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Position effects influence HIV latency reversal


The main obstacle to curing HIV is the presence of latent proviruses in the bodies of infected patients. The partial success of reactivation therapies suggests that the genomic context of integrated proviruses can interfere with treatment. Here we developed a method called Barcoded HIV ensembles (B-HIVE) to map the chromosomal locations of thousands of individual proviruses while tracking their transcriptional activities in an infected cell population. B-HIVE revealed that, in Jurkat cells, the expression of HIV is strongest close to endogenous enhancers. The insertion site also affects the response to latency-reversing agents, because we found that phytohemagglutinin and vorinostat reactivated proviruses inserted at distinct genomic locations. From these results, we propose that combinations of drugs targeting all areas of the genome will be most effective. Overall, our data suggest that the insertion context of HIV is a critical determinant of the viral response to reactivation therapies.

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Figure 1: Principle of B-HIVE technology.
Figure 2: Insertion landscapes of barcoded viruses.
Figure 3: Quantification of the expression of individual barcoded viruses by B-HIVE.
Figure 4: Location of latent proviruses.
Figure 5: Effects of latency-reversing agents on individual proviruses.
Figure 6: Model for the differential effect of latency-reversing agents.

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We would like to thank A. Jordan (IBMB, Barcelona) for providing research material, P. Stienen and L. Carey for their critical feedback on the manuscript and the genomics core facility of the CRG for their technical support. This research was supported by the Government of Catalonia and the Spanish Ministry of Economy and Competitiveness (Plan Nacional BFU2012-37168, Centro de Excelencia Severo Ochoa 2013–2017 SEV-2012-0208). J.P.M. and A.M. were supported by a grant from the Spanish Ministry of Economy and Competitiveness and FEDER (SAF2013-46077-R). E.Z. and G.J.F. are supported by the European Research Council (Synergy Grant 609989).

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Authors and Affiliations



Conceptualization, G.F.; methodology, H.C. and J.M.; software, G.F. and E.Z., formal analysis, G.F., investigation, G.F., H.C., E.Z. and J.M.; resources, G.F.; data curation, E.Z.; writing of original draft manuscript, G.F., H.C. and J.M.; writing, manuscript review and editing, E.Z. and A.M.; visualization, G.F., H.C. and E.Z.; supervision, G.F., project administration, G.F. and A.M.; funding acquisition, G.F. and A.M.

Corresponding author

Correspondence to Guillaume J Filion.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Barcoding PCR and validation of barcoded HIV library.

(A) Structure of the primers with their position on the construct. The forward primer contains a stretch of 20 random nucleotides at the 5’ end that will constitute the barcode, followed by a T7 promoter. The reverse primer contains the sequence of the Illumina sequencing primer PE1.0 at the 5’ end. Both primers are phosphorylated to allow the ligation of the PCR products. (B) Schematic of the reaction. The divergent primers amplify the whole backbone. Because the random nucleotides are different between primers, half of the products contain a non-double stranded end. The T4 polymerase removes the 3’ overhangs and generates blunt products by using the barcode as a template. Finally, the blunt products are self ligated. (C) Structure of the HIV construct after the barcoding PCR. The net result is the addition of a 20 nucleotide barcode that is unique for every ligation product, flanked by the Illumina primer PE1.0 and the T7 promoter. This design has two purposes: it allows sequencing PCR and RT-PCR products directly without additional steps of library preparation, and it also allows quantifying barcode abundance by T7 PCR (see Figure S4). (D) 10 clones randomly selected in a barcoded library were confirmed by PCR. Only the clones and barcoded HIV library containing an inserted product showed a 566 bp PCR product. (E) 10 clones were validated by Sanger sequencing. Each clone contained a T7 promoter (nucleotides with red background), a 17-22 nucleotide barcode and the sequence of the Illumina PE1.0 primer (nucleotides with blue background). Clone #4 could not be sequenced.

Supplementary Figure 2 Efficiency of two independent HIVBCD infections in Jurkat cells and FACS isolation of GFP(+) and GFP(–) infected cells.

(A) Infection efficiency of non-barcoded minimal HIV construct derived from plasmid pEV731 (open bar) and barcoded HIV (HIVBCD) in two independent infections (Rep1 and Rep2) in Jurkat cells were checked by measuring GFP expression 48 hours post infection by FACS analysis. The success of infection is similar with and without barcode. Representative FACS profiles for sorting GFP(+) cells 4 days post infection (B to E), and for separating GFP(+) from GFP(-) cells 21 days post infection (F to I). (B and F) Jurkat T cells were selected by FSC-A versus SSC-A (pink line-gated region). (C and G) Singlets were selected by FSC-A versus FSC-H (pink line-gated region). (D and H) Live cells (pink line-gated region) were selected by DAPI negative on a SSC-A versus DAPI-A (wavelength: 450/40). (E and I) GFP(+) cells (4 days post infection) as well as GFP(+) and GFP(-) cells (21 days post infection) were acquired by GFP fluorescence (wavelength: 525/50) versus autofluorescence (AF, wavelength: 585/15).

Supplementary Figure 3 Validation of the mapping step of B-HIVE.

(A) Expected results from genomic DNA digested by BplI and HpyCH4III. The size of digested products is expected to lie between 0.5 and 1 kb. Lanes 1, 2 and 3: before BplI and HpyCH4lll digestion; lanes 4, 5 and 6: after BplI and HpyCH4lll digestion. Lanes 1 and 4: uninfected cells; lanes 2 and 5: genomic DNA from cells infected by non-barcoded HIV; lanes 3 and 6: genomic DNA from cells infected by HIVBCD. (B) Expected results from the preparation of samples from B-HIVE. 2.0% (wt/vol) agarose gel displaying a PCR smear corresponding to different integrations in cells infected by HIVBCD (lane 12). Uninfected cells (lane 10), cells infected by non-barcoded HIV (lane 11) and no ligase controls (lanes 7, 8 and 9) did not yield any PCR product. (C) A PCR smear after the first inverse PCR was confirmed by Sanger sequencing. Nucleotides with blue background represent the Illumina sequencing primer. Nucleotides with red background represent the sequence of a T7 promoter. Nucleotides with yellow background represent a barcode. Nucleotides with grey background present sequence from a HIV vector. The sequence after the ligation point was confirmed to human DNA sequence from clone RP3-336H9 on chromosome 6p by NCBI BLAST ( White box: Partial of barcoded HIV 5’LTR; blue arrow: illumina universal TruSeq adaptor; multicolor box: barcode; red arrow: T7 promoter.

Supplementary Figure 4 Template-switch T7 PCR.

(A) Sketch of the key steps in template switch T7 PCR. 1. The 5-cutter restriction enzyme HpyCH4III was chosen to fragment genomic DNA between 500 bp to 1 kb. 2. In vitro ssRNA was transcribed from the T7 promoter inserted next to the barcode. Template switch reverse transcription was further performed with the RT primer annealed on the HIV 5’LTR together with the template switching oligonucleotide (TSO). 3. A primer annealing to Illumina PE1.0 and the TSO were used for DNA amplification. (B) Left: in vitro transcribed ssRNA was validated by RT-PCR. Only the reaction containing reverse transcriptase yielded a correct 198 bp RT-PCR product; no PCR product was detected from the reaction without reverse transcriptase. Right: only the reaction containing reverse transcriptase yielded a correct 105 bp PCR product after T7 PCR was detected. (C) The scatter plot shows individual HIV expression measured by B-HIVE against expression measured by T7 PCR (a.u: arbitrary units).

Supplementary Figure 5 Expression of HIV versus expression of endogenous genes.

Scatter plot where each dot represents a provirus inserted in a protein-coding gene. The X axis shows the expression of the gene measured in transcripts per million (tpm) and the Y axis shows the expression of barcoded HIV proviruses measured by B-HIVE. The expression of HIV does not show any dependence on the expression of the endogenous genes (Pearson correlation 0.07, P = 0.005).

Supplementary Figure 6 Drug treatment of the nonlatent GFP(+) population and comparison of drug activities for all HIV insertions.

(A) Left: evolution of the percentage of GFP(+) cells in the non latent population upon PHA, VOR or control DMSO treatment. Dotted lines correspond to experiments where the drug is removed after 24 hours. The percentage remains high throughout the whole time period of 10 days. Right: fluorescence intensity of the GFP(+) cells. (B) Quantification of viral copy number in the non latent population for two independent infections (Rep1 and Rep2). qPCR quantification of viral copies using hHBB as a reference, average of four values. Bar pairs show the value at day 1 next to the value at day 10. The error bars show the estimated standard deviation of the measurements. The treatment is indicated below the bars with the same legend as in panel (A). (C) Each panel is a scatter plot representing the mRNA tag counts of the same provirus (average of two replicates) in different conditions (PHA, VOR or DMSO). Left: first independent infection; Right: second independent infection. The expression of the proviruses is similar when they receive the same treatment, but it is variable when the treatment is different.

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Supplementary Figures 1–6 and Supplementary Tables 1 and 2 (PDF 1482 kb)

Supplementary Data Set 1

Feature summary of barcoded proviruses in Jurkat cells (TXT 278 kb)

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Chen, HC., Martinez, J., Zorita, E. et al. Position effects influence HIV latency reversal. Nat Struct Mol Biol 24, 47–54 (2017).

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