Hematopoietic stem and progenitor cells are a distinct HIV reservoir that contributes to persistent viremia in suppressed patients

Long-lived reservoirs of persistent HIV are a major barrier to a cure. CD4+ hematopoietic stem and progenitor cells (HSPCs) have the capacity for lifelong survival, self-renewal, and the generation of daughter cells. Recent evidence shows that they are also susceptible to HIV infection in vitro and in vivo. Whether HSPCs harbor infectious virus or contribute to plasma virus (PV) is unknown. Here, we provide strong evidence that clusters of identical proviruses from HSPCs and their likely progeny often match residual PV. A higher proportion of these sequences match residual PV than proviral genomes from bone marrow and peripheral blood mononuclear cells that are observed only once. Furthermore, an analysis of near-full-length genomes isolated from HSPCs provides evidence that HSPCs harbor functional HIV proviral genomes that often match residual PV. These results support the conclusion that HIV-infected HSPCs form a distinct and functionally significant reservoir of persistent HIV in infected people

Nonrandom Packaging of Host RNAs in Moloney Murine Leukemia Virus

Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced env mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA(Met) were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that env mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that Ψ(−) mRNAs and some other host RNAs may be specifically excluded from assembly sites

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

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 env 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 CD4high 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

Hematopoietic Stem and Progenitor Cells Are a Distinct HIV Reservoir that Contributes to Persistent Viremia in Suppressed Patients

Summary: Long-lived reservoirs of persistent HIV are a major barrier to a cure. CD4+ hematopoietic stem and progenitor cells (HSPCs) have the capacity for lifelong survival, self-renewal, and the generation of daughter cells. Recent evidence shows that they are also susceptible to HIV infection in vitro and in vivo. Whether HSPCs harbor infectious virus or contribute to plasma virus (PV) is unknown. Here, we provide strong evidence that clusters of identical proviruses from HSPCs and their likely progeny often match residual PV. A higher proportion of these sequences match residual PV than proviral genomes from bone marrow and peripheral blood mononuclear cells that are observed only once. Furthermore, an analysis of near-full-length genomes isolated from HSPCs provides evidence that HSPCs harbor functional HIV proviral genomes that often match residual PV. These results support the conclusion that HIV-infected HSPCs form a distinct and functionally significant reservoir of persistent HIV in infected people. : HIV causes an infection that persists even when optimal therapy is used. Zaikos et al. provide evidence that HIV-infected progenitor cells from the bone marrow can amplify virus through normal cellular growth pathways in some treated people. Keywords: HIV, latency, reservoir, virus, persistence, clonal, hematopoietic, human, defective, infectiou

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

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

CD4^high HSPCs include progenitors with multi-lineage potential.

<p>A. Flow cytometric analysis of differentiation markers expressed on bone marrow HSPCs purified as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006509#ppat.1006509.g001" target="_blank">Fig 1A</a>. For the two right-most panels, numbers indicate percentage of total CD34⁺ events in each sort falling into that gate. B. Summary table of frequencies for each phenotypic gate as shown in A. Lineage outputs based upon 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>]. (Abbreviations: 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) C. Summary graphs depicting the percentage of each subset of the total CD34⁺ cells in each sort. Cells were isolated from cord blood (n = 5, circles) or bone marrow (n = 2, squares). For three experiments (2 cord blood and 1 bone marrow), lineage-positive cells were physically or analytically excluded from analysis (open symbols). Mean ± standard deviation is indicated; 2-tailed Student’s t-test (*<i>p</i><0.05, ***<i>p</i><0.001, ****<i>p</i><0.0001).</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

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