shRNAs shhtatsf1-a, shhtatsf1-b and shhtatsf1-c were designed to target htatsf1 transcript [NCBI RefSeq_RNA: NM_014500.3] at the sequences GCT ACA TAT CAG GCC AAT TAT, GCG CAT CTA GTT CTA CCG CAA and CTG CAA CTG GAA TGG CGT T, respectively (Additional file
1). These target sites were selected from sequences suggested by The RNAi Consortium (
http://www.broad.mit.edu/genome_bio/trc/rnai.html). All shRNAs were designed to contain a loop sequence derived from miR-31. G:U mismatches were incorporated at the 3’ end of the anti-guide strand of some shRNAs to decrease thermodynamic stability of this end of the hairpin stem and favour selection of the intended guide strand. RNA Pol III U6 shRNA expression cassettes were generated by a two-step PCR approach described previously
. These were cloned into pTZ57R/T (Fermentas). Construct sequence was confirmed by automated cycle sequencing.
Several previously developed constructs were used as controls in experiments: a mock pTZU6+1 (U6) construct with no shRNA sequence
; a shRNA negative control, shHBVx-5, which targets an irrelevant site in hepatitis B virus (HBV) X protein
; and, two positive controls, shLTR-U5 and shTAT, which are named after the location of their target sequences within HIV-1 transcripts and were initially developed based on subtype B molecular clone HXB2 [GenBank: K03455]
[31, 32]. shRNA shpsip1-a was adapted from a guide strand previously shown to inhibit LEDGF/p75 expression
 that targets the p75 isoform of psip1 transcript [NCBI RefSeq_RNA: NM_033222.2] at the sequence GAC AGC ATG AGG AAG CGA A.
Cell culture and transfections
HeLa-derived TZM-bl cells (NIH AIDS Research and Reference Reagent Program), which express the HIV receptor CD4 and coreceptor CCR5 and contain a luciferase reporter driven by a Tat-inducible LTR promoter derived from pSG3.1 [GenBank: L02317]
[26–28], were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% heat-inactivated fetal calf serum (FCS), at 37°C and 5% CO2. HEK293T, HeLa and SupT1 cells (NIH AIDS Research and Reference Reagent Program), the latter a non-Hodgkin’s T cell lymphoma suspension cell line expressing high levels of surface CD4
, were maintained in the same media.
Transfections were carried out using 1 μl Lipofectamine2000 (Invitrogen) to 1 μg DNA, according to manufacturer’s instructions. Medium was changed 5 h post-transfection. Where appropriate, a plasmid with constitutive eGFP expression (pCI-eGFP) was cotransfected followed by fluorescence microscopy 48 h later to verify equivalent transfection efficiencies
Dual luciferase reporter assay
To generate psiCheck target plasmids, with all shRNA target sites for each cellular factor adjacent to one another, complementary oligonucleotides were treated with polynucleotide kinase (Promega), annealed and cloned directly into the XhoI-NotI sites of psiCheck2. An EcoRV site was inserted within each annealed dsDNA insert to facilitate screening. The oligonucleotides used for psiCheck htatsf1 were: TCG AGA TAT CGC TAC ATA TCA GGC CAA TTA TGC GCA TCT AGT TCT ACC GCA AAC TGC AAC TGG AAT GGC GTT GC; and, CTA GAT GCG CAT AAT TGG CCT GAT ATG TAG CGA TAT CGG CCG CAA CGC CAT TCC AGT TGC AGT TTG CGG TAG AA; and, for psiCheck psip1 were: TCG AGA TAT CAG ACA GCA TGA GGA AGC GAA GCA GCT ACA GAA GTC AAG ATT GC; and, GGC CGC AAT CTT GAC TTC TGT AGC TGC TTC GCT TCC TCA TGC TGT CTG ATA TC. Target constructs psiCheck HBVx
 and psiCheck LTR
 have been described previously.
HeLa or HEK293T cells were seeded at 5.0 × 104 and 1.2 × 105 cells per well, respectively, in a 24-well culture plate and transfected 24 h later with 500 ng shRNA expression construct, 100 ng of psiCheck target reporter construct and 10 ng pCI-eGFP, in triplicate. Firefly and Renilla luciferase activities were determined 48 h later using the Dual Luciferase Reporter Assay System (Promega) and a Veritas dual-injection luminometer (Turner Biosystems), according to manufacturer’s instructions. Renilla: firefly luciferase activity ratios were normalised to the U6 control mean.
Quantitative RT-PCR of cellular factor mRNAs
TZM-bl cells were seeded at 5.0 × 104 cells per well in a 24-well culture plate and transfected 24 h later with 500 ng shRNA expression construct and 10 ng pCI-eGFP, in triplicate. Total TZM-bl cellular RNA was extracted using TriReagent (Sigma-Aldrich) 48 h later, or from stably transduced SupT1 cells cultured for periods equivalent to days 0, 10 and 20 of the HIV-1 replication assay (see below). Total RNA was subjected to DNase treatment (Promega) and random-primed reverse-transcription using the SuperScript III reverse transcriptase (RT) (Invitrogen). cDNA was analysed for target mRNA expression relative to β-actin mRNA (actb) transcript NM_01101.2 using the SensiMix Lite Kit (Quantace) with the following primers: htatsf1 forward AGTGGGACCTGGACAAAAAGG; htatsf1 reverse GTT CCG GGG CTT TTT CTT GTG; psip1 forward GCT GAA CAA AGA CAG CAT GAG GA; psip1 reverse ATT GCT CTC CCC GTT ATG TTG TG; actb forward AGG TCA TCA CCA TTG GCA ATG AG; and, actb reverse TCT TTG CGG ATG TCC ACG TCA. The qPCR was performed in a Carousel-based Lightcycler V.2 System (Roche) with the following parameters: denaturation at 95°C for 10 min, 50 cycles of denaturation at 95°C, annealing at 60°C and extension at 72°C, each for 10 s. Amplification cycles were followed by melting curve analysis to verify the specificity of the PCR products. No RT controls were included for each sample and no cDNA controls for each primer set. Target mRNA: actb ratios were normalised to the mean expression ratio of U6-transfected samples.
TZM-bl cells were seeded at 1.5 × 105 cells per well in a 6-well culture plate and transfected 24 h later with 2 μg shRNA expression construct and 10 ng pCI-eGFP. Cells were harvested 72 h post-transfection and lysed with RIPA buffer. Total protein was quantified using the BCA Protein Assay Kit (Pierce). A ladder composed of IgG-binding proteins ranging from 22 to 120 kDa in size and 80 μg of samples were resolved on a 12% polyacrylamide gel. Protein was transferred to a PVDF membrane (Millipore) and probed with rabbit polyclonal antibodies to Tat-SF1 (a gift from M. Garcia-Blanco) at 1:100 and β-actin (GenWay Biotech) at 1:1,000. The latter was used to quantify loading of samples. HRP-conjugated donkey anti-rabbit IgG secondary antibody (GenWay Biotech) was used at a dilution of 1:25,000 and proteins were detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce). Images were acquired with a G-BOX (Syngene). Levels of target protein are reported relative to levels of β-actin and normalised to the shHBVx-5 control.
SupT1 cells were similarly analysed by Western blot, with the exception that cells were harvested after culture periods equivalent to days 0 and 20 of the HIV-1 replication assay (see below). Day 20 samples were prepared in duplicate. Mean target protein expression relative to levels of β-actin are reported normalised to the U6 mock at each time point.
Northern blot analysis of shRNA guide strand processing
TZM-bl cells were seeded at 2 × 106 cells in a 60 cm2 culture dish and transfected with 20 μg shRNA expression plasmid 24 h later. Total cellular RNA was isolated from TZM-bl cells 48 h post-transfection, or SupT1 cells, using TriReagent (Sigma-Aldrich). Thirty micrograms of RNA was resolved on urea denaturing 15% polyacrylamide gels and blotted onto nylon membranes. RNA molecular weight markers were run alongside the cellular RNA. Blots were hybridised to DNA oligonucleotide probes of complementary sequence to hairpin-derived guide strands and, therefore, of the same sequence as the shRNA target sequences (see above).
For analysis of TZM-bl RNA, the RNA ladder and DNA probes were labelled at their 5’ ends with [γ-32P] ATP and T4 polynucleotide kinase. To quantify loading of the TZM-bl RNA, an oligonucleotide sequence complementary to U6 small nuclear RNA was used of the following sequence: TAG TAT ATG TGC TGC CGA AGC GAG CA. Following hybridisation, blots were exposed to an imaging plate and viewed on a FLA-7000 phosphorimager (Fujifilm), stripped and reprobed.
For SupT1 RNA analysis, levels of 5S rRNAs on the ethidium bromide-stained polyacrylamide gel verified equal loading of the samples. The RNA ladder and DNA probes were labelled at their 3’ ends with the DIG Oligonucleotide 3’-end Labelling Kit according to manufacturer’s instructions (Roche). Following hybridisation, chemiluminescence detection of bound probes was enabled by incubation of the membranes with alkaline phosphatase-conjugated anti-DIG antibody, incubation with CDP-Star (Roche) and image acquisition with a G-BOX (Syngene).
TZM-bl cells were seeded at 3 × 104 cells per well on CELLocate microgrid coverslips (Eppendorf) in a 24-well culture plate. Cells were transfected with 500 ng shRNA expression constructs 24 h later, in duplicate. Another subset of cells was treated with 500 nM trichostatin-A 80 h post-seeding as a positive control. Seventy-two hours post-transfection, or 16 h post-trichostatin-A treatment, apoptosis was quantified using the TACS Annexin V-FITC Apoptosis Detection Kit (R&D Systems). Fluorescence images were acquired for two fields of view per well on an Axiovert 100 M microscope with image capture by AxioVision 2.0.5 software (Carl Zeiss Microimaging). Fluorescence was quantified using ImageJ 1.40 g (developed by W. Rasband, NIH) and reported normalised to the U6 mock.
MTT assay for cell viability
TZM-bl cells were seeded at 1 × 104 cells per well in a 96-well culture plate. Cells were either transfected with 100 ng shRNA expression construct, or treated with 10, 100 or 500 nM trichostatin-A, 24 h later, in triplicate. A further 48 h later, 0.1 mg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltretrazolium bromide (MTT) was added to each well. Cells were incubated at 37°C for 1 h, media removed and formazan precipitates resuspended in 200 μl DMSO. Absorbance at 570 nm, with a reference wavelength of 655 nm, was determined in a Model 680 microplate reader (BioRad) and reported normalised to the cell control, which was not transfected or treated with TSA.
TZM-bl cells were seeded at 3 × 104 cells per well in a 24-well culture plate and transfected with 500 ng shRNA expression construct or 1 μg of the double-stranded RNA polyinosinic:polycytidylic acid (poly(I:C) (Sigma-Aldrich) as a positive control, in triplicate. Total RNA was extracted using TriReagent (Sigma-Sldrich) 48 h post-transfection and subject to DNase treatment, reverse transcription and qPCR, as described above. Primers used to amplify interferon-β mRNA (ifnb1) were: forward TCC AAA TTG CTC TCC TGT TGT GCT; and, reverse CCA CAG GAG CTT CTG ACA CTG AAA A. ifnb1:actb expression ratios were normalised to the mean expression ratio of U6-transfected samples.
Virus preparation and propagation
1.2 × 106 HEK293T cells were seeded in a 25 cm2 culture flask and transfected 24 h later, using PolyFect transfection reagent (Qiagen), with 4 μg of subtype B molecular clone p81A-4 (HIV-1p81A-4) (NIH AIDS Research & Reference Reagent Program)
[29, 30]. Media was replaced 24 h later. A further 24 h later, media was collected, filtered, made up to 20% FCS, aliquoted and stored at −80°C.
Median tissue culture infectious dose (TCID50) was determined using the Spearman-Karber method
[41, 42]. TZM-bl and SupT1 cells were seeded at 1 × 104 cells per well in a 96-well culture plate and infected with various dilutions of virus, in triplicate, 24 h later. For TZM-bl cells, infections were carried out in the presence of 15 μg/ml DEAE-D. Cells were washed with PBS 24 h post-infection, referred to as day 0. For TZM-bl cells, luciferase activities were determined in cell lysates 48 h post-infection using the Bright-Glo Luciferase Assay System (Promega). Samples were considered luciferase positive if the luminescence signal was greater than that of the mean of the no virus samples plus two standard deviations. SupT1 cells were incubated for 7 days post-washing and both day 0 and day 7 culture supernatant samples were analysed for the HIV-1 antigen p24 by ELISA using the HIV antigen mAb Kit (Murex Biotech). Samples were classed as positive if the A450 was greater than the absorbance of the kit’s negative control + 0.50.
HIV-1 replication in TZM-bl reporter cells
TZM-bl cells were seeded at 5 × 104 cells per well in a 24-well culture plate and transfected 24 h later with 500 ng shRNA expression constructs and 10 ng pCI-eGFP, in triplicate. Cells were infected with either FV5 or HIV-1p81A-4 at a TCID50 of 1000/ml 24 h later in the presence of 15 μg/ml DEAE-D. Cells were washed with PBS 24 h post-infection. Forty-eight hours post-infection, 100 μl of culture supernatant was removed and stored at −80°C for subsequent analysis of p24 levels using the HIV antigen mAb Kit (Murex Biotech). Another 100 μl of culture supernatant was used to infect additional TZM-bl cells, seeded at 5 x 104 cells per well in a 24-well culture plate the preceeding day, in the presence of 15 μg/ml DEAE-D. Tat-induced luciferase activities were determined in cell lysates 48 h post-infections using the Bright-Glo Luciferase Assay System (Promega). Data are reported normalised to the U6 mock.
Generation of shRNA-expressing SupT1 cell lines
shRNA expression cassettes were excised from pTZ plasmids by digestion with EcoRI and AccI and cloned into the EcoRI and ClaI sites of second generation lentivector pLVTH (Addgene plasmid 12262, deposited by D. Trono)
, which encodes a GFP reporter. Lentiviruses were generated from the shRNA-expressing lentivectors by transfecting 3.6 × 106 HEK293T cells in a 60 cm2 culture dish 24 h later with 5 μg shRNA-expressing lentivector, 3.8 μg psPAX2 and 2.5 μg pMD2.G (Addgene plasmids 12260 and 12259, respectively, both deposited by D. Trono). Culture media collected 24 and 48 h post-transfection was pooled, filtered and stored at −80°C. Lentiviruses were titred based on non-linear regression of the number of GFP+ SupT1 cells following transduction with various dilutions of lentivirus. This was determined using a FACSCalibur flow cytometer (BD Biosciences) to acquire 5 × 103 events per sample with analysis by FlowJo 9.1 (Tree Star). SupT1 cells were gated based on forward and side scatter characteristics and GFP+ cells determined from that subset by comparison of transduced with untransduced cells.
SupT1 cells were seeded at 3 × 105 cells per 75 cm2 culture flask and incubated with lentivirus at a MOI of 0.15. Cells were cultured for 5 days prior to harvest and fluorescence activated cell sorting (FACS) on a FACSCalibur. Sorted GFP+ cells were concentrated by centrifugation and cultured in DMEM with 20% FCS, 100 U/ml penicillin, 100 μg.ml streptomycin, 50 μg/ml tetracycline, 100 μg/ml ampicillin, 170 μg/ml chloramphenicol, 50 μg/ml kanamycin and 100 μg/ml ciprofloxacin for 1 week. Sorted cell lines were cultured for a further week without antibiotics and stocks made. The proportion of GFP+ SupT1 cells in each cell line was determined by flow cytometry and FlowJo analysis (Tree Star) based on the acquisition of 5 × 103 events immediately prior to sorting (pre-sort) and freezing (post-sort). Thawed SupT1 cell lines were cultured for 5 days prior to seeding in all subsequent experiments.
HIV-1p81A-4 replication in SupT1 cell lines
SupT1 cell lines with shRNA expression were seeded at 2 × 104 cells per well in a round-bottomed 96-well culture plate and immediately infected with HIV-1p81A-4 at a TCID50 of 50/ml in duplicate. Mock SupT1 cells with the U6 promoter but no shRNA expression were cultured both with and without infection as controls. Twenty-four hours post-infection, cells were washed with PBS, resuspended in 350 μl media and pelleted prior to removal of 150 μl media as day 0 samples. Cells were resuspended with replacement of the media removed. Cells were pelleted and another 150 μl media sample removed seventy-two hours post-infection (day 2). Samples were removed in the same fashion on days 4, 7, 10, 14 and 17. All samples were stored at −80°C prior to analysis of p24 content by ELISA (Murex Biotech). Dilutions of the kit positive control were used to generate a standard curve of p24 levels from which absolute levels of p24 in the experimental samples were determined.
Nuclear run-on analysis of htatsf1 transcription
SupT1 cell lines expressing either shhtatsf1-a or shLTR-U5 were cultured for periods equivalent to days 0 and 20 of the HIV-1p81A-4 replication assay before harvesting of cell nuclei, in triplicate. Nuclear run-on was performed as previously described
, with modification to use biotin-tagged transcripts
. Biotinylated RNA was isolated using Dynabeads MyOne Streptavidin C1 beads (Invitrogen), prior to reverse transcription and qPCR. htatsf1:actb transcription ratios were normalised to the mean expression ratio of day 0 samples.
Proliferation of SupT1 cell lines
SupT1 cell lines were analysed by flow cytometry after culture for periods equivalent to days 0 and 20 of the HIV-1p81A-4 replication assay (see below). The proportion of GFP+ SupT1 cells in each population was determined following acquisition of 5 × 103 events on a FACSCalibur (BD Biosciences) and analysis using FlowJo 9.1 (Tree Star). SupT1 cell lines with shRNA expression were also seeded at 5 × 104 cells per well in a 12-well plate in quadruplicate. After 20 days culture, cellular DNA was extracted and quantified by NanoDrop (Thermo Fisher Scientific), in duplicate.
Data are expressed as the mean ± the standard error of the mean (SEM). Statistical difference was considered significant (*) when p <0.05. Data were analysed using non-linear regression, unpaired t-test, one-way ANOVA, followed by Dunnett’s multiple comparison post-tests, and two-way ANOVA, followed by Bonferroni post-tests, where appropriate, using Prism 4.0c (GraphPad Software).