Exploring histone loading on HIV DNA reveals a dynamic nucleosome positioning between unintegrated and integrated viral genome

Significance The biology of HIV DNA, from its synthesis to its integration into the host genome, remains poorly understood. Here we show that in the nucleus, histones are rapidly loaded on newly synthesized unintegrated HIV DNA. Interestingly, the chromatin architecture around the HIV long terminal repeat (LTR) is different in unintegrated and integrated HIV DNA. Specifically, a nucleosome present only on the DNase hypersensitive site of unintegrated HIV DNA contributes to the transcriptional silencing of unintegrated HIV DNA by preventing RNAPII recruitment.

infection (hpi), and subjected to flow cytometry analysis using a MACSQuant Analyzer (Miltenyi Biotec). Data were analyzed using FlowJo software.

Luciferase assay
Jurkat cells were infected with HIV-Luc IN wt or IN D116A at the multiplicity of infection of 0.5. After 3 hours, the cell culture medium was changed. Subsequently, TSA (sigma: 0.5 µM) was added in the medium at 32 hpi, and cells were lysed in passive lysis buffer (Promega) at 48 hpi. Insoluble proteins were pelleted by centrifugation at 16,300 g, and supernatant was subjected to Luciferase assay system (Promega). The protein concentration was measured by Pierce BCA protein assay kit (Thermo Fisher Scientific). Luminescence was measured by TriStar LB 941 (Berthold technologies), and normalized by the protein concentration. For experiments using primary CD4 T cells, CD4 T cells activated for 3 days were infected with HIV-Luc IN wt or IN D116A at the multiplicity of infection of 0.5. After 3 hours, the cell culture medium was changed. TSA (0, 0.125, 0.5, 2, 8 µM) was added in the medium at 32 hpi, and cells were lysed in passive lysis buffer at 48 hpi.

Quantification of viral DNA
Jurkat cells were infected with VSV-G pseudotyped HIV-1 containing the dual-color reporter at the multiplicity of infection of 0.2 (calculated by GFP expression) in the presence or absence of 1 µM raltegravir (Selleckchem). After 3 hours, the cell culture medium was changed. Cells were collected at 3, 6,9,12,24, and 48 hpi, DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen) and subjected to qPCR. For quantification of total HIV DNA, 25 ng DNA was used in each reaction (3,4). For quantification of 2LTR circle, 100 ng DNA was used in each reaction. Integrated HIV DNA was quantified by Alu-PCR method as described previously (4), using 100 ng extracted DNA. Total HIV DNA, 2LTR circle, and integrated HIV DNA were normalized by bglobin. Primer sequences were listed in SI Appendix table S2. For experiments using PF74, differentiated THP-1 cells were treated with VLP-Vpx, and infected with VSV-G pseudotyped HIV-1 containing the dual-color reporter in the presence or absence of 2 µM PF74 (Sigma). The cell culture medium was changed 3 hours later, and cells were collected at 24 hpi. DNA was then extracted using the DNeasy Blood and Tissue Kit (Qiagen) and subjected to qPCR. All drugs were added in the medium for the entire duration of infection.

ChIP assay
Jurkat cells were infected with VSV-G pseudotyped HIV-1 containing the dual-color reporter at the multiplicity of infection of 0.2 (calculated by GFP expression) in the presence or absence of 1 µM raltegravir (Selleckchem) or nevirapine (Sigma). For experiments using primary CD4 Tcells, CD4 T cells activated for 3 days were infected with VSV-G pseudotyped NL4-3 IN wt or IN D116A at the multiplicity of infection of 0.4. After 3 hours, the cell culture medium was changed. Cells were collected at 9 and 48 hpi, and fixed with 1% formaldehyde for 10 min at room temperature. Chromatin immunoprecipitation reactions were performed using SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the protocol provided by the manufacturer. 13-16 µg of chromatin was digested with MNase (Roche) and reacted with 4 µg of respective antibodies. qPCRs were performed using specific primers (SI Appendix table S3) For experiments using PF74, differentiated THP-1 cells were treated with VLP-Vpx and infected with VSV-G pseudotyped HIV-1 containing the dual-color reporter in the absence or presence of 2 µM PF74 (Sigma). The cell culture medium was changed 3 hours later. Cells were collected at 24 hpi, and fixed with 1% formaldehyde for 10 min at room temperature. Chromatin immunoprecipitation reactions were performed using a ChIP-IT PBMC Kit (Active Motif) according to the protocol provided by the manufacturer, except for the nucleus preparation step. Fixed cells were directly lysed by ChIP buffer (Active Motif), and sonicated with Bioruptor Pico (Diagenode). 30-40 µg of sonicated chromatin were reacted with 4 µg of anti-Histone H3 antibody. qPCRs were performed using specific primers (SI Appendix table S3). The following antibodies were used for ChIP: anti-histone H3 (Abcam, ab1791), anti-histone H2B (Abcam, ab1790), anti-histone H3ac (Millipore, 06-599), anti-histone H3K4me3 (Abcam, ab8580), and anti-RNAPII (Santa Cruz Biotechnology, F12 sc-55492) antibody.

Capture MNase-seq
Jurkat cells (1x10 7 ) were infected with VSV-G pseudotyped NL4-3 IN wt or IN D116A at the multiplicity of infection of 0.2. For experiments using primary CD4 Tcells, CD4 T cells (0.5x10 7 ) activated for 3 days were infected with VSV-G pseudotyped NL4-3 IN wt or IN D116A at the multiplicity of infection of 0.2. After 3 hours, the cell culture medium was changed. Cells were collected at 9 and 48 hpi, and native mononucleosomes were prepared as previously described (5). Briefly, 2x10 6 cells were treated with hypotonic buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100) for 15 min on ice, followed by centrifugation. Pellets were washed with 300 μl of MNase digestion buffer (10 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, 3 mM CaCl2). The nuclei were resuspended with 100 μl of MNase digestion buffer, and treated with 2.1 U of MNase at 37 °C for 4 min. Reactions were terminated by adding 150 μl of stop buffer (20 mM EDTA, 20 mM EGTA, 0.4% SDS, and 0.5 mg/ml proteinase K), and incubate at 55 °C for 6 hours or overnight. DNA was then extracted by phenol-chloroform extraction, followed by ethanol precipitation. Mononucleosome-sized DNA was separated by 2% agarose gel electrophoresis, and purified using QIAGEN gel extraction kit. To obtain sonicated fragments, DNA was extracted from nuclei washed with MNase digestion buffer using DNeasy Blood and Tissue Kit (Qiagen), and sonicated with Bioruptor Pico (Diagenode) to produce an average DNA fragment size of 100-300 bp. Libraries were prepared using NEXTflex Rapid DNA-Seq kit2.0 (BIOO SCIENTIFIC) using 100 ng input DNA according to the protocol provided by the manufacturer. To capture library DNAs containing HIV-1 sequence, the custom-designed capture bait system, consisting of 804 biotinylated RNAs targeting entire pNL4-3 and pHRET (2) except for the sequence of the vector, was designed according to the manufacturer's instructions (Agilent Technologies). Library DNAs (600-800 ng) were concentrated using a vacuum concentrator at below 45°C, and reconstitute with H2O to obtain 3.4 μl total volume. Library DNAs were then hybridized with capture bait RNAs according to the protocol provided by the manufacturer (SureSelect XT Target Enrichment, Agilent Technologies). Briefly, SureSelect Block Mix were added to library DNAs, and were heated at 95°C for 5 min, followed by an incubation at 65°C for more than 5 min. Subsequently, Capture Library Hybridization Mix, containing Custom Design SureSelect XT Capture Libraries and SureSelect RNase Block, was mixed with library DNAs, and was incubated at 65°C for 24 hours. To capture the hybridized DNA, MyOne Streptavidin T1 Dynabeads (Life Technologies) were mixed with hybridized library DNAs. After incubation at 25°C for 30 min with mixing at 1400 rpm, beads were washed with SureSelect Wash Buffer according to SureSelect XT Target Enrichment protocol. After washing, 30 μl H2O was added to beads. Post-capture PCR was carried out using DNAs bound to beads via biotinylated-RNAs (16 cycles using NEXTflex primer mix2 (BIOO SCIENTIFIC)). Captured MNase-seq libraries were paired-end sequenced on Illumina HiSeq 2500.

Processing and analysis of MNase-seq data
Paired-end Mnase-seq reads were trimmed of adapter sequences (cutadapt 1.8.3) using Trim Galiore tool (Trim Galore 0.5.0) together with a quality control step (FastQC v0.11.7). Clean reads were aligned on viral HIV DNA pNL4-3 sequence (GenBank: AF324493.2) using Burrows-Wheeler Aligner (BWA 0.7.15, http://bio-bwa.sourceforge.net/). The aligned reads were filtered and sorted using Samtools 1.8. Duplicated reads (e.g. due to PCR) were removed using picard tools (picard-2.18.2). When visualizing reads onto LTRs, multiple alignment option was chosen to keep reads aligning onto such repeated sequences. In this instance, multiple reads were normalized to the number of copies of viral genome detected in cells. For specific detection of nucleosome profiles, alignment were performed using Deeptools bamCoverage (deeptools-3.0.2, python-3. (i.e. evaluating differences weighted by the mean signal) to compare nucleosome-centered signals. Differences between conditions (2x2 replicates) were validated statistically using the NormR R package designed for differential analysis of sequencing data. The diffR function was applied to compare samples coverage with the following configuration: "countConfiguration<-countConfigPairedEnd(binsize = 50 ,mapq = 0, shift = 0, midpoint=T, tlenFilter = c(130, 200))" allowing us to detect systematically differences at the nucleosome scale. Differences detected were filtered with a FDR < 1e-3. Differential heatmaps were then filtered to represent only loci where decreases or increases were significantly validated with a FDR < 1e-3.

Data and software availability
The accession number for the data reported in this paper is GEO (GSE139557(GSE135551 and GSE139556)). Software and Algorithms were listed in SI Appendix table S4.             (16)