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