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Droplet-microfluidics-assisted sequencing of HIV proviruses and their integration sites in cells from people on antiretroviral therapy

Nature Biomedical Engineering volume 6, pages 1004–1012 (2022)Cite this article

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Abstract

The human immunodeficiency virus (HIV) integrates its genome into that of infected cells and may enter an inactive state of reversible latency that cannot be targeted using antiretroviral therapy. Sequencing such a provirus and the adjacent host junctions in individual cells may elucidate the mechanisms of the persistence of infected cells, but this is difficult owing to the 150-million-fold higher amount of background human DNA. Here we show that full-length proviruses connected to their contiguous HIV–host DNA junctions can be assembled via a high-throughput microfluidic assay where droplet-based whole-genome amplification of HIV DNA in its native context is followed by a polymerase chain reaction (PCR) to tag droplets containing proviruses for sequencing. We assayed infected cells from people with HIV receiving suppressive antiretroviral therapy, resulting in the detection and sequencing of paired proviral genomes and integration sites, 90% of which were not recovered by commonly used nested-PCR methods. The sequencing of individual proviral genomes with their integration sites could improve the genetic analysis of persistent HIV-infected cell reservoirs.

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Fig. 1: Application of SIP-seq to ART-treated individuals.
Fig. 2: Demonstration and validation of SIP-seq with HIV-infected cell line samples.
Fig. 3: SIP-seq of HIV in ART-treated participant CD4+ T cells after clonal expansion.
Fig. 4: SIP-seq of HIV proviruses and flanking sequences from CD4+ cells directly from infected individuals.

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Data availability

The sequence data have been deposited in the Sequencing Read Archive under BioProject accession number PRJCA006195. All other data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

Code availability

Custom scripts and functions are available at https://github.com/abateLab.

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Acknowledgements

This work was supported by a Chan Zuckerberg Biohub grant to A.R.A., a National Institutes of Health grant (R01 HG008978) to A.R.A., a National Institutes of Health grant (U01 AI129206) to A.R.A. and E.A.B. and National Institutes of Health grants (R01 AI125026 and R33 AI122361) to J.I.M. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

  1. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA

    Chen Sun, Leqian Liu, Xiangpeng Li, Yifan Liu, Peng Xu & Adam R. Abate

  2. Virus Persistence and Dynamics Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Liliana Pérez & Eli A. Boritz

  3. Department of Microbiology, University of Washington, Seattle, WA, USA

    James I. Mullins

  4. Department of Medicine, University of Washington, Seattle, WA, USA

    James I. Mullins

  5. Department of Global Health, University of Washington, Seattle, WA, USA

    James I. Mullins

  6. Department of Laboratory Medicine, University of Washington, Seattle, WA, USA

    James I. Mullins

  7. California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, CA, USA

    Adam R. Abate

  8. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Adam R. Abate

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Contributions

C.S. and A.R.A. conceived the project. C.S., L.L., X.L., Y.L. and P.X. performed the experiments. C.S. sequenced the samples and analysed the data. C.S. and A.R.A. wrote the initial draft of the manuscript. L.P. assisted with patient sample processing. J.I.M. and E.A.B. revised the manuscript. All authors read, reviewed and approved the manuscript.

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Correspondence to Adam R. Abate.

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Nature Biomedical Engineering thanks Angela Ciuffi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Source data

Source Data for Fig. 4

Sequences to generate the phylogenetic trees in Fig. 4, and the chimaeric read information to determine the integration sites.

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Sun, C., Liu, L., Pérez, L. et al. Droplet-microfluidics-assisted sequencing of HIV proviruses and their integration sites in cells from people on antiretroviral therapy. Nat. Biomed. Eng 6, 1004–1012 (2022). https://doi.org/10.1038/s41551-022-00864-8

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