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The BFV indicator cell line (BFVL) is a modified BHK-21 cell line that contains a luciferase reporter gene under the control of the BFV LTR promoter (Guo et al., 2011). BICL, a second reporter cell line also based on the BHK-21 cell line, contains an EGFP gene integrated into the genomic DNA driven by the BFV LTR (Ma et al., 2007). HEK 293T, BHK-21, BICL, and BFVL cells were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco, Gr and Island, USA) supplemented with 10% (v/v) fetal bovine serum, 50 IU/mL penicillin, and 50 μg/mL streptomycin at 37 ℃ in a 5% CO2 atmosphere. HEK 293T, BHK-21, and BICL cells were transfected using polyethylenimine (PEI; Polysciences, Warrington, USA) per the manufacturer's protocol.
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A single infectious BFV clone containing the BFV3026 proviral genome in pBluescript SK-(pBS-BFV) was used as the starting point for all mutations (Bing et al., 2014). An Nde I restriction site was used to divide the full-length BFV genome into two segments, S1 and S2, and mutations were introduced in the S2 segment. To strea-mline the polymerase chain reaction (PCR) -based site-directed mutagenesis, S2 was subcloned into the smaller pMD18T vector. PCR-based site-directed mutagenesis was used to introduce two stop codons using the primers ΔBTas-upper and ΔBTas-lower (Table 1). The desired mutations were confirmed by DNA sequencing. The mutated S2 fragment was then used to replace the original S2 fragment in the full-length infectious clone pBS-BFV. The final BTas-deficient clone was termed pBS-BFV-ΔBTas. For "add back" assays to recover BTas func-tion when cells were transfected with pBS-BFV-ΔBTas, wild-type BTas was expressed from the plasmid pcDNA3.1-BTas (Tan et al., 2008a). pcDNA3.1-BTas and BFV clones containing the lysine-to-arginine (K→R) mutations that were previously shown to inhibit acetylation of BTas (K66R, K109R, K110R, and K66/109/110R [also called 3M]) were generated using PCR-based site directed mutagenesis (Chang et al., 2011). The primers used for the K→R mutations are shown in Table 1. DNA sequencing was used to confirm that the desired sequence was achieved for all constructs.
Primers Sequence (5′-3′) ΔBTas upper GTAAGGAAGACCTCATAT A AT AACTTGCTTGTACCAΔBTas lower TGGTACAAGCAAGTT A TT ATATGAGGTCTTCCTTACK66R upper GAATTATAAACTTCATCTCA G ACGAGATGAACTTAGAGGAGK66R lower CTCCTCTAAGTTCATCTCGT C TGAGATGAAGTTTATAATTCK109R upper GTCATTAGAAACA G AAAGGGGCCATACK109R lower GTATGGCCCCTTT C TGTTTCTAATGACK110R upper CATTAGAAACAAAA G GGGGCCATACATGK110R lower CATGTATGGCCCC C TTTTGTTTCTAATGK109/110R upper GTCATTAGAAACA G AAG GGGGCCATACK109/110R lower GTATGGCCCC C TTC TGTTTCTAATGACNote: The mutant nucleotides are indicated with boxes. The underlined nucleotides indicate the positions of the mutated codons. Table 1. Primers used in this study
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Cell lysates were separated by SDS-PAGE. The separated proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (GE, Freiburg, Germany) by electroblotting for 1 h at 100 V. The PVDF membranes were blocked in 5% non-fat milk-PBS for 45 min at room temperature and subsequently incubated with primary antibody for 90 min. The primary antibodies against BTas (Tan et al., 2008b), Bet, and Gag (Wang et al., 2010) were generated in our lab and have been described previously. The secondary antibody was either a goat anti-mouse or goat anti-rabbit IgG conjugated to peroxidase. The membranes were incubated with secondary antibodies for an additional 45 min. Protein signals were detected by chemiluminescence (Millipore, Billerica, USA).
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The infectivity of each BFV infectious clones was analyzed using two different BFV indicator cell lines. BICL cells were transfected with pBS-BFV infectious clones, and the GFP signals were detected at different times post-transfection. BHK-21 cells or BICL cells were transfected with pBS-BFV plasmids. The transfected cells were harvested and then co-cultured with the BFVL reporter line. After 48 h co-cultivation, the co-cultured cells were harvested and subjected to luciferase assays according to the manufacturer's protocol (Promega, Madison, USA). The luciferase activity was normalized to the uninfected value. At least three independent experiments were performed, and the data were presented as means ± standard deviations.
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HEK 293T cells were transfected with either wild-type or mutant BTas plasmid and either the LTR-Luc or IP-Luc plasmid, which expressed firefly luciferase under the control of the BFV LTR or IP promoter, respectively. Transfection efficiency was normalized to the expression of the pCMV-β -gal plasmid, which expresses β -Gal from the CMV promoter and was co-transfected with the BTas-and luciferase-expressing plasmids. Luciferase activity was measured 48 h post-transfection and normalized to β -gal activity.
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BICL cells were plated at a density of approximately 2.0–2.5×105 per well in six-well plates in DMEM medium. Twenty-four hours later, the cells were transfected with pBS, pBS-BFV, or infectious clones containing the mutant BTas genes. BICL cells were passaged when they reached approximately 95% confluency. The GFP signal in the cells was assessed by fluorescence microscopy (Olympus Ⅸ-71, Tokyo, Japan) at 2, 4, and 6 days post-transfection to detect BFV transactivation. BICL cells transfected for 2, 4, and 6 days were also collected and co-cultured with BFVL cells.