Cell culture and transfection
HeLa cells and 293T cells were grown in Dulbecco’s modified Eagle’s medium (GIBCO, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FCS; Sigma-Aldrich, St. Louis, MO, USA). Human PBMCs were isolated on a Ficoll (Lymphosepal; Immuno-Biological Laboratories, Minneapolis, MN, USA) gradient from a healthy donor. Monocytes were selected from PBMCs using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and a MACS separation column (Miltenyi Biotec) with a Quandro MACS separation unit (Miltenyi Biotec) according to manufacturer instructions. Monocytes were cultured at the desired density in 6- or 24-well plates and grown in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% heat-inactivated FCS (Culture Biosciences, San Francisco, CA, USA), 5% AB serum (Sigma-Aldrich), and 10 ng/ml macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA) for 1 week until they spontaneously differentiated into mature macrophages.
Plasmid transfection was performed using FuGENE HD (Promega, Madison, WI, USA) or Lipofectamine2000 (Invitrogen). siRNA transfection was performed using Lipofectamine2000 or Lipofectamine RNAi MAX (Invitrogen). siRNA and plasmid co-transfection was performed using Lipofectamine2000. siRNA transfection for macrophages was performed using Lipofectamine RNAi MAX. Macrophages were transfected with 50 nM siRNA in Opti-MEM (GIBCO). At 4-h post-transfection, macrophages were washed and cultured in RPMI-1640 medium containing 10% heat-inactivated FCS, 5% AB serum, and 10 ng/ml M-CSF for 20 h. Cells were subjected to subsequent rounds of transfection, washing, and culturing as described.
The expression vector pME18Neo encoding Flag-tagged WT Vpr (pME18Neo/Flag-Vpr) and pGEX-6P-3 encoding GST-tagged Vpr (pGEX-6P-3/GST-Vpr) have been described previously [57, 58]. The molecular clone vectors pNL4-3-Luc-env(−) and pNL4-3-Luc-env(−)vpr(−) and an expression construct for VSV-G (pVSV-G) were kindly gifted by Dr. Ishizaka (Department of Intractable Diseases, National Center for Global Health and Medicine). For construction of the vector pME18Neo/Flag-Vpr-IRES-ZsGreen1 and the control vector pME18Neo/Flag-IRES-ZsGreen1, a fragment containing an IRES sequence and a ZsGreen1-coding sequence was amplified by polymerase chain reaction (PCR) using the primers 5'-CCCAAACTTAAGCTTGGTACCGA-3' and 5'-TAGCGGCCGCTCAGGGCAAGGCGGAGCCGGAG-3' and pRetroX-IRES-ZsGreen1 (Clontech Laboratories, Mountain View, CA, USA) as a template. The PCR fragment was subcloned into pME18Neo/Flag-Vpr and pME18Neo/Flag at the NotΙ site.
For construction of the expression vector pCAGGS encoding HA-tagged HIP1 (pCAGGS/HA-HIP1), human HIP1 mRNA was amplified by reverse transcription (RT)-PCR from RNA derived from HeLa cells. RT was performed with an oligo-dT primer, and PCR was performed using the primers 5'-AAAGATATCGGATCGGATGGCCAGCTCCATGAAGCAGGTGCCCAA-3' and 5'-AAAGCGGCCGCCTATTCTTTTTCGGTTACCACTTC-3'. The PCR fragment was subcloned into the pCAGGS/HA vector between the EcoRV and NotΙ sites. For construction of the pCAGGS/HA-siR-HIP1 vector, the mutant was generated using standard PCR mutagenesis techniques using pCAGGS/HA-HIP1 as a template.
siRNAs against 256 genes were prepared as an siRNA mini-library , and those targeting HIP1 and DCAF1 were designed using BLOCK-iT RNAi Designer (Invitrogen). The siRNA forward sequences targeting HIP1 were 5'-CACAGACCUUCUGGUCUGUUGUCAA-3' for siRNA#1 and 5'-GGAGCUAAUGGUGUGUUCUCAUGAA-3' for siRNA#2. The siRNA forward sequence targeting DCAF1 was 5'-CCCUGGGUGAUUGGCACCAAUUAUA-3'.
293T cells were co-transfected with the indicated vectors, and at 48-h post-transfection, the cells were lysed with lysis buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.5% NP-40] supplemented with a protease-inhibitor cocktail (Roche, Basel, Switzerland) for 30 min on ice. The lysates were centrifuged at 15,000 rpm for 5 min, and the supernatants were collected and mixed with Anti-FLAG M2 agarose beads (Sigma-Aldrich) and incubated at 4°C for 18 h with gentle rotation. The beads were washed five times with lysis buffer, and the bound proteins were eluted using the FLAG peptide (Sigma-Aldrich). Eluted proteins were fractionated by 6% and 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed western blot.
Protein expression and purification
Recombinant GST or GST-Vpr was expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL cells (Stratagene, San Diego, CA, USA). Expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 16°C for 24 h, followed by lysis with BugBuster reagent (Novagen; Merck, Kenilworth, NJ, USA) according to manufacturer instructions. The lysate was cleared by centrifugation, and the soluble fraction was mixed with glutathione-Sepharose 4 FastFlow beads (GE Healthcare, Pittsburgh, PA, USA), which were centrifuged and washed with BugBuster reagent and phosphate-buffered saline (PBS).
To express and purify HA-HIP1, 293T cells were transfected with pCAGGS/HA-HIP1 using FuGene HD transfection reagent (Promega), and at 48-h post-transfection, the cells were collected and lysed with wash buffer. The lysates were centrifuged at 15,000 rpm for 5 min, and the supernatants were collected, mixed with Anti-HA agarose beads (Sigma-Aldrich), and incubated at 4°C for 1 h with gentle rotation. The affinity beads were washed with wash buffer twice, and HA-HIP1 was eluted using the HA peptide (Sigma-Aldrich).
Purified HA-HIP1 was incubated with GST or GST-Vpr preadsorbed onto glutathione-Sepharose 4 FastFlow beads at 4°C for 2 h in wash buffer. The beads were then washed with wash buffer five times, and bound proteins were eluted by incubation with sample buffer for SDS-PAGE at 100°C for 5 min. Eluted proteins were fractionated by 6% SDS-PAGE for western blotting.
Cells were lysed for 30 min on ice in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.1% SDS supplemented with a protease-inhibitor cocktail (Roche). Lysates were mixed with SDS-PAGE sample buffer and boiled for 5 min. Protein concentrations were determined with a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Waltham, MA, USA) using bovine serum albumin as a standard. Equal amounts of total protein were examined by western blotting using the following antibodies: anti-Flag monoclonal antibody (mAb; M2; Sigma-Aldrich), anti-Flag polyclonal Ab (Sigma-Aldrich), anti-β-actin mAb (Sigma-Aldrich), anti-HA mAb (MBL International, Woburn, MA, USA), anti-HIP1 mAb (Novus Biologicals, Littleton, CO, USA), anti-VprBP polyclonal Ab (Proteintech, Rosemont, IL, USA), horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Amersham Biosciences, Little Chalfont, UK), and HRP-conjugated goat anti-rabbit IgG (Amersham Biosciences). Signals were visualized after treatment with SuperSignal West Pico chemiluminescent substrate (Pierce; Thermo Fisher Scientific).
Analysis of cell cycle profiles by CELAVIEW RS100
HeLa cells were plated in 24-well polystyrene plates. To analyze the effect of siRNA transfection on Vpr-induced G2 arrest, the cells were co-transfected with siRNAs and either pME18Neo/FVpr-IRES-ZsGreen1 or pME18Neo/Flag-IRES-ZsGreen1 and cultured for 48 h. To analyze the effect of HIP1 overexpression on the inhibition of Vpr-induced G2 arrest following HIP1 knockdown, the cells were co-transfected with siRNAs, pME18Neo/FVpr-IRES-ZsGreen1, and pCAGGS/HA-siR-HIP1 or pCAGGS/HA and cultured for 48 h. To analyze the effect of HIP1 knockdown on etoposide-induced G2 arrest, the cells were transfected with siRNAs. At 24-h post-transfection, the cells were treated with 10 μM etoposide and cultured for 24 h. These cells were fixed and stained with 3.6% formaldehyde containing 5 μg/ml Hoechst33342 for 10 min at room temperature and then washed three times with PBS. For each sample, at least 200 ZsGreen1-positive cells were observed and analyzed using a CELAVIEW microscope (RS100; Olympus, Tokyo, Japan).
Analysis of cell cycle profiles by flow cytometry
HeLa cells were co-transfected with siRNAs and either pME18Neo/Flag-Vpr-IRES-ZsGreen1 or pME18Neo/Flag-IRES-ZsGreen1 as a control and cultured for 48 h. The cells were harvested and fixed with 1% formaldehyde, followed by 70% ethanol. Fixed cells were incubated in PBS containing RNase A (50 μg/ml) at 37°C for 20 min and then stained with propidium iodide (40 μg/ml). For each sample, at least 7,000 cells were analyzed using a FACS Calibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA) with CELL Quest software (Becton Dickinson). Ratios of the numbers of cells in G1 and G2/M phases (G2/M:G1 ratios) were calculated using ModFit LT software (Verity Software House, Topsham, ME, USA).
Viral stock and viral infection of macrophages
To generate viral stocks, 293T cells were co-transfected with pNL4-3-Luc-env(−) or pNL4-3-Luc-env(−)vpr(−) and pVSV-G using FuGENE HD (Promega), and the virus was harvested at 48-h post-transfection. HIV-1 titers were measured using an anti-p24 enzyme-linked immunosorbent assay kit (Ryukyu Immunology, Okinawa, Japan).
Primary macrophages in 24-well plates were inoculated with VSV-G pseudotyped reporter viruses [NL-Luc-E−R+ (VSV-G) or NL-Luc-E−R− (VSV-G); 4 ng of p24 antigen], cultured for 6 days, harvested, and lysed in luciferase assay substrate (Promega). Infectivity was determined by measurement of luciferase activity.
Statistical analyses were performed by Prism 8.0 (GraphPad software, San Diego, CA, USA). For two-group comparisons, two-tailed Student′s t-test was used. Data are presented as mean ± SD and were considered statistically significant when the P value was <0.05.