Proviral sequence classification in all analyzed HIV-1-infected cells from 3 study participants.

(A) Pie charts reflecting proportions of proviruses classified as intact or defective. All proviruses identified by single-genome, near-full-length, next-generation sequencing and by counting amplification products in agarose gel electrophoresis were included. The total number of individual sequences included is listed below each pie chart. (B) Pie charts indicating proportions of intact and defective proviruses detected once (classified as non-clonal) and detected multiple times (classified as clonal). The total number of proviral sequences identified by single-genome, near-full-length, next-generation sequencing is listed below each pie chart. (C) Virograms summarizing individual HIV-1 proviral sequences aligned to the HXB2 reference genome from each participant; color coding reflects the classification of proviral sequences.

Distribution of total, intact, and defective HIV-1 proviruses in individual tissue compartments.

Bar diagrams reflect relative frequencies of total (A), intact (B), and defective (C) proviruses in all analyzed tissues in study participants 1-3. The total number of individual proviral sequences determined by single-genome, near-full-length, next-generation sequencing and by counting amplification products in agarose gel electrophoresis from each tissue site of each participant is listed aside each bar. The red bars reflect samples with detectable proviral sequences; grey bars reflect samples at limit of detection for proviral sequences, calculated as 0.5 (single genome near–full-length PCR) copies per maximum number of cells tested without target identification (see Materials and Methods for details). N.d. (not done) indicates that the samples were not available from the indicated tissue sites.

Dissemination of HIV-1-infected cells across multiple anatomical tissues in participants 1 and 2.

(A and B): Circular maximum likelihood phylogenetic trees (A) and circus plots (B) of intact proviral sequences from participant 1 (P1) and participant 2 (P2). HXB2, reference HIV-1 sequence. Color coding reflects tissue origins. Each symbol reflects one intact provirus. Clonal intact sequences, defined by complete sequence identity, are indicated by blue arches in (A) and by internal connections in (B). (C) Circos plots reflecting the clonality of defective proviral sequences from participant 1 (left panel) and participant 2 (right panel). Each symbol reflects one defective provirus. Clonal sequences, defined by complete sequence identity, are highlighted. Color-coded arches around the plots indicate types of defects in HIV-1 genomes.

Dissemination of HIV-1-infected cells across CNS tissues.

(A, C, and E) Circular maximum likelihood phylogenetic trees of all proviral sequences derived from CNS tissues of the 3 study participants (A, participant 1; C, participant 2; E, participant 3). Color coding reflects tissue origins. Clonal sequences, defined by complete sequence identity, are indicated by blue arches. (B, D, and F) Circos plot reflecting the clonality of all proviral sequences isolated from CNS tissues of 3 participants (B, participant1; D, participant 2; F, participant 3). Each symbol reflects one provirus. Clonal sequences, defined by complete sequence identity, are highlighted. Color-coded arches around the plots indicate types of proviral sequences.

HIV-1 tropism analysis of proviral sequences.

(A) Pie charts indicating the proportions of all proviruses from each participant with CCR5-tropic or non– CCR5/CXCR4-tropic V3 envelope sequences are shown. The total number of proviral sequences included in this analysis is listed below each pie chart. (B) The proportions of proviruses with CCR5-tropic or non-CCR5/CXCR4-tropic V3 env sequences in each tissue from participant 2 are shown. (C) Pie charts indicating the proportions of all non– CCR5-tropic proviruses from participant 2 are shown. Color coding reflects tissue origins. The total number of analyzed proviral sequences is listed below the pie chart. HIV-1 tropism was computationally inferred using Geno2pheno (https://coreceptor.geno2pheno.org/). HIV-1 tropism was classified as “CCR5” if the false-positive rate (FPR) predicted by Geno2pheno was >2% and “CXCR4” if FPR was <2%.