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

Antiviral Activities of Dead-Box RNA Helicases

Arthropod-borne RNA viruses pose a threat to human health throughout the world, but few treatments are available. A better understanding of the interactions between viruses and host cells will enable the development of new ways to combat these infections. During entry and replication, viral RNAs are exposed to host RNA-binding proteins with diverse functions. Some of these interactions have been studied, but many remain undefined. To understand how host RNA-binding proteins contribute to cellular defenses, we sought to characterize the antiviral functions of conserved DEAD-box helicase proteins identified by targeted screening in insect and mammalian host cells. Focusing on alphavirus and flavivirus infection, we assessed the effect of siRNA silencing of DDX23, DDX24, and DDX56 on infection by a panel of RNA viruses, and used CLIP-Seq to assess binding interactions of DDX56 on CHIKV and DENV RNAs during infection of human cells. We found a specific enrichment of DDX56 binding within the CHIKV nsP4 gene encoding the viral RNA-dependent RNA polymerase, which led us to investigate the role of DDX56 on initial steps of viral RNA translation and replication as an entering CHIKV genome launches replication within a host cell. We found that depletion of DDX56 improves stability of CHIKV genomes and increases levels of viral proteins, suggesting that DDX56 is capable of exerting an antiviral effect at the earliest stages of infection. The region of the CHIKV nsP4 gene bound by DDX56 is adjacent to a known structural element, and RNA folding algorithms predict structure in the DDX56 binding region as well. Taken together, these results suggest that DDX56 control of alphavirus infection likely involves recognition of a structural element that results in the CHIKV genome being targeted for degradation rather than launching replication. In contrast, CLIP-Seq analysis of DDX56 interactions with DENV RNA revealed lower and more uniform binding along the length of the viral genome, suggesting a less specific interaction and possibly a distinct antiviral mechanism. Together, these studies highlight the sensitivity of viral RNA to cellular factors shortly after entry and uncover additional virus-host interactions contributing to the control of infection

Antiviral Activities of Dead-Box RNA Helicases

Arthropod-borne RNA viruses pose a threat to human health throughout the world, but few treatments are available. A better understanding of the interactions between viruses and host cells will enable the development of new ways to combat these infections. During entry and replication, viral RNAs are exposed to host RNA-binding proteins with diverse functions. Some of these interactions have been studied, but many remain undefined. To understand how host RNA-binding proteins contribute to cellular defenses, we sought to characterize the antiviral functions of conserved DEAD-box helicase proteins identified by targeted screening in insect and mammalian host cells. Focusing on alphavirus and flavivirus infection, we assessed the effect of siRNA silencing of DDX23, DDX24, and DDX56 on infection by a panel of RNA viruses, and used CLIP-Seq to assess binding interactions of DDX56 on CHIKV and DENV RNAs during infection of human cells. We found a specific enrichment of DDX56 binding within the CHIKV nsP4 gene encoding the viral RNA-dependent RNA polymerase, which led us to investigate the role of DDX56 on initial steps of viral RNA translation and replication as an entering CHIKV genome launches replication within a host cell. We found that depletion of DDX56 improves stability of CHIKV genomes and increases levels of viral proteins, suggesting that DDX56 is capable of exerting an antiviral effect at the earliest stages of infection. The region of the CHIKV nsP4 gene bound by DDX56 is adjacent to a known structural element, and RNA folding algorithms predict structure in the DDX56 binding region as well. Taken together, these results suggest that DDX56 control of alphavirus infection likely involves recognition of a structural element that results in the CHIKV genome being targeted for degradation rather than launching replication. In contrast, CLIP-Seq analysis of DDX56 interactions with DENV RNA revealed lower and more uniform binding along the length of the viral genome, suggesting a less specific interaction and possibly a distinct antiviral mechanism. Together, these studies highlight the sensitivity of viral RNA to cellular factors shortly after entry and uncover additional virus-host interactions contributing to the control of infection

Antiviral Activities of Dead-Box RNA Helicases

Arthropod-borne RNA viruses pose a threat to human health throughout the world, but few treatments are available. A better understanding of the interactions between viruses and host cells will enable the development of new ways to combat these infections. During entry and replication, viral RNAs are exposed to host RNA-binding proteins with diverse functions. Some of these interactions have been studied, but many remain undefined. To understand how host RNA-binding proteins contribute to cellular defenses, we sought to characterize the antiviral functions of conserved DEAD-box helicase proteins identified by targeted screening in insect and mammalian host cells. Focusing on alphavirus and flavivirus infection, we assessed the effect of siRNA silencing of DDX23, DDX24, and DDX56 on infection by a panel of RNA viruses, and used CLIP-Seq to assess binding interactions of DDX56 on CHIKV and DENV RNAs during infection of human cells. We found a specific enrichment of DDX56 binding within the CHIKV nsP4 gene encoding the viral RNA-dependent RNA polymerase, which led us to investigate the role of DDX56 on initial steps of viral RNA translation and replication as an entering CHIKV genome launches replication within a host cell. We found that depletion of DDX56 improves stability of CHIKV genomes and increases levels of viral proteins, suggesting that DDX56 is capable of exerting an antiviral effect at the earliest stages of infection. The region of the CHIKV nsP4 gene bound by DDX56 is adjacent to a known structural element, and RNA folding algorithms predict structure in the DDX56 binding region as well. Taken together, these results suggest that DDX56 control of alphavirus infection likely involves recognition of a structural element that results in the CHIKV genome being targeted for degradation rather than launching replication. In contrast, CLIP-Seq analysis of DDX56 interactions with DENV RNA revealed lower and more uniform binding along the length of the viral genome, suggesting a less specific interaction and possibly a distinct antiviral mechanism. Together, these studies highlight the sensitivity of viral RNA to cellular factors shortly after entry and uncover additional virus-host interactions contributing to the control of infection

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

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