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Research Article

Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors

Sean C. Patro, Leah D. Brandt, Michael J. Bale, Elias K. Halvas, Kevin W. Joseph, Wei Shao, Xiaolin Wu, Shuang Guo, Ben Murrell, Ann Wiegand, Jonathan Spindler, Castle Raley, Christopher Hautman, Michele Sobolewski, Christine M. Fennessey, Wei-Shau Hu, Brian Luke, Jenna M. Hasson, Aurelie Niyongabo, Adam A. Capoferri, View ORCID ProfileBrandon F. Keele, Jeff Milush, Rebecca Hoh, Steven G. Deeks, Frank Maldarelli, Stephen H. Hughes, John M. Coffin, Jason W. Rausch, John W. Mellors, and Mary F. Kearney
PNAS first published November 27, 2019 https://doi.org/10.1073/pnas.1910334116
Sean C. Patro
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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  • For correspondence: sean.patro@nih.gov john.coffin@tufts.edu
Leah D. Brandt
bDepartment of Medicine, University of Pittsburgh, Pittsburgh, PA 15213;
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Michael J. Bale
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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Elias K. Halvas
bDepartment of Medicine, University of Pittsburgh, Pittsburgh, PA 15213;
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Kevin W. Joseph
bDepartment of Medicine, University of Pittsburgh, Pittsburgh, PA 15213;
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Wei Shao
cLeidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702;
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Xiaolin Wu
cLeidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702;
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Shuang Guo
cLeidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702;
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Ben Murrell
dDepartment of Microbiology, Tumor and Cell Biology, Karolinska Institute, 171 65 Stockholm, Sweden;
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Ann Wiegand
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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Jonathan Spindler
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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Castle Raley
cLeidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702;
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Christopher Hautman
cLeidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702;
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Michele Sobolewski
bDepartment of Medicine, University of Pittsburgh, Pittsburgh, PA 15213;
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Christine M. Fennessey
eAIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702;
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Wei-Shau Hu
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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Brian Luke
cLeidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702;
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Jenna M. Hasson
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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Aurelie Niyongabo
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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Adam A. Capoferri
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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Brandon F. Keele
eAIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702;
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  • ORCID record for Brandon F. Keele
Jeff Milush
fDepartment of Medicine, University of California, San Francisco, CA 94143;
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Rebecca Hoh
fDepartment of Medicine, University of California, San Francisco, CA 94143;
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Steven G. Deeks
fDepartment of Medicine, University of California, San Francisco, CA 94143;
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Frank Maldarelli
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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Stephen H. Hughes
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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John M. Coffin
gDepartment of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111;
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  • For correspondence: sean.patro@nih.gov john.coffin@tufts.edu
Jason W. Rausch
hBasic Research Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702
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John W. Mellors
bDepartment of Medicine, University of Pittsburgh, Pittsburgh, PA 15213;
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Mary F. Kearney
aHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702;
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  1. Contributed by John M. Coffin, October 18, 2019 (sent for review June 27, 2019; reviewed by Cynthia A. Derdeyn, James A. Hoxie, and Morgane Rolland)

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Significance

To develop a cure for HIV-1, it is necessary to understand how infected cells persist despite treatment. Integrated HIV DNA (proviruses) can be distinguished by their sites of integration into the host genome and by their proviral sequence. We applied multiple-displacement amplification (MDA) to single proviruses isolated from blood and lymph nodes to determine their integration sites and full-length sequences. We found that identical subgenomic HIV-1 sequences can result from either clonal expansion or from genetic bottlenecks that occurred with transmission or with the emergence of drug resistance mutations. Furthermore, we show this MDA approach can be used to infer intact ancestral HIV-1 sequences from the archives of defective ones, providing an approach for investigations of HIV-1 evolution in vivo.

Abstract

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

  • HIV persistence
  • proviral structure
  • integration site analysis

Footnotes

  • ↵1To whom correspondence may be addressed. Email: sean.patro{at}nih.gov or john.coffin{at}tufts.edu.
  • Author contributions: S.C.P., B.F.K., S.G.D., F.M., S.H.H., J.M.C., J.W.R., J.W.M., and M.F.K. designed research; S.C.P., L.D.B., M.J.B., E.K.H., K.W.J., X.W., S.G., A.W., J.S., C.R., C.H., M.S., C.M.F., J.M.H., A.N., A.A.C., J.M., and R.H. performed research; S.C.P., M.J.B., K.W.J., W.S., X.W., S.G., B.M., W.-S.H., B.L., B.F.K., S.G.D., F.M., S.H.H., J.M.C., J.W.R., J.W.M., and M.F.K. analyzed data; and S.C.P., E.K.H., F.M., S.H.H., J.M.C., J.W.M., and M.F.K. wrote the paper.

  • Reviewers: C.A.D., Emory University; J.A.H., University of Pennsylvania; and M.R., US Military HIV Research Program and Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc.

  • Competing interest statement: J.W.M. is a consultant to Gilead Sciences, Merck Research Laboratories, Janssen Pharmaceuticals, and AccelevirDx, and a share option holder of Co-Crystal, Inc. B.F.K. and J.A.H. are co-authors on an October 2015 article. The remaining authors have no potential conflicts.

  • Data deposition: All sequence data reported in this paper have been deposited publicly in the GenBank database (accession nos. MK147632–MK147695, MK147878–MK148024, and MN691959–MN692189). All integration site data reported in this paper have been deposited in the Retroviral Integration Database (https://rid.ncifcrf.gov/), and all NFL genome sequences reported in this paper have been deposited in the Proviral Sequence Database (https://psd.cancer.gov/) using the PubMed ID of this paper.

  • This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1910334116/-/DCSupplemental.

Published under the PNAS license.

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Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors
Sean C. Patro, Leah D. Brandt, Michael J. Bale, Elias K. Halvas, Kevin W. Joseph, Wei Shao, Xiaolin Wu, Shuang Guo, Ben Murrell, Ann Wiegand, Jonathan Spindler, Castle Raley, Christopher Hautman, Michele Sobolewski, Christine M. Fennessey, Wei-Shau Hu, Brian Luke, Jenna M. Hasson, Aurelie Niyongabo, Adam A. Capoferri, Brandon F. Keele, Jeff Milush, Rebecca Hoh, Steven G. Deeks, Frank Maldarelli, Stephen H. Hughes, John M. Coffin, Jason W. Rausch, John W. Mellors, Mary F. Kearney
Proceedings of the National Academy of Sciences Nov 2019, 201910334; DOI: 10.1073/pnas.1910334116

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Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors
Sean C. Patro, Leah D. Brandt, Michael J. Bale, Elias K. Halvas, Kevin W. Joseph, Wei Shao, Xiaolin Wu, Shuang Guo, Ben Murrell, Ann Wiegand, Jonathan Spindler, Castle Raley, Christopher Hautman, Michele Sobolewski, Christine M. Fennessey, Wei-Shau Hu, Brian Luke, Jenna M. Hasson, Aurelie Niyongabo, Adam A. Capoferri, Brandon F. Keele, Jeff Milush, Rebecca Hoh, Steven G. Deeks, Frank Maldarelli, Stephen H. Hughes, John M. Coffin, Jason W. Rausch, John W. Mellors, Mary F. Kearney
Proceedings of the National Academy of Sciences Nov 2019, 201910334; DOI: 10.1073/pnas.1910334116
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