Cells

GHOST28 and 293FT (Invitrogen) cells were cultured in DMEM, 10% fetal bovine serum (FBS) (HyClone) and antibiotics. Peripheral blood mononuclear cells were isolated from Institutional Review Board (IRB)-approved buffy coats from normal donors. CD14+ cells were isolated by two sequential positive selections with anti-human CD14 magnetic beads (Miltenyi). Purity was at least 99%. CD14+ cells were cultured in RPMI medium, 10% FBS, antibiotics and HEPES in the presence of recombinant human GM-CSF at 10 ng ml−1 and IL-4 at 50 ng ml−1 (eBioscience). Fresh media was added at day 3, and cells were stimulated or infected at day 4. To isolate CD11c+ dendritic cells, CD14-depleted peripheral blood mononuclear cells were further depleted using biotin-labelled antibodies against CD3, CD16, CD19 and CD56 and streptavidin magnetic beads (Miltenyi). The negative fraction was stained and sorted on a FACSAria (BD Biosciences) as CD3−CD14−CD16−CD19−CD20−CD56−HLA-DR+CD11c+ (Supplementary Table 2). Purity was at least 98%. Total CD4+ T cells were isolated using human CD4 magnetic beads (Miltenyi). Naive CD4+ T cells were further sorted as CD4+CD25−CD45RA+CD45RO−.

T-cell clones

T-cell clones were expanded as previously described29. The clone DR4LI15 (LGLNKIVRMYSPTSI) was obtained from N. Bhardwaj and the clones B81TL9 (TPQDLNTML) and B14DA9 (DRFYKTLRA) were obtained from B. Walker.

Reagents

LPS and poly(I:C) were from Sigma and were used at 1 µg ml−1 and 10 µg ml−1, respectively. Curdlan (CM-Curdlan) was form Wako and was used at 1 µg ml−1. Cyclosporin A and FK506 were from Calbiochem. AZT, raltegravir, lopinavir, saquinavir and tipranavir were obtained through the NIH AIDS Research & Reference Reagent Program. SCY (SCYX00011867717) is similar to SCY-635 and was a gift from Scynexis.

Infection and stimulation

At day 4 of MDDC differentiation, cells were harvested, counted and resuspended in their own media at a concentration of one million per ml with 5 µg ml−1 polybrene, and 100 µl was aliquoted in round-bottomed 96-well plates. For infection, 50 µl of media or SIV-VLP(G) was first added. One-hundred microlitres of media or dilutions of various HIV-1-derived viral preparations were then added. Saquinavir, tripranavir, AZT and raltegravir were added at 10 µM. Neutralizing anti-IFN-α and anti-IFN-β were added at 20 µg ml−1. B18R (eBioscience) was added at 0.2 µg ml−1. IL28RA-Fc (R&D Systems) was added at 1 µg ml−1. Neutralizing anti-IFNAR was added at 1 µg ml−1. For shRNA-transduced dendritic cells, cells were harvested, counted and resuspended in fresh media containing GM-CSF, IL-4 and 1 µg ml−1 polybrene at a concentration of one million per ml. One-hundred microlitres was aliquoted in round-bottomed 96-well plates and 100 µl of media or virus was added.

Western blot analysis

Cells were lysed in 1% NP-40, 50 mM Tris pH 8, 120 mM NaCl, 4 mM EDTA, 50 mM NaF, 1 mM NA 3 VO 4 and a protease inhibitor cocktail (Roche). Total lysates were resolved on SDS–PAGE, transferred to polyvinylidene fluoride (PVDF) membranes and probed with primary antibodies and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare).

Cytoplasmic and nuclear fractionation

MDDCs were harvested at 24 h after infection or treatment. 4 × 106 cells were washed once with room temperature PBS, gently pelleted and resuspended in 400 µl of cold cytoplasmic lysis buffer (CL buffer) containing 10 mM HEPES pH 7.9, 10 mM sodium potassium, 1.5 mM magnesium chloride, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 2 mM sodium β-glycerophosphate, 5 mM sodium fluoride, complete EDTA-free protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (SIGMA P2850). Cells in cold CL buffer were immediately pelleted at 4 °C, the supernatant was discarded, 40 µl CL was added, and buffer and cells were gently resuspended by slow pipetting and soft flicking and left on ice for 15 min. Two and a half microlitres of 10% NP-40 was added and cells were lysed by gentle flicking. Nuclei were pelleted at 15,900g for 5 min at 4 °C. Forty microlitres of supernatant was harvested and saved as the cytoplasmic fraction, and remaining liquid was discarded. Forty microlitres of cold nuclear lysis buffer (NL buffer) containing 420 mM sodium chloride, 20 mM HEPES pH 7.9, 1.5 mM magnesium chloride, 0.2 mM EDTA, 25% glycerol and protease and phosphatase inhibitors as in CL buffer was added. Nuclei were resuspended by vigorous flicking and incubated on ice for 15 min, with occasional flicking. Nuclei were vortexed for 10 s and sonicated for 10 min in a 4 °C bath sonicator (30 s on, 30 s off). The nuclear lysate was cleared by centrifugation at 15,900g for 5 min at 4 °C, and the resulting supernatant was saved as the nuclear extract. Western blot loading buffer with dithiothreitol was added to the cytoplasmic and nuclear extracts, and the samples were heated at 70 °C for 15 min. Ten microlitres of each sample was run on a 7.5% SDS–PAGE gel and transferred to PVDF membrane (Roche). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and probed with primary antibody overnight while rocking at 4 °C, washed six times for 5 min with TBST, probed with secondary HRP-conjugated antibody (GE Healthcare) for one hour at 19 °C (room temperature), washed six times for 5 min in TBST, and incubated with ECL reagents (Pierce Pico or Pierce Femto). Chemiluminescence signal was visualized using Kodak film.

Quantitative bioassay for IFNs

293FT and THP-1 cells were infected with HIV-GFP(G) and SIV-VLP(G) or transfected with poly(I:C) or total RNA from NDV-infected A549 cells harvested in Trizol (Invitrogen) 8 h after infection using lipofectamine 2000 (Invitrogen). NDV viral stock was produced by inoculating 10-day-old embryonated chicken eggs (Charles River). CD4+ T cells were expanded with 5 µg ml−1 phytohemagglutinin-L (Sigma) and 10 U ml−1 human IL-2 for 4 days and infected with 100 haemagglutinin (HA) units ml−1 of Sendai virus (Charles River) or infected with HIV-GFP(G) and SIV-VLP(G). Media were replaced after 24 h and culture supernatants were harvested after another 24 h. Cell culture supernatants were ultraviolet-irradiated to inactivate traces of Sendai virus. Supernatants were assayed for IFN activity using a recombinant COS-1 cell line, which carries a luciferase reporter containing multiple repeats of IFN-stimulated response element (ISRE). In brief, the reporter cells were exposed to cell culture supernatants for 8 h to overnight and assayed for luciferase activities, which were then translated to IFN activities by using a standard curve generated from a serial dilution of human IFNα-2a.

Microarray analysis

MDDCs were infected with HIV-GFP(G), SIV-VLP(G), both, or treated with LPS. Cells were harvested after 48 h and a subset was analysed by flow cytometry. RNA was prepared with TRIZOL and microarray data generation was done using standard protocols on Human Genome U133A 2.0 arrays (Affymetrix). Microarray analysis was performed using the Bioconductor package in R and Genespring GX10 (Agilent). Probes were filtered based on at least a twofold change in expression and P < 0.05. Promoter analysis was performed using PRIMA30 in EXPANDER31. TLR pathway analysis was performed using SPIKE32.

Quantitative PCR

Quantitative PCR analysis was performed as described33 using the standard curve method or the ΔC T method (for primer sets see Supplementary Table 3).

Plasmids

HIV-GFP, which is env−vpu−vpr−vif−nef− , with the GFP open reading frame in place of nef, has already been described34. HIV-GFP ΔRev was generated by mutating the start codon of rev. HIV-GFP PTAP− was generated by mutating the PTAP motif in p6 to LIRL. HIV-GFP ΔGag was generated by inserting a stop codon after seven amino acids of Gag. Vpx–Vpr fusion protein was generated by fusing SIVmac251 Vpx with HIV-1 NL4-3 Vpr using the linker ANYAAAAAAADPS in pIRES2-EGFP (Clontech). LKO1gfp was generated by replacing the puroR open-reading frame in pLKO1puro35 with the EGFP coding region. shRNAs were designed as described previously (Supplementary Table 4), except that a partial mir30 sequence CTGTGAAGCCACAGATGGG was used for the loop. shRNAs were then cloned as described35. T54A/N57A, Q63A/Q67A, G89V and the parental vector pLaiΔEnv-GFP3 are env–nef– , with GFP in place of Nef, and were previously described12. HDV-IRES-RFP is described elsewhere36. HIV-2 ROD9 Δenv GFP was generated from an HIV-2 ΔEnv construct37 by inserting the GFP-coding sequence in Nef, thus disrupting Nef. All plasmid DNA was prepared with Invitrogen HiPure plasmid kit. Plasmid DNA did not induce dendritic cell maturation, and viral-producing cells were washed after DNA transfection.

Virus production

Viral particles were produced by transfection of 293FT cells with 3 µg DNA and 8 µl TransIT-293 (Mirus Bio); for shRNA vectors, we used 0.4 µg CMV-VSVG, 1 µg pCMV-ΔR8.91 and 1.6 µg shRNA; for SIV-VLP(G), 0.4 µg CMV-VSVG and 2.6 µg pSIV3+ (ref. 38); for HIV-GFP(G), 0.4 µg CMV-VSVG and 2.6 µg HIV-GFP; for HIV2 ROD9 Δenv GFP(G), 0.4 µg CMV-VSVG and 2.6 µg HIV2 ROD9 ΔEnv GFP; for NL4-3-ΔE-EGFP, 0.4 µg pCMV-VSVG and 2.6 µg pNL4-3-ΔE-EGFP39. HIV-GFP ΔRev and HIV–GFP PTAP− were produced with 0.4 µg CMV-VSVG, 0.5 µg pCMV-ΔR8.91 and 2.1 µg HIV plasmid. HIV-GFP ΔGag and HIV-1 capsid mutants were produced with 0.4 µg CMV-VSVG, 1 µg pCMV-ΔR8.91 and 1.6 µg HIV plasmid. R5-GFP is NL4-3 encoding for the BAL envelope and GFP in Nef34,36. One day after transfection, media was removed, cells were washed out once and fresh media was added. Viral supernatants were harvested one day later and filtered at 0.45 µM. In some experiments, p24 concentration was measured by p24 enzyme-linked immunosorbent assay (ELISA).

RNAi

Synthetic short interfering RNA can be delivered in MDDCs by electroporation, but this is highly and rapidly toxic and renders difficult the interpretation of dendritic cell activation, which can be altered by the presence of apoptotic or necrotic cells. In addition, we have not been able to achieve significant knockdown using synthetic siRNA with lipid-based reagents in MDDCs. In fact, fluorescently labelled siRNA appeared to be simply endocytosed with all the lipid-based reagents that we tested (data not shown). We thus used shRNA vectors. Using this method, we routinely obtained >90% transduction efficiency, alleviating the need for cell sorting or selection. Five-million freshly isolated CD14+ cells were cultured in 5 ml of media containing GM-CSF, IL-4, and 1 µg ml−1 polybrene. One millilitre of SIV-VLP(G) supernatant and 2.5 ml of shRNA vector supernatant were added to cells. At days 1 and 3, 2 ml of fresh media was added. At day 4, cells were transduced at more than 90% based on GFP expression and were used for further infections and stimulation as above.

HIV-specific T-cell-clone stimulation

Forty-eight hours after infection of MDDCs, 105 rested HIV-specific T-cell clones were added in the presence of GolgiStop (BD Biosciences). Where indicated, T cells were activated with 50 ng ml−1 PMA (Sigma) and 0.5 µg ml−1 ionomycin (Sigma). Cells were incubated for 6 h and processed for intracellular staining33.

Naive T-cell proliferation assay

Forty-eight hours after infection, naive T cells were labelled with CFSE (eBioscience) as descried previously18. Dendritic cells were infected with dilutions of SIV-VLP(G) and pLaiΔEnv-GFP3(G) wild type (indicated as HIV-GFP(G)) or G89V. AZT was added at the time of infection and SCY was added from 3 h to 8 h after infection. Forty-eight hours after infection, half of the dendritic cells were processed for surface staining and cytometry. The other half were washed with media and resuspended in fresh media without cytokines. Twenty-thousand T cells were mixed with dendritic cells at a dendritic cell to T cell ratio of 1:5 and 1:15. Cells were stimulated by dilutions of anti-CD3 (OKT3 hybridoma supernatant, approximately 1–100 ng ml−1) in a total volume of 150–200 µl in round-bottomed 96-well plates. Cells were harvested and analysed by flow cytometry at day 4 or day 5 after activation.

Trans-enhancement

105 day 4 MDDCs were infected with dilutions of HDV-IRES-RFP(G) and SIV-VLP(G) in 96-well round-bottomed plates in the presence of 5 µg ml−1 polybrene. Type-I-IFN-neutralizing antibodies and recombinant proteins were maintained throughout the experiment in some samples. Media was replaced after 24 h. Another 24 h later, half of the cells were processed for surface staining and RFP and CD86 expression were measured by flow cytometry. The other half was mixed with a preparation of replication- competent R5–GFP and 5 × 105 CD4+ T cells at day 4 after activation with PHA-L (Sigma) and IL-2, in the absence of polybrene. GFP expression in CD4+ T cells was measured another 48 h later.