Volume 21, Issue 3, Pages (October 2017)

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Volume 21, Issue 3, Pages 813-822 (October 2017) Identification of Genetically Intact HIV-1 Proviruses in Specific CD4+ T Cells from Effectively Treated Participants Bonnie Hiener, Bethany A. Horsburgh, John-Sebastian Eden, Kirston Barton, Timothy E. Schlub, Eunok Lee, Susanne von Stockenstrom, Lina Odevall, Jeffrey M. Milush, Teri Liegler, Elizabeth Sinclair, Rebecca Hoh, Eli A. Boritz, Daniel Douek, Rémi Fromentin, Nicolas Chomont, Steven G. Deeks, Frederick M. Hecht, Sarah Palmer Cell Reports Volume 21, Issue 3, Pages 813-822 (October 2017) DOI: 10.1016/j.celrep.2017.09.081 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 813-822DOI: (10.1016/j.celrep.2017.09.081) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Full-Length Individual Proviral Sequencing Assay (A) Primer binding sites in HIV-1 5′ and 3′ LTR regions used by full-length individual proviral sequencing (FLIPS) to amplify full-length (defective and intact) HIV-1 proviruses via nested PCR. (B) Process used to identify genetically intact and defective de novo assembled proviruses. (C) Percentage of defective and intact proviruses within the total pool of sequenced proviruses. (D) Percentage of intact proviruses sequenced for each participant. (E) Percentage of PCR reactions positive for amplified product with increasing copies of pWT/BaL. Data are represented as mean ± SD (n = 2). Cell Reports 2017 21, 813-822DOI: (10.1016/j.celrep.2017.09.081) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Comparison of Intact Proviruses between Cell Subsets (A) Number of intact proviruses per million cells in each cell subset. (B) Proportion of each cell subset to the total pool of CD4+ T cells. (C) Contribution of each CD4+ T cell subset to the overall intact reservoir. Data are represented as mean ± confidence interval (CI) for all graphs. Confidence intervals are calculated from one-sample t tests, except for TCM in (A), whose confidence interval upper bound is calculated from the product of the mean proportion of HIV+ cells in the TCM subset, and the upper bound of the confidence interval for the proportion of proviruses that are intact for the TCM subset. See also Figure S1. Cell Reports 2017 21, 813-822DOI: (10.1016/j.celrep.2017.09.081) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Representative Phylogenetic Tree of HIV-1 Sequences from a Participant on Long-Term ART Participant 2026. Horizontal lines adjacent to tips represent the individual contig sequence aligned to HXB2. Red asterisks denote intact sequences; black box indicates identical intact sequences. See also Data S1A–S1E. Cell Reports 2017 21, 813-822DOI: (10.1016/j.celrep.2017.09.081) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Comparison of FLIPS to Other Assays (A) Frequency of cells containing either intact or integrated HIV-1 DNA. (B) Number of intact proviruses identified using the FLIPS assay compared to simulated SPS of gag-pol and env regions. Data are represented as mean ± confidence interval (CI) for all graphs. Cell Reports 2017 21, 813-822DOI: (10.1016/j.celrep.2017.09.081) Copyright © 2017 The Author(s) Terms and Conditions