Protein A beads were purchased from Sigma (St. Louis, MO, USA). Anti-Flag M2 monoclonal antibody (MAb) was obtained from Stratagene (Agilent Technologies, Santa Clara, CA, USA). Anti-HA rabbit antibody, anti-HA mouse antibody, anti-β-actin C4 mouse antibody, horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA).
Plasmids and proteins
pBIV-LTR-Luc and pHIV-LTR-Luc plasmids were constructed by subcloning BIV LTR or HIV LTR into a pGL3-basic luciferase reporter vector (Promega, Madison, WI, USA) upstream of the luciferase gene. The BIV LTR and HIV LTR were provided by Dr. Charles Wood (University of Nebraska Lincoln). pCMV-HA-BHEXIM1 and pCMV-HA-HHEXIM1 were generated by inserting BHEXIM1 or HHEXIM1 cDNA into the expression vector, pCMV-HA (Clontech, Mountain View, CA, USA). BHEXIM1 deletion mutants were created by PCR and cloned into pCMV-HA. The mammalian expression plasmid, Myc-BTat, was provided by Dr Matjaz Barboric (University of California). pcDNA3.1(+)-HTat (1-86 aa) was constructed by inserting HTat (1-86 aa) into the pcDNA3.1(+) vector (Invitrogen, CA, USA). The pcDNA-B-cyclin T1 (aa residues 1-272 of bovine cyclin T1) was a kind gift from Dr. Alan Frankel (University of California) and was subcloned into the pCMV-Tag2B vector (Stratagene). The pGEX6p-1 (Amersham Bioscience, Pittsburgh, PA, USA) and pETH (Novagen, Darmstadt, Germany) vectors were used to construct bacterial expression plasmids for GST- and His-fusion proteins. Escherichia coli BL21 (DE3) strain cells were transformed with these constructs to express GST-B-cyclin T1, His-BHEXIM1, and His-BTat proteins. The proteins were purified with glutathione Sepharose 4B beads (GE healthcare, Pittsburgh, PA, USA) or His SepharoseTM 6 (GE healthcare) according to the respective manufacturer’s instructions.
Cells and viruses
293 T, HeLa, fetal bovine lung (FBL) cells were primary cells isolated from fetal bovine lung by our laboratory; canine thymus cell line (Cf2Th), which is a BIV permissive cell line, was kindly provided by Prof. Jin-Ming Gao (Peking Union Medical College); and BIV baby hamster kidney indicator cells, BIVL, which harbor the BIV LTR-luciferase reporter, were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 2 mmol/L glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin sulfate. All of the cells were cultured at 37°C in 5% CO2. The R29 strain of BIV was provided by Dr. Charles Wood (University of Nebraska Lincoln). Tissue culture infectious dose endpoint (TCID50) of BIV as BIV titers was evaluated by BIVL cells. Confluent BIVL cells on 96-well plates were infected with 25 μL of 10-fold serial dilutions of stock viruses with eight parallel wells for each dilution. Then the cells were incubated at 37°C in 5% CO2 for 48 h. Examination of luciferase activity was performed and the TCID50 was calculated with the Reed-Muench method.
RNA isolation and RT-PCR
Total RNA was isolated from FBL cells using Trizol Reagent (Gibco) according to the manufacturer’s instructions. Fifteen micrograms of total RNA was used for cDNA synthesis using Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) (Promega). BHEXIM1 sequences were amplified by polymerase chain reaction (PCR) using the following primers: forward, 5′- GTGGAATTCAAATGGCCGAGCCACT-3′; reverse, 5′- AGCCTCGAGCTAGTCTCCAAAGTTG-3′.
Small hairpin RNA (shRNA) treatments
The pSIREN-RetroQ (Clontech) was used to construct the shRNA-BHEXIM1 plasmid, which targets the CAGCGATGAGGACTTTATG sequence. A control shRNA targeting sequence GACAGAACCAGAGGATAGA, which does not pair with any eukaryotic mRNA, was used. The lentiviral shRNA system was used in this study as previously described. Briefly, stock lentiviruses were produced by transfecting 293 T cells. Supernatants were harvested at 48 h post-transfection and were then stored at -80°C. To generate the BHEXIM1 downregulated cells, FBL cells were infected with stock lentiviruses for 12 h and then the virus was washed away. At 48 h after infection, puromycin (2 μg/mL) was added to the cultures to select for BHEXIM1-downregulated cells.
The amounts of BHEXIM1 and GAPDH mRNA in shRNA-BHEXIM1 or shRNA-control treated FBL cells were examined by real-time PCR. Real-time PCR was performed using the IQ5 Multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and SYBR green real-time PCR master mix (Toyobo, Osaka, Japan) according to the manufacturers’ instructions. The sequences of the primer pairs were as follows: BHEXIM1 forward (5′-ACGACACCAGCGATGAGGAC-3′) and reverse (5′-TCCAGCCGCAGCCGATTATT-3′); BIV capsid protein-CA forward (5′-AAGGAGCCGTACACAGACTT-3′) and reverse (5′-TTCTGGAGCCGCCATACCTT-3′); and GAPDH, which was used as an internal control, forward (5′-AACGGCACAGTCAAGGCAGA-3′) and reverse (5′-TCGGCAGAAGGTGCAGAGAT-3′). The specificity of the amplification reaction was verified by melting-curve analyses. Threshold cycle (C
) values were calculated automatically as the cycle when the sample fluorescence exceeded a threshold level corresponding to 10 standard deviations (SD) of the mean of the baseline fluorescence. The level of BHEXIM1 and CA expression was normalized to GAPDH using the 2-ΔΔCT method.
Cf2Th cells were plated in 12-well plates (1 × 105/well) and cultured at 37°C for 20 h before transfection using polyethylenimine (PEI; sigma). The total DNA in each transfection mixture was normalized using empty vector DNA. pCMV-β-gal plasmid DNA (0.1 μg) was included in each transfection. Cells were harvested at 48 h after transfection. The luciferase activity was measured using a commercial assay system (Promega). The cells were rinsed with PBS and lysed with lysis buffer (Promega). The cell lysates were then clarified from the insoluble materials by centrifugation at 12 000 rpm for 3 min. The clarified lysate (5 μL) was mixed with the luciferin reagent (50 μL) and luciferase activity was measured in a chemiluminescence measurement machine (Promega). Simultaneously, 50 μL clarified lysate was mixed with the same volume (50 μL) of o-Nitrophenyl-β-D-Galactopyranoside (ONPG) buffer (4.4 mM ONPG, 4.3 mM MgCl2-6H2O, 164 mM Na2HPO4-12H2O, 36 mM NaH2PO4-2H2O, 0.684% β-mercaptoethanol). The activity of β-galactosidase was then examined at OD405 nm with a Precision Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) until the results ranged between 0.200 and 0.800. The results were used as an internal control to normalize the efficiency between transfections by the formula: (each well of luciferase activity) / (each well of β-galactosidase activity - the background of β-galactosidase activity). The luciferase activity of each well divided by the control well was expressed as the relative luciferase activity (fold). Each experiment was repeated at least three times.
Cf2Th cells plated in 12-well plates (1 × 105/well) were transfected with various plasmids. Fourty-eight hours post-transfection, the cells were harvested and washed twice with 1× phosphate buffered saline. The cells were then lysed with NP-40 lysis buffer (0.5% NP-40, 0.1% Triton X-100, 0.1% sodium deoxycholate, 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA) containing 1% PMSF for 30 min on ice. After centrifugation at 10 000 × g for 10 min at 4°C, the supernatants were collected, and protein concentrations were measured using the Bio-Rad Protein Assay reagent (Catalog No. #500-0006). The samples (30 μg/lane) were separated by 12% polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) by electroblotting for 1 h at 100 V, 4°C. Following incubation in 5% nonfat milk (in 1× phosphate-buffered saline) for 45 min at room temperature, the membrane was blotted with primary antibody for 90 min at room temperature and then incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated with peroxidase.
Co-immunoprecipitation (Co-IP) and Pulldown assays
For the co-immunoprecipitation assay, a total of 1 × 107 293 T cells in 100-mm-diameter dishes were transfected with various plasmids using the PEI reagent. Cells were harvested at 48 h after transfection; lysed in 600 μL lysis buffer (50 mM Tris-HCl [pH8.0], 150 mM NaCl, 1% NP-40, and 1 mM phenylmethylsulfonyl fluoride); sonicated; and centrifuged for 15 min at 4°C, 10 000 × g. The supernatant was incubated with antibody at 4°C for 2 h. Thirty microliters of protein A beads was then added, and the mixture was incubated for an additional 2 h at 4°C. Immunocomplexes were washed five times with the lysis buffer and then boiled in SDS loading buffer. The precipitated proteins were detected by SDS-PAGE and immunoblotting. For the GST pulldown assay, purified GST and GST-fusion proteins were immobilized on glutathione Sepharose 4B beads, and then incubated with purified His-fusion proteins. After extensive washing, the beads were boiled in SDS loading buffer and the proteins were subjected to SDS-PAGE and immunoblotting.