CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo
Fig 10
Identical HIV proviral genomes in CD4-negative progeny.
A and C. Diagrammatic representation of clonal, defective proviral genomes from the indicated donor 431000 cell types. Colored bars indicate regions of identity. Dotted lines indicate location of deletion. White space indicates region where no sequence information is available. mRNA is cell associated from part B. B, (upper panel) Flow cytometric analysis of purified CD4-negative and CD4+ populations isolated from donor 431000 PBMCs. (Lower panels) Summary graphs of HIV-1 RNA isolated from supernatant (left) and cells (right) following treatment with PMA and ionomycin. Dashed line indicates limit of detection. *p<0.05 D and E. Provirus found in CD4-negative progeny can be recovered in two independent donations separated by months. D. Flow cytometric plots showing purity of CD4-negative lineages. “Pre” indicates the cell population post CD4-bead depletion and prior to fluorescence activated cell sorting (FACS). “Post” indicates the cell populations following FACS. Numbers in the upper right corner indicate the frequency of cells in that quadrant. The frequency of CD4+ cells that were also CD3+ by gating was 0% (see also Table 5). E. Phylogenetic tree showing genetic relationships amongst amplicons from two separate donations (408 and 428). Arrow indicates location of identical 693 bp gag amplicon from CD8 cells. (The region of identity extended through the non-translated LTR region of the amplicon, which was not included in the phylogenetic analysis.) Red lines indicate identical sequences. Scale indicates nucleotide substitutions per site. 89.6, BaL, YU-2, HXB2 and NL4-3 are subtype B HIVs. 84ZR085 (84ZR) and 94UG114 (94UG) are subtype D HIV molecular clone outgroups [32]. Phylogenetic analysis was performed by maximum likelihood method using MEGA7 [33] and history was inferred based on the Hasegawa-Kishino-Yano model [34]. The tree with the highest log likelihood is shown. Abbreviations: PBMC, unfractionated peripheral blood mononuclear cells; BMMC, bone marrow mononuclear cell (column flow-through).