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

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 noway to l...

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Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 116; no. 51; pp. 25891 - 25899
Main Authors:	Patro, Sean C., Brandt, Leah D., Bale, Michael J., Halvas, Elias K., Joseph, Kevin W., Shao, Wei, Wu, Xiaolin, Guo, Shuang, Murrell, Ben, Wiegand, Ann, Spindler, Jonathan, Raley, Castle, Hautman, Christopher, Sobolewski, Michele, Fennessey, Christine M., Hu, Wei-Shau, Luke, Brian, Hasson, Jenna M., Niyongabo, Aurelie, Capoferri, Adam A., Keele, Brandon F., Milush, Jeff, Hoh, Rebecca, Deeks, Steven G., Maldarelli, Frank, Hughes, Stephen H., Coffin, John M., Rausch, Jason W., Mellors, John W., Kearney, Mary F.
Format:	Journal Article
Language:	English
Published:	United States National Academy of Sciences 17-12-2019
Subjects:	Anti-Retroviral Agents - therapeutic use Antiretroviral agents Antiretroviral therapy Base Sequence Biological Sciences Cell Line Cloning Deoxyribonucleic acid DNA DNA structure DNA, Viral - genetics Drug resistance Drug Resistance, Viral Gene sequencing Gene therapy Genomes HIV HIV Infections - virology HIV-1 - genetics Human immunodeficiency virus Humans Integration Leukocytes (mononuclear) Leukocytes, Mononuclear - virology Lymph nodes Lymph Nodes - virology Lymphocytes Lymphocytes T Medicin och hälsovetenskap Mutation Nucleotide sequence Peripheral blood mononuclear cells Proviruses Proviruses - genetics Reconstruction Replication Virus Integration - genetics Virus Integration - physiology Virus Replication - genetics Viruses proviral structure integration site analysis HIV persistence
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Summary:	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 noway 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 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) 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.
ISSN:	0027-8424 1091-6490 1091-6490
DOI:	10.1073/pnas.1910334116