Irreversible Proviral Silencing in Myeloid Cells

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ABSTRACT: In the pursuit of strategies with which to cure HIV-1 infection, most of the attention has been on mechanisms that govern HIV-1 persistence in CD4+ T cell reservoirs and on approaches to limit persistence in those reservoirs. We recently provided evidence [1] that macrophages enable viral persistence in HIV-1-infected individuals on effective ART. We showed that rebound viremia from individuals undergoing an analytic treatment interruption (ATI) comprised highly macrophage-tropic viruses and we used a novel virion immunocapture method to demonstrate that those macrophage-tropic viruses in post-ATI plasma had a myeloid cell origin. Furthermore, Molecular Clock analysis indicated that macrophage-tropic viruses in plasma pre-dated the treatment interruption as opposed to having been created during the treatment interruption interval. Given these lines of evidence that myeloid cells can serve as viral reservoirs under effective ART, we set about establishing a latency model in primary macrophages. In the pursuit of this model of macrophage latency, we observed that NF-kB inhibition, originally intended to promote synchronous latency, led to irreversible loss of proviral competence- proviruses were refractory to latency reactivating agents (LRAs) even though host cell functions remained intact. On the basis of these observations, we hypothesize that the provirus is epigenetically modified under conditions where NF-kB is limiting and that sustained transcriptional activity is required to counteract epigenetic silencing processes of the host cell. In this proposal, we will assess the mechanisms governing loss of proviral competence. Aim 1: Assess correlates of proviral silencing in NF-kB-depleted macrophages. We will determine the stage in the viral replication cycle at which proviruses are rendered inactive and, through heterokaryon formation, whether dominant negative factors mediate irreversible proviral silencing by NF-kB inhibition. We will examine whether inactive proviruses exhibit evidence of epigenetic modification and methylation and whether inhibiting methylation can maintain proviral responsiveness to reactivation in the face of NF-kB inhibition. Aim 2: Examine whether the proviral silencing created under conditions of NF-kB inhibition can be extended to infected CD4+ T cells from ART-suppressed subjects and to other primary macrophage populations. We will assess whether NF-kB inhibition can irreversibly impede HIV-1 reactivation in CD4+ T cells from individuals on effective ART and whether this proviral silencing can also be achieved in macrophages that are physiologically representative of the major myeloid cell compartments including lung (alveolar macrophages), liver (Kupffer cells), and Central Nervous System (microglia). If the proposed studies are successful, they will provide the rationale for in vivo studies using established SHIV/macaque models of macrophage reservoirs and NF-kB antagonists currently in clinical use.