The identification and eradication of long-lived cellular reservoirs is necessary to cure HIV and eliminate the need for lifelong therapy. We have previously demonstrated that HIV can infect multipotent HSPCs, establishing both active and latent infections (Carter et al., 2010
). Here we demonstrate that, similar to infection of T cells, infection of multipotent HSPCs depends on CD4. However, based on the panel of Envs we tested, robust infection of primitive HSPCs capable of generating multilineage colonies in soft agar only occurs with CXCR4- or dual-tropic viruses. Blockade of CXCR4 dramatically reduced infection of multipotent hematopoietic cells by dual-tropic HIVs. In contrast, the R5-tropic HIVs we tested had only minimal infectivity in multipotent HSPCs and blockade of CCR5 had no effect on infection of multipotent cells by dual-tropic HIVs.
The simplest explanation for the inability of CCR5-bearing viruses to infect multipotent HSPCs is that CCR5 is not expressed at high enough levels to support infection. An alternative hypothesis is that engagement of R5-tropic Envs with the CCR5 chemokine receptor affects the ability of multipotent HSPC to form colonies or is toxic to the cells. This hypothesis is less likely because dual-tropic HIVs able to bind both CXCR4 and CCR5 can infect cells with a multipotent phenotype. In addition, we have detected minimal CCR5 expression and signaling in response to CCR5 ligands on human CD34+CD133+ cells.
HIV infection of multipotent HSPCs could lead to the presence of viral genomes in multiple hematopoietic lineages. However, HIV is primarily detected in myeloid and T cells, but not in B cells. This apparent enigma may be explained by the fact that active infection of HSPCs leads to the upregulation of markers of apoptosis and rapid depletion of infected cells from the culture (Carter et al., 2010
). Thus, the lack of evidence for HIV genomes in B cells in infected people may be due to the fact that active HIV infection kills early HSPCs, preventing the development of infected cells in some lineages (Carter et al., 2010
). Latent infection can also occur in HSPCs, but induction of differentiation may induce viral activation and subsequent cell death (Carter et al., 2010
We have also determined that HIV infects HSCs that are capable of stable, multilineage engraftment in irradiated NOD/SCID IL-2Rγnull
mice. All of the mice that were successfully transplanted with infected HSPCs demonstrated multilineage engraftment of infected, GFP+
human cells. These results have significant implications for viral persistence because HSCs are capable of long-term self-renewal in vivo
. Thus, latently infected HSCs would persist indefinitely, forming a long-term viral reservoir. Additionally, based on our prior results (Carter et al., 2010
), active infection can trigger cell death in multipotent HSPCs. If a sufficient number of HSPCs were infected, the subsequent death of these cells could disrupt the entire hematopoietic cascade. Over many years, this disruption could impact the function of the bone marrow. Unfortunately, the toxicity of wild type HIV precluded us from generating sufficient numbers of infected cells to test whether wild type virus from patient samples infects cells in vivo
that have the capacity to engraft. The experiments presented here were only possible with the use of replication defective HIV constructs that do not express additional cytotoxic HIV proteins following integration.
The findings in this study appear to conflict with previous reports indicating that HSCs are resistant to infection with HIV-1 as well as with lentiviral constructs pseudotyped with HIV Envs (Shen et al., 1999
; Weichold et al., 1998
; Zhang et al., 2007
). The mechanism by which HIV-1 infection of HSCs is purported to be blocked has been inconsistent: one study found that the block was solely at the level of entry due to insufficient expression of viral receptors and that VSVG-pseudotyped viral particles could efficiently infect HSCs (Shen et al. 1999
), whereas another group found that there was a post-entry block to infection mediated by p21 (Zhang et al. 2007
). The apparent difference between our findings and those of previous groups can be explained by low rates of infection (typically less than 2%) that rapidly decline over time because of the cytoxitcity of the virus. Such infection rates are too low to yield detectable infection with many of the non-flow cytometric assays used in previous studies, especially those that require that the cells be cultured for more than a couple days. The use of recently optimized culture conditions for HSPCs (Zhang et al., 2008
) has allowed us to increase infection rates in these cells due to improved health of the cells. The previously described blocks to infection likely contribute to the low infection rates that we observe in HSPCs, but importantly, we show that these blocks are not absolute and that X4-tropic HIVs can infect HSCs at a low but significant rate.
In sum, we have shown that multipotent HSPCs and HSCs can be infected by HIV and that this infection is primarily accomplished by CXCR4-tropic HIVs. The infection and destruction of multipotent HSPCs may contribute to the more rapid decline in CD4 counts associated with CXCR4-tropic HIV isolate emergence. Alternatively, as infected HSCs could create an extremely long-lived reservoir of virus, preferential infection of these cells by CXCR4-tropic virus could provide a reservoir for the emergence of CXCR4-tropic isolates late in disease: as other viral reservoirs are depleted, CXCR4-tropic virus from the HSC and HSPC reservoir could begin to predominate. In addition, our demonstration that HIV can infect cells capable of stably engrafting for months in the xenograft model indicates that HIV can infect HSCs that are capable of self-renewal and, if the integrated viral genome is latent, that it can be maintained and even expanded by cell division.
Based on these data, there should be a renewed focus on primitive hematopoietic progenitors as an important reservoir for HIV that will require eradication to improve the treatment of HIV-infected people. Our data suggesting that a subset of HSCs and other primitive hematopoietic progenitors could function as a latent reservoir for HIV raise the possibility that combining HIV therapies with approaches to activate HSCs might deplete this reservoir by triggering the apoptosis of infected HSCs.