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HIV-1 infects multipotent progenitor cells causing cell death and establishing latent cellular reservoirs - pdf of full article attached
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Nature Medicine
Published online: 7 March 2010 | doi:10.1038/nm.2109
Christoph C Carter1,2,7, Adewunmi Onafuwa-Nuga3,7, Lucy A McNamara4,5, James Riddell IV3, Dale Bixby3, Michael R Savona3,6 & Kathleen L Collins1,2,3,4
HIV causes a chronic infection characterized by depletion of CD4+ T lymphocytes and the development of opportunistic infections. Despite drugs that inhibit viral spread, HIV infection has been difficult to cure because of uncharacterized reservoirs of infected cells that are resistant to highly active antiretroviral therapy (HAART) and the immune response. Here we used CD34+ cells from infected people as well as in vitro studies of wild-type HIV to show infection and killing of CD34+ multipotent hematopoietic progenitor cells (HPCs). In some HPCs, we detected latent infection that stably persisted in cell culture until viral gene expression was activated by differentiation factors. A unique reporter HIV that directly detects latently infected cells in vitro confirmed the presence of distinct populations of active and latently infected HPCs. These findings have major implications for understanding HIV bone marrow pathology and the mechanisms by which HIV causes persistent infection.

Despite the host immune response and treatment with HAART, HIV causes a persistent infection. Viral persistence is due in part to latent HIV reservoirs in resting CD4+ T cells1 that do not express viral proteins but can be induced to active infection by a variety of stimuli. However, recent studies of viral genetics have revealed that additional reservoirs probably exist2.
HPCs have been considered as a possible reservoir, but it has been difficult to establish that these cells are infected by HIV3, 4, 5, 6 because they are difficult to maintain in culture, and indirect measurements of infection may be confounded by contamination with other cell types. Here we used flow cytometry and recently developed culture conditions7 that have allowed us to conclude that a proportion of HPCs become infected after exposure to HIV both in vivo and in vitro.
HIV infects and is cytotoxic to HPCs

To assess the susceptibility of HPCs to HIV, we examined intracellular expression of the HIV capsid protein Gag in purified bone marrow CD34+ cells treated with an HIV molecular clone derived from peripheral blood (89.6)8. 89.6 is a dual tropic virus that can use either CCR5 or CXCR4 as a co-receptor to enter cells. In this case, we used an envelope-deleted molecular clone (89.6∇E) complemented with 89.6 envelope (89.6∇Eenv89.6) (Fig. 1). Three days after infection, 6% of CD34+ HPCs expressed intracellular HIV Gag (Fig. 2a). Antiretroviral treatment blocked Gag expression (Fig. 2a), and experiments with five other HIVs yielded similar results (Supplementary Fig. 1a). As previously reported for HIV-infected T cells9, 10, infected CD34+ cells downmodulated major histocompatibility complex class I (Fig. 2b).
HPCs are a heterogeneous collection of cells that include multipotent HPCs and stem cells (HSCs). Multipotent HPCs have a Lin-CD34+CD133+CD38- surface phenotype, where 'Lin' represents markers of specific hematopoietic lineages. After treatment with wild-type HIV 89.6 (Fig. 1b), both Lin+ and Lin- cells expressed intracellular Gag (Fig. 2c).
A time-course analysis revealed that Gag+ cells were lost rapidly in culture (Fig. 2d and Supplementary Fig. 1b). Moreover, infected cells showed high annexin V reactivity (Fig. 2e), and a high fraction of Gag+ cells had light scatter properties of dead cells (Supplementary Fig. 1c). Cell death required active viral gene expression, as transduction of the cells with a reporter virus (Fig. 1c) pseudotyped with an HIV envelope did not result in cell loss unless the HIV long terminal repeat (LTR) actively expressed HIV genes (Supplementary Fig. 1d).
Multipotent HPCs are susceptible to HIV infection
To assess the developmental capacity of infected HPCs, we used a minimal HIV genome (HIV-7SF-GFP, Fig. 1d) pseudotyped with 89.6 Env, which 'tagged' infected cells without causing cell death. We found that a proportion of CD34+ cells were infected (GFP+) (1-6% in replicate experiments (for example, Fig. 3a; initial sort purity shown in Supplementary Fig. 2a)), and a more primitive subset of these cells (CD34+CD38-CD133+) had a similar infection rate (Fig. 3b). Infection of CD133+ HPCs purified from bone marrow yielded similar results (Supplementary Fig. 2f,g). These infection rates were comparable to those of the fraction of CD34+ cells expressing both HIV co-receptors (Supplementary Fig. 3a,b).
CD133+ HPCs from umbilical cord blood (UCB) infected with HIV-7SF-GFPenv89.6 generated GFP+ colonies of erythroid (CFU-E), myeloid (CFU-M and CFU-GM) and multilineage (CFU-GEMM) origin, indicating that HIV can infect multipotent HPCs (Fig. 3c). Quantification revealed similar numbers of total colonies from uninfected and infected cells (Fig. 3d). We obtained similar results with a full-length HIV reporter (89.6-SI∇E-SF-GFP, Fig. 1e) that did not express HIV genes because of an LTR mutation (Fig. 3e,f).
Induction of latent HIV from infected HPCs
To assess latent infection, we asked whether induction of differentiation with phorbol 2-myristate 13-acetate (PMA) induced viral gene expression. In these experiments, we used bone marrow-derived HPCs (99.5% CD34+, Supplementary Fig. 2b) infected with a replication-defective HIV (HXB-ePLAP (Fig. 1f)) pseudotyped with the VSV-G envelope (HIV HXB-ePLAPenvVSV-G). This virus expresses a marker protein, placental alkaline phosphatase (PLAP). We found that PMA treatment increased the number of cells expressing PLAP 12-fold (Fig. 4a) and resulted in more viral particle production (Supplementary Fig. 4a) than treatment of cells with DMSO alone. Bone marrow immunodepleted for CD34+ cells was not viable under these conditions (Supplementary Fig. 4b).
We found similar numbers of integrated genomes in the presence or absence of PMA (Fig. 4b), indicating that PMA-induced gene expression was not due to effects on integration. Consistent with these results, the integrase inhibitor raltegravir blocked initial infection but not PMA-induced gene expression (Supplementary Fig. 5). We obtained similar results when the replication-defective PLAP-expressing HIV was pseudotyped with a bona fide HIV envelope (HXB-ePLAPenv89.6), albeit with lower infection rates (Fig. 4c).
We infected purified bone marrow-derived HPCs (98% CD34+, Supplementary Fig. 2d) with wild-type HIV 89.6 (Fig. 1b) and cultured them with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α) to induce myeloid differentiation11. Treatment of infected HPCs with GM-CSF and TNF-α resulted in rapid release of HIV into the culture supernatant (Fig. 4d). In contrast, bone marrow mononuclear cells (BMMCs) immunodepleted for CD34+ cells did not release HIV (Fig. 4d) and rapidly died (Supplementary Fig. 4b-d,f). Flow cytometric analysis of the cells confirmed that treatment with GM-CSF and TNF-α stimulated intracellular HIV Gag expression (Fig. 4e) and that cells cultured with GM-CSF and TNF-α acquired myeloid markers (CD83) (Fig. 4f).
To assess the stability of latent HIV in HPCs, we infected CD34+ bone marrow-derived HPCs (99% pure, Supplementary Fig. 2e) with wild-type HIV 89.6. After 7 d, when the culture was uniformly Gag negative, we added GM-CSF and TNF-α to half of the cells. Addition of GM-CSF and TNF-α resulted in a resurgence of HIV gene expression compared with the untreated culture (Fig. 4g,h). We obtained similar results with a wild-type virus that uses only CXCR4, although, as expected, there was less viral spread in the differentiated myeloid cells (Supplementary Fig. 6b). The spread of infection in the culture was inhibited by treatment with the antiretroviral drug raltegravir, and supernatant from infected cells could be used to infect T cell lines (Supplementary Fig. 6).
Direct detection of latency
To detect latent infection in situ without inducing changes in the infected cells, we developed a new latency reporter virus (HIV 89.6-∇E-SF-GFP (Fig. 1c)) that expresses GFP independently of the HIV LTR. Infection of T cells with HIV 89.6-∇E-SF-GFPenv89.6 yields some cells expressing Gag and others expressing only GFP (Fig. 5a). Confirming that GFP+Gag- cells were latently infected, we found that CD4 downmodulation, which occurs only when HIV Nef, Vpu or Env is expressed12, occurred in Gag+ but not GFP+Gag- cells (Fig. 5a). In contrast, when we infected cells with a virus that expresses GFP from the HIV LTR (89.6-∇E-IRES-GFP, Fig. 1g), the GFP-expressing cells downmodulated CD4 (Fig. 5a). We obtained similar results with peripheral blood mononuclear cells infected with 89.6∇E-SF-GFPenv89.6 (Fig. 5b). Moreover, PMA and ionomycin treatment of Jurkat cells infected with the reporter virus increased Gag+ cell frequency and lowered GFP+Gag- cell frequency (Fig. 5c).
We observed separate populations of Gag+ and GFP+ cells in UCB-derived CD34+ HPCs infected with the latency reporter virus, indicating that active and latent infection occurred in this cell type (Fig. 5d). In culture, the Gag+ cells were rapidly lost, whereas the GFP+Gag- \cells persisted for at least 20 d (Fig. 5e and Supplementary Fig. 1d). Analysis of these cells revealed that many had a cell surface phenotype consistent with primitive HPCs (CD34+Lin- or CD34+CD38-) (Fig. 5f).
Evidence that CD34+ bone marrow cells are infected in vivo
We obtained samples from HIV-infected people with high viral loads (donors 1-6, Supplementary Table 1) and found that we could detect Gag+CD34+ cells in three of six freshly isolated samples (Fig. 6a and Supplementary Table 1). When we cultured the cells in GM-CSF and TNF-α, we could detect Gag expression in samples from all six donors (Fig. 6b,c). In contrast, donor BMMCs specifically depleted of CD34+ cells did not express Gag after culturing (Fig. 6c,d). The addition of the anti-HIV drug raltegravir, which inhibits new in vitro infection in T cells (Supplementary Fig. 7), partially suppressed the induction of Gag expression (Fig. 6c,d), confirming that a component of the infection we observed was the result of viral spread. We obtained similar results from a donor (number seven) who had undetectable viral loads for 2 years (Fig. 6e and Supplementary Table1).
Using a real-time PCR assay for integrated HIV DNA, we detected viral genomes in freshly isolated CD34+ cells from four of nine donors on HAART with undetectable viral loads for longer than 6 months (44%) (Fig. 6f,g). In these donors, we detected 40 (donor 7), 3.1 (donor 12), 39 (donor 14) and 2.5 (donor 15) HIV genomes per 10,000 CD34+ cells. We detected HIV genomes in BMMCs immunodepleted of CD34+ cells only for donor 12, for whom we detected1.2 HIV genomes per 10,000 CD34- cells. The limit of detection for this assay varied by donor but was approximately 1 genome per 10,000 cells, owing to the limited number of CD34+ cells obtained from each donor. Thus, it is likely that the proportion of donors in which we detected HIV genomes underestimates the percentage of HIV+, HAART-treated individuals harboring integrated HIV genomes in CD34+ cells.
Long-lived cellular reservoirs of latent HIV genomes are a major obstacle to viral eradication. Here we demonstrate that HIV can infect hematopoietic progenitor cells in vivo and in vitro to cause an active, cytotoxic infection as well as a latent infection that can be induced to active infection by cytokine treatment.
Our finding that HIV infects HPCs with an immature phenotype has clear ramifications for HIV disease, because some of these cells may be long lived and could carry latent HIV for extended periods of time. Although further studies are needed to show that CD34+ stem cells are infected, our detection of HIV genomes in HPCs isolated from people effectively treated with HAART for more than 6 months confirms that HIV targets some long-lived HPCs. One might expect these results to predict the presence of identifiable proviral records in differentiated lineages that are known not to be susceptible. However, we show that actively infected HPCs are rapidly killed. Therefore, we expect latently infected HPCs will be killed by viral activation shortly after differentiation is induced.
Further studies are now needed to show that residual circulating virus in individuals on HAART is derived in part from HPCs, as previously demonstrated for resting memory T cells2. Additionally, studies examining the factors influencing HIV infection and latency in CD34+ cells, as well as limiting-dilution experiments to determine the fraction of proviral genomes in these cells that can be reactivated, would further understanding of this viral reservoir.
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