293T cells were maintained in Dulbecco's modified Eagle medium/high modified (with 4500 mg/l dextrose and 4 mM L-glutamine) medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% (vol/vol) heat-inactivated cosmic calf serum (CCS; Hyclone, Tauranga, New Zealand) and 100 U/ml of penicillin/streptomycin (P/S) (Invitrogen). MT-2 and Jurkat cells (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Invitrogen) supplemented with 10% vol/vol heat-inactivated fetal calf serum (FCS; Invitrogen) and P/S. HeLa cells (American Type Culture Collection [ATCC], CCL-2) and chicken fibroblast cell line DF1 (ATCC, CRL-12203) were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 1.5% sodium bicarbonate and P/S.
Virus production and purification
HIV virus stocks were produced by poly(ethylenimine) (PEI; Polysciences Inc., Warrington, PA, USA) co-transfection of 293T cells with the full length HIV-1 plasmid DNA pNL4.3 (obtained through the National Institutes of Health AIDS Reagents Program from Dr. Malcolm Martin ) and either the EGFP-Vpr fusion protein expressing plasmid pEGFP-Vpr or the mCherry-Vpr fusion protein expressing plasmid mCherry-Vpr (a kind gift from Prof. Tom Hope, Northwestern University, Chicago) to produce HIVGFP-Vpr and HIVmCh-Vpr.. Cells were counted and plated at 1 × 106 cells per plate onto 10 cm tissue-culture plates in 6 ml of media. Twenty-four hours later cells were transfected with 2.5 μg of plasmid DNA (1.875 μg of pNL4.3 and 0.625 μg of pEGFP-Vpr) at a 9:1 PEI:DNA ratio. Twelve hours post-transfection cells were washed twice with phosphate buffered saline (calcium and magnesium free; PBS-) and fresh media added. Supernatants were collected 36 hours post-transfection and cellular debris removed by sequential filtration through 0.8 μm and 0.45 μm sterile syringe filters (Sartorius, Goettingen, Germany) . Virus particles were then concentrated by ultracentrifugation through a 20% sucrose cushion using an L-90 ultracentrifuge (SW-41 rotor, Beckman Coulter, Fullerton, CA, USA) at 100,000 × g for 1 h at 4°C. Pellets were then resuspended in Benzonase buffer [20 mM Tris–HCl pH 8.0, 2 mM MgCl2, 20 mM NaCl] and treated with 90 units/ml Benzonase (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37°C to remove any contaminating plasmid DNA. The concentrated viral stocks were quantified using the Vironostika HIV-1 antigen (p24 CA) MicroELISA assay (bioMèrieux, Marcy l'Etoile, France) according to the manufacturer’s instructions and frozen in single-use aliquots at −80°C.
Infection of lymphoid cells
Synchronized infections were performed as described previously . MT-2 or Jurkat cells were spinoculated with 300 ng p24 CA, as determined by Vironostika HIV-1 antigen (p24 CA) MicroELISA assay (bioMèrieux, Marcy l'Etoile, France) virus per 1 × 106 cells for 2 h at 1,200 × g in either 12 or 24 well plates at a non fusion-permissive temperature (15°C). After spinoculation, cells were washed twice with PBS- to remove unbound virus and incubated with warm media at 37°C, 5% CO2 to initiate infection.
Removal of extracellular plasma membrane proteins and un-entered virus using pronase
As part of the experiment using the RT inhibitor AZT pronase treatment was used 20 minutes post-infection to remove all proteins from the outside of the cells, thus removing any bound but un-entered virions from the cell surface. Twenty minutes post-spinoculation cells were washed and resuspended in ice-cold Hank's Balanced Salt Solution (HBSS, Invitrogen) containing 2 mg/ml of protease from Streptomyces griseus (pronase E; Sigma-Aldrich) for 10 min on ice. Cells were then washed extensively with HBSS containing 10% FCS then fresh media added and the cells incubated at 37°C.
Reverse-transcription inhibition using Zidovudine (AZT)
The RT inhibitor Zidovudine (AZT) was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. AZT was added to cell culture supernatants at the point of infection at either 0.1 or 10 μg/ml as separate conditions. AZT was maintained in the cell culture supernatant throughout the infection.
Modulation of miRNA levels via synthetic nucleic acids
Introduction of synthetic nucleic acids to DF1 or HeLa cells were carried out using the transfection reagent Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, polyinosinic-polycytidylic acid (pIC) was diluted to 10 times the intended final concentration in media, and Lipofectamine was diluted into media at 1 μL / cm2 of well area. After 5 min the Lipofectamine and pIC were combined 1:1, incubated for 15 min and then added to cell culture as per manufacturer’s instructions (Invitrogen).
Quantitative PCR for HIV-1 reverse transcription products
Quantification of HIV-1 reverse transcription products and standardization of cell numbers was performed using quantitative PCR (qPCR). Cells were harvested at various time points post-infection and lysed in PCR lysis buffer containing 10 mM Tris [pH 8.0], 50 mM KCl with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 mg/ml proteinase K (Roche, Basel, Switzerland). Samples were incubated at 56°C for 2 h before the proteinase K was inactivated by heating to 95°C for 10 min. Samples were then stored at −20°C. Quantitative PCR was performed on an MX3000P QPCR machine (Agilent, Santa Clara, CA, USA). Each PCR reaction contained 1× Brilliant II SYBR Green Master mix (Agilent), 400 nM each primer and 5 μl of cell lysates (1:10 dilution) in a 15 μl reaction volume. The HIV-1 specific primers M667 (sense; 5′-GGCTAACTAGGGAACCCACTG-3′) and AA55 (antisense; 5′-CTGCTAGAGATTTTCCACACTGAC-3′) were used to detect early HIV-1 cDNA ([−]strong-stop DNA). The HIV-1 specific primers M667 (sense; 5′-GGCTAACTAGGGAACCCACTG-3′) and M661 (antisense; 5′-CCTGCGTCGAGAGATCTCCTCTGG-3′) were used to detect intermediate HIV-1 reverse transcription products (LTR/gag, post 2nd strand transfer). HIV-1 PCR conditions were an initial denaturation at 95°C for 15 min followed by 40 rounds of cycling at 95°C for 10 s, then 60°C for 30 s. Cell numbers were standardized for the human CCR5 gene using the primers LK46 (sense; 5′-GCTGTGTTTGCGTCTCTCCCAGGA-3′) and LK47 (antisense; 5′-CTCACAGCCCTGTGCCTCTTCTTC-3′). CCR5 PCR conditions were an initial denaturation at 95°C for 15 min followed by 40 rounds of cycling at 95°C for 20 s, 58°C for 40 s and 72°C for 40 s.
Quantitative reverse transcription-PCR (qRT-PCR) analyses of miRNA
RNA was harvested using Tri-reagent (Sigma-Aldrich) according to the manufacturer’s instructions. One μg of extracted RNA was subjected to DNase treatment using a DNase 1 kit (Sigma-Aldrich) according to the manufacturer’s instructions. The DNase treated RNA was then polyadenylated with 300 units of polyadenylase polymerase to a final volume of 20 μl and incubated at 37°C for 30 min, then 95°C for 5 min similar to the method by Shi et al. . The miRNA was reverse-transcribed to complimentary DNA (cDNA) using a Superscript III first strand synthesis kit (Invitrogen) according to the manufacturer’s instructions then diluted to 1:20. qRT-PCR was carried out using Sybr Green (applied biosystems) and the comparative threshold cycle method, to derive fold change, previously described by Bannister et al., (2011). The forward primer sequence miR146 (5’GCG TGA GAA CTG AAT TCC ATG GG) and the endogenous control miR5.8S (5’ TGG GAA TAC CGG GTG CTG T) were individually amplified with a universal reverse primer (5’ GAG GCG AGC ACA GAA TTA ATA CGA C) to generate Ct values for analyses similar to Bannister et al. .
O-FISH detection of HIV-1 nucleic acids and miR146
Cells were fixed with either 4% formaldehyde in PBS- at 4°C overnight (HIV analyses) or in 300 μl methanol for 20 min at 20°C (miR146 analyses) and then washed twice in PBS-. Fixed cells were mounted onto slides using a Cytospin II machine (Shandon, Astmoore, UK) at 67 × g for 5 minutes. The slides were removed and air-dried overnight in the dark. Slides were either processed immediately or stored in sealed bags at −20°C until needed. All reactions were performed as open-droplet reactions with a droplet volume of 15 μl. Cells were rehydrated in PBS- for 5 minutes and permeabilised with 0.2% Triton for 6 minutes, then washed twice with PBS- before being treated with 0.005% pepsin (Sigma-Aldrich) in 0.01N HCl for 1 min(pepsin treatment was omitted in the miR146 analyses) and then again washed twice with PBS-. O-FISH probe stocks were then diluted to a final concentration of 500 nM in hybridisation buffer (10 mM TRIS [pH7.4], 600 mM NaCl, 1 mM EDTA, 10 nM DTT, 0.1% SDS, 50% formamide) and added to the cells, which were incubated in a humidity chamber at 37°C for 1 hour. Cells were washed twice with PBS- and blocked in blocking buffer (Olink Bioscience, Uppsala, Sweden) at 37°C for 30 minutes. Probe binding was detected via incubation with an anti-biotin primary antibody (Sigma-Aldrich), diluted 1:500 for HIV-1 nucleic acids or 1:1500 for miR146 in antibody diluent (Olink Bioscience) in a humidity chamber at room temperature for 30 minutes. Detection of primary antibody binding was carried out using Duolink II anti-mouse PLA probes and detection kit (Olink bioscience) as per the manufacturer’s instructions. After O-FISH detection, the cells were counterstained with Hoechst 33258 (Invitrogen) and then mounted in Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA, USA) or mounted directly using fluromount with DAPI (Olink Bioscience) for miRNA microscopy.
O-FISH Probes used for RNA and cDNA detection
Probes were designed complementary to different parts of the HIV-1 RNA genome, to HIV-1 cDNA synthesised at various stages of the reverse-transcription process, or to miR146. Negative sense probes pol (5’ CTG TCA GTT ACA TAT CCT GCT TTT CC 3’) and U5/PBS (5’ CGG GCG CCA CTG CTA GAG ATT TTC 3’) were used to detect positive sense HIV-1 RNA. Positive sense probes gag (5’ ATG GGT GCG AGA GCG TCG GTA TTA AG 3’) and strong stop (5’ TGT GAC TCT GGT AAC TAG AGA TCC CT 3’) were used to detect negative sense HIV-1 cDNA. The miRNA probe (5’ CCC ATG GAA TTC AGT TCT C) was used to detect miR146a. All probes had a biotin molecule conjugated to their 5’ end.
Image acquisition and analysis
For HIV O-FISH analysis, images were acquired using either a DeltaVision-RT (Applied Precision, Issaquah, WA, USA) or Zeiss Axio Observer Z1 (Zeiss) microscope. Images taken on the DeltaVision-RT were acquired in a z-series on a charge-coupled device (CCD) camera (CoolSnap HQ; Photometrics, Tucson, AZ, USA) through either a 60X 1.42 numerical aperture (NA) or a 100X 1.4 NA oil immersion lens. Reference brightfield images were also acquired. Images were deconvolved using softWoRx deconvolution software (Applied Precision) before analysis. All images taken on the Zeiss Axio Observer Z1 were taken in a z-series on a charge-coupled device (CCD) camera (AxioCam MRm Rev. 3, Carl Zeiss, Germany) through a 100X 1.30 NA oil immersion lens. Reference differential interference contrast images (DIC) were also acquired. Images of a minimum of 5 fields were taken per slide for analysis for all experiments. Cell images were analysed by quantification of fluorescent signal using Bitplane Imaris software (Bitplane AG, Zurich, Switzerland). For miRNA analyses, similar procedures were used but the samples were imaged using a Leica (Leica Microsystems, Sydney) SP5 confocal microscope. Fluorescence and DIC images were collected and all images were taken with the same microscope parameters. Quantification of O-FISH signal events was performed using the spots function in Imaris (Bitplane AG, Switzerland). O-FISH signals were designated to be at least 0.5 μm in diameter and intensity was determined on an experimental basis for each set of slides. The total number of O-FISH signals was divided by the number of nuclei for each image. The mean for each sample was then calculated from an average of between 13.4 and 30 cells per time point and condition from at least five randomly acquired images (resulting in data being derived from a total of between 120 and 160 cells per panel in each figure) and is reported as ‘signals per cell’.
Statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Paired two-tailed t-tests were used to compare variance between two sets of observations as indicated.