Culture of HEK 293 T, HeLa-CD4 and C8166 cells and their optimal culture conditions have been described previously [12, 18].
Plasmids and virus production
The REAF-EGFP expression plasmid was generated by PCR amplifying the open reading frame from HeLa-CD4 cDNA. The vector used was pEGFP-C3 (Clontech). The infectious molecular clone for HIV-189.6 was obtained from the Centre for AIDS Research (NIBSC, UK). Plasmid construct HIV-189.6Δenv was generated from the HIV-189.6 molecular clone using overlap extension PCR. Clones were confirmed by plasmid sequencing (Source BioScience). Primers for all constructs are available upon request. Virus stocks were prepared from infectious full-length or pseudotyped HIV clones by polyethylenimine (Polysciences) or Lipofectamine 2000 (Invitrogen) transfection of HEK 293 T cells. HIV-2CBL-23 and SIV stocks were grown in C8166 cells.
Preparation of protein lysates
Prior to analysis on SDS-PAGE gels, cell pellets were lysed in ice cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate) containing protease inhibitor cocktail (Roche Applied Sciences) and 5 mM of sodium fluoride, β-glycerophosphate and sodium orthovanadate.
Western blot and immunoprecipitations
SDS-PAGE separated proteins, immobilised on nitrocellulose membrane (Novex) were detected with the primary rabbit polyclonal antibody against REAF (RbpAb-RPRD2), rabbit pAb-GAPDH, rabbit pAb-PABP or rat pAb-tubulin (Abcam) followed by corresponding horseradish peroxidise-conjugated goat α-rabbit/rat antibody (Abcam). Proteins were visualised using a chemiluminescence kit (ECL/ECL Prime, GE Healthcare).
Oligo (dT) immunoprecipitation
Prior to oligo (dT) addition, control samples were treated with DNase (10U) or increasing amounts of an RNaseA/H cocktail (250-2500U RNaseA, 20U RNaseH) and incubated at 37°C for 15 min. DNase (DNA-free™ Kit; Invitrogen) was physically removed from the appropriate sample according to manufacturer’s instructions. Ribonucleoprotein properties of REAF were then investigated by mixing lysates prepared as above with oligo (dT) magnetic beads (Invitrogen). After 15 min at 4°C the oligo (dT)-protein complexes were washed extensively with lysis buffer and TE. The complex was eluted into 1× PAGE loading buffer and analysed by Western blot.
Viral RNA IP and RT-qPCR analysis
Immunoprecipitation of viral RNA was performed as previously reported with slight modifications . Briefly, HEK 293 T cells were transfected with either pEGFP-C3 or REAF-EGFP and HIV-189.6 molecular clone DNA using Lipofectamine 2000 (Invitrogen). Cells were washed 2× with ice cold PBS then 2× with ice cold swelling buffer (25 mM HEPES, 1.5 mM MgCl2, 85 mM KCl, pH 8.0) and lysed in ice cold lysis buffer (25 mM HEPES, 1.5 mM MgCl2, 85 mM KCl, 0.02% NP-40, 1% Triton X-100, pH 8.0) containing RNaseOUT (500U/ml, Invitrogen), protease inhibitor cocktail (Roche Applied Sciences) and 10 mM of sodium fluoride, sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate. After centrifugation, the lysate was incubated with 2U of DNase (Ambion) for 5 min at 37°C and pre-cleared with Protein A/G-coupled rabbit IgG beads for 30 min at 4°C. One tenth of the volume was kept for total cellular RNA input, one tenth for Western blotting input and the rest mixed with Protein A/G-coupled REAF Ab beads and incubated for 1 hr at 4°C. The bead bound complexes were washed 2× with lysis buffer, 2× with wash buffer (25 mM HEPES, 1.5 mM MgCl2, 85 mM KCl, 0.25% Triton X-100, pH 8.0) containing 0.1U/ml RNaseOUT, 2× in wash buffer supplemented with 0.4 M NaCl followed by 2 washes with wash buffer supplemented with 0.1U/ml RNaseOUT and 0.1% v/v SDS. An aliquot of the complex was kept for Western blotting while the rest was incubated with DNase for 10 min followed by two phenol:chloroform:isoamyl alcohol (50:49:1) and one chloroform extraction. After precipitation with ethanol and glycogen carrier (Ambion), the RNA pellet was resuspended in DEPC-treated water and reverse transcribed using Superscript III First Strand Synthesis System (Invitrogen) according to manufacturer’s instructions. The resulting cDNA was used in qPCR analysis with primers for RU5 and late HIV-1 DNA as previously described . Primers are available upon request. This qPCR was repeated in a standard PCR machine for a non-saturating number of amplification cycles. These PCR products were resolved on a 4% agarose gel for visual confirmation.
siRNA transfection and infection with replication competent virus
HeLa-CD4 cells were seeded at 2.5 × 104 cells/well in 24-well plates. siRNA transfection (30nM) was performed using HiPerfect (QIAGEN) according to the manufacturer’s instructions. 72 hr after siRNA transfection, cells were challenged with virus (MOI 0.2) for up to 5 hr. Infection was assessed up to 48 hr by intracellular p24 staining or qPCR analysis. When required, reverse transcriptase inhibitor AZT (100 μM) and proteasomal inhibitor MG132 (10 μM) were added 1 hr before infection.
In situ immunostaining for p24 antigen
Infected cells were fixed with cold (−20°C) methanol:acetone (1:1), washed with PBS then immunostained for p24 using mouse anti-HIV-1 p24 monoclonal antibodies EVA365 and 366 (NIBSC, UK) (1:50) or anti-SIV p27 monoclonal antibodies (1:500) (to detect SIV infected cells), as previously described . Infected cells were blue (regarded as foci of infection (FFU/ml)) and quantitated by light microscopy.
First round Alu-PCR
DNA was extracted at various time points after infection with the QIAamp DNA Blood Mini Kit (QIAGEN). Integrated HIV-1 DNA was measured by nested PCR, as previously described .
qPCR for HIV-1 DNA
The isolated DNA was subjected to qPCR to determine the number of early (negative strand strong stop DNA, -sssDNA), RU5 and late (gag) transcripts, normalised for cell number by genomic GAPDH as previously described .
cDNA synthesis and mRNA analysis
Total HeLa-CD4 RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN) and cDNA was synthesised with Superscript III First Strand Synthesis System (Invitrogen), according to manufacturer’s instructions. The cDNA produced was subjected to qPCR as previously described . Primer sequences used to determine REAF mRNA levels are available upon request.