CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo

<div><p>Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified <i>env</i> alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4^high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.</p></div

Evidence for transmission of proviral genomes from multipotent CD4⁺ HSPCs to differentiated peripheral blood cells.

<p>A. Flow cytometric plots showing purity of CD4-negative lineages containing provirus identical to HSPC-derived provirus. “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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.t005" target="_blank">Table 5</a>). B and C. Phylogenetic trees showing genetic relationships amongst amplicons. HIV RNA shown is cell-associated (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.g010" target="_blank">Fig 10B</a>). Arrows indicate location of identical amplicons shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.g010" target="_blank">Fig 10</a>. Red lines indicate identical sequences. Scale indicates nucleotide substitutions per site. ACH2, 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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.ref032" target="_blank">32</a>]. Phylogenetic analysis was performed by maximum likelihood method using MEGA7[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.ref033" target="_blank">33</a>] and history was inferred based on the Hasegawa-Kishino-Yano model [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.ref034" target="_blank">34</a>]. The tree with the highest log likelihood is shown. Abbreviations: PBMC, unfractionated peripheral blood mononuclear cells; BMMC, bone marrow mononuclear cell (column flow-through).</p

Summary of donor <i>env</i> amplicons in Sort 1 and 2 HSPCs.

<p>Summary of donor <i>env</i> amplicons in Sort 1 and 2 HSPCs.</p

Analysis of <i>env</i> amplicons isolated from HIV⁺ donors.

<p>Analysis of <i>env</i> amplicons isolated from HIV⁺ donors.</p

Summary of Env phenotypes with V3 regions matching HSPC-associated <i>env</i> sequences.

<p>Summary of Env phenotypes with V3 regions matching HSPC-associated <i>env</i> sequences.</p

Targeting of intermediate progenitors by CCR5-tropic Envs is a conserved property extending to a transmitted/founder virus.

<p>A. Schematic of HIV-7/SF-GFP construct and HIV envelope plasmid used to construct pseudotyped viruses used in C-E. B. Summary table of envelope proteins used to pseudotype HIV-7/SF-GFP virus. C. Representative flow plots of cord blood-derived CD133-sorted cells expanded for four days, transduced with the indicated virus and harvested 3 days post-infection for flow cytometric analysis. In each right panel, GFP⁺ cells were overlaid onto plots of the total cell population and the percentage of GFP⁺ cells in the CD133^high and CD133^low regions is indicated. Gates were determined based on isotype control antibody staining (top panel). D. Summary graph of CD133 MFI for experiments performed as in C. Results are compiled from 11 cord blood experiments. Mean ± standard deviation is indicated; <i>n</i>≥3 for each envelope. 2-tailed Student’s t-test indicates significance for each HIV envelope with respect to VSV-G (**<i>p</i> < 0.01,***<i>p</i> < 0.001, ****<i>p</i> < 0.0001). E. Data from (D) compiled by tropism. Mean ± standard deviation is indicated; one-way ANOVA, <i>p</i> = 0.0002, with Tukey’s Multiple Comparisons Test indicated (**<i>p</i> < 0.01 and ***<i>p</i> < 0.001).</p

Expression of HIV receptors in progenitor cells.

<p>A. Affymetrix array data of gene expression in human bone marrow stem and differentiated cell types extracted from a published data set [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.ref012" target="_blank">12</a>] accessed via the NCBI Gene Expression Omnibus database (GSE42519). Left panel, heat map representing the z-score (scaled for each column) of the log transformed gene expression data. Right panel, relative expression levels of HIV receptors in each subset. The original microarray data went through background correction, normalization, and log transformation via the RMA method. For the right panel, data was converted from log transformed to linear. B. RNA seq analysis of mouse bone marrow and differentiated cell types. Left panel, heat map representing the z-score (scaled for each column) of the log transformed gene expression data. Right panel, relative expression levels of HIV receptors in each subset. Mean ± SEM is shown. (Abbreviations: FC, fold change; HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythrocyte progenitor; PM, promyelocyte; MY, myelocyte; MM, metamyelocytes; BC, band cell; PMN, polymorphonuclear cells; GR, granulocyte; ProB, pro-B cell; PreB, pre-B cell; BM, unfractionated bone marrow. FPKM, Fragments Per Kilobase Of Exon Per Million Fragments Mapped) (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001, 2-tailed unpaired t test).</p

HIV isolation from CD4-negative lineages from a subset of ten donors.

<p>HIV isolation from CD4-negative lineages from a subset of ten donors.</p

Sort 2 HSPCs are depleted for HSCs.

<p>A. Diagrammatic representation of HSPC purification. FT, column flow-through; CB, cord blood; BMMC, bone marrow mononuclear cells. B. Representative flow cytometric analysis of HSPC samples from an HIV⁺ individual. C. Gates for each HSPC population phenotype and lineage output according to Doulatov <i>et al</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.ref009" target="_blank">9</a>]. (HSC, hematopoietic stem cell; MPP, multipotent progenitor; MLP, multilymphoid progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte/erythrocyte progenitor; GMP, granulocyte/monocyte progenitor; B-NK, B and NK cell progenitor; MDC, macrophage and dendritic cell; EMK, erythroid and megakaryocyte; G, granulocyte.) D. Representative flow cytometric analysis of differentiation markers expressed on bone marrow HSPCs purified as described in A. For the two right-most panels, numbers indicate percentage of total CD34⁺ events from each sort falling into that gate. E. Flow cytometric plot comparing relative numbers of HSCs (CD34⁺CD38^- cells that are also CD90⁺) in Sort 1 versus Sort 2. F-I. Summary graph showing the relative frequency of the indicated progenitor in each sort, <i>n</i> = 3 uninfected donors. To facilitate comparison, results were normalized to Sort 2 (F and G) or Sort 1 (H—I). Mean ± standard deviation is indicated. J. Summary plots of methylcellulose colony formation assays from three uninfected donors. Mean ± standard deviation is indicated. Colony forming unit (CFU)-E, erythroid; CFU-GM, granulocyte/macrophage and CFU-GEMM, multilineage. (*<i>p</i><0.05 and **<i>p</i><0.01, 2-tailed Student’s t-test).</p

HSPCs with greater CD4 expression are preferentially infected by HIV Envs.

<p>A. Representative flow cytometry plots and gating strategy for cord blood-derived CD133-sorted cells infected with virus containing the indicated envelope protein and harvested 3 days post-infection. Gating for CD4 was determined by the inclusion of 1% of cells stained with an isotype control antibody (gray). For GFP plots, numbers indicate the percentage of GFP⁺ events. B. Summary graphs depicting the ratio of infected cells in CD4^high versus CD4^low/- subsets of cord blood-derived HSPCs infected and analyzed as in part A. For SV16, two replicates had 0.0% infection in the CD4^low/- gate leading to an undefined ratio, so 30.0 was used as a conservative estimate of the ratio. Data from 11 uninfected cord blood experiments. Mean ± standard deviation is indicated; n≥3 for each envelope. Results were compared to infection by VSV-G pseudotyped viruses and <i>p</i> values were determined using 2-tailed Student’s t-test (***<i>p</i><0.001, ****<i>p</i><0.0001). C. Data from B but compiled by tropism. Mean ± standard deviation is indicated; one-way ANOVA, <i>p</i><0.0001, with Tukey’s Multiple Comparisons Test indicated (***<i>p</i><0.001 and ****<i>p</i><0.0001).</p