HIV infected and uninfected cells oil red O staining was performed. THP-1 cells were differentiated into macrophages by treatment with 50 ng/ml PMA for 5 days then infected with HIV AD8 (moi, 0.01) or Bal (moi, 0.001). On day 10 post infection, cells were loaded with cholesterol by incubating with 50μg/ml Ac-LDL (Biomedical Technologies Inc., Stoughton, MA) for 48 h followed by 30μg/ml apoA-I stimulation for 18 hours. Differentiated THP-1 cell-differentiated macrophage cells were also infected with Ad-Cav-1 or Ad-GFP at an moi (multiplicity of infection) of 100. Twenty-four hours later they were infected with VSV pseudotyped HIV pSG3Δenv or pNL4-3.Luc.R-E- at an moi of 3 and incubated for 5 days. Oil red O staining was performed as previously described . Briefly, cells were rinsed with PBS, followed by fixation with 3.7% paraformaldehyde for 60 min. The cells were stained using freshly prepared Oil red O (Sigma) working solution at room temperature for 10min. Intensity of cell staining was observed using a light microscope.
Virus infectivity assay
To test Cav-1’s influence on HIV-1 infectivity, TZM-bl cells were infected with virus harvested from Cav-1 treated cells and the infectivity levels were measured by luciferase activity. MDMs were first infected with adenovirus expressing Cav-1 or GFP at an moi of 100 in serum free medium for 6 hours. The cells were then washed and incubated in serum-containing medium over-night, after which cells were infected with HIV AD8 at an moi of 0.1 for 6 hours, at which point they were washed and refreshed with new medium. On day 6 post infection, supernatants were subjected to RT assay or titered using the indicator TZM-bl cell line. Virus amounts were normalized with level of infectivity being assayed by measuring luciferase within TZM-bl cells . Normalized amounts of virus were used for subsequent infections.
Determination of cholesterol content and cholesterol replenishment assay
Equivalent amounts of virions were quantified by p24 assay and tested for cholesterol content using the Amplex Red cholesterol Assay Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. To replenish cholesterol virus amounts were also normalized by p24 assay and incubated in 0.5mM (2-Hydroxypropyl)-ß-Cyclodextrin solution (Sigma Aldrich) with 1.5mM cholesterol (Sigma Aldrich) at 37°C for 1 hour. These quantified and normalized amounts of virus were used to infect TZM-bl cells and monitored for luciferase activity.
U87 cells were transfected with pcDNA3.1 and pCZ-vector (mock), pcDNA3.1SF2Nef and pCZ-vector (Nef), pCZ-cav-1 and pcDNA3.1 (Cav-1), pcDNA3.1SF2Nef and pCZ-cav-1 (Nef plus Cav-1), pCI NL4-3 NefG2A-HA (NefG2A) and pCZ-vector, or pCI NL4-3 NefG2A-HA and pCZ-cav-1 (NefG2A plus Cav-1). Twenty-four hours after transfection cell culture medium was replaced with serum free medium containing 2 μCi/mL [3H] cholesterol and 1.5% BSA and incubated for 36 hours. Radioisotope-containing medium was then removed and cells were washed twice with PBS and cultured for an additional 18 hours in serum free medium in the presence or absence of 50 μg/ml ApoA-l (Biomedical Technologies Inc., Stoughton, MA). Cholesterol content was measured in the cell free media as well as within cells after lysing using 0.1N NaOH. ApoA-l specific cholesterol efflux was determined using the formula: apoA-l specific efflux = % cholesterol efflux with apoA-l - % cholesterol efflux without apoA-l (blank); cholesterol efflux= [cpm(supernatants)/cpm(supernatants+cells)] ×100%. HDL mediated cholesterol efflux is also examined by incubating cells for 18 hours in the presence or absence of 50 μg/ml HDL (Biomedical Technologies Inc., Stoughton, MA).
To determine cholesterol efflux from macrophages, MDMs were first infected with Ad-Cav-1 or Ad-GFP at an moi of 50 for 24 hours, which was followed by infection of pseudotyped HIV pSG3Δenv (psHIVwtNef) or pNL4-3.Luc.R-E- (psHIVΔNef). Five days post infection cells were then labeled with 1 μCi/mL [3H] cholesterol for 48 hours and apoA-l mediated cholesterol efflux was determined as described above. Similarly cholesterol efflux from THP-1 cell-differentiated macrophages was determined 21 days after infecting with HIV AD8 at an moi of 0.001. Cholesterol efflux was also determined 14 days after THP-1 cell-differentiated macrophages infected with an moi of 0.001, 0.01, or 0.1. In addition, primary macrophages (MDMs) were infected with AD8 or ADnefmut HIV with an moi 0.01 and then cultured cells were subjected to cholesterol efflux assay 15 days after infection. ABCA-1 expression was determined by Western blots in MDMs 14 days after co-infection with AD8 or ADnefmut HIV and with Ad-Cav-1 or Ad-GFP. Inhibition of HIV replication was performed by treating infected cells with 5 uM azidothymidine (AZT) (Sigma-Aldrich, St. Louis, MO).
Immunoprecipitation and Immunoblotting analyses
U87 cells were transfected with pCZ-Cav-1 and HA-tagged Nef (pCI NL4-3 Nef-HA-WT) or HA-tagged NefG2A (pCI NL4-3 NefG2A-HA), followed by incubation of medium containing cholesterol (30μg/ml) for 48 hours. Cells were then treated with apoA-I (20μg/ml) for 30 min. Cells were put on ice, washed twice with cold PBS and total cellular protein was extracted in lysis buffer (50 mM Tris pH 7.5,100 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100, 10 mM NaF, 1 mM phenylmethyl sulfonyl fluoride, and 1 mmol/L vanadate) with a complete protease Inhibitor mixture (Roche Diagnostics, Indianapolis, IN). The concentration of extracted protein was determined and adjusted to 1 ug/ul. A total of 500 ul was used for each immunoprecipitation, to which 2μg of antibodies (anti-Cav-1 or anti-HA) or normal IgG were added. The mixtures were incubated at 4°C overnight. Following the overnight incubation, 25 μl of protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added and the mixtures were then rotated for 2 hours at 4°C. The beads were harvested by centrifugation and washed five times with lysis buffer. Loading buffer was added and boiled for 5 min. The samples were subjected to SDS-PAGE and analyzed by immunoblotting as described previously . The primary antibodies used for immunoblotting were rabbit polyclonal anti-Cav-1(Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-Nef, rabbit Nef antiserum, and human monoclonal anti-Gag (NIH AIDS Research and Reference Reagent Program), goat polyclonal anti-HA (Genescript), mouse monoclonal anti-ABCA1 (abcam), and ß-actin protein antibody (Sigma, St. Louis, MO). The secondary antibodies were HRP-linked anti-rabbit, anti-mouse (Cell Signaling Technology, Inc., Danvers, MA), anti-human IgG (Sigma, St. Louis, MO) or anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
Student’s t test was applied to analyze the differences between sets of data. All analyses were performed with SPSS 12.0.1 for Windows, and were considered significant at p ≤ 0.05.