The biomolecular mechanisms controlling latent HIV-1 infection, despite their importance for the development of a cure for HIV-1 infection, are only partially understood. For example, ex vivo studies have recently shown that T cell activation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, but the molecular biology of this phenomenon is unclear. We demonstrate that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of T cell receptor (TCR)/CD3 activation-inert latently infected T cells. RNA-level analysis identified extensive transcriptomic differences between uninfected, TCR/CD3 activation-responsive and -inert T cells, but did not reveal a gene expression signature that could functionally explain TCR/CD3 signaling inertness. Network analysis suggested a largely stochastic nature of these gene expression changes (transcriptomic noise), raising the possibility that widespread gene dysregulation could provide a reactivation threshold by impairing overall signal transduction efficacy. Indeed, compounds that are known to induce genetic noise, such as HDAC inhibitors impeded the ability of TCR/CD3 activation to trigger HIV-1 reactivation. Unlike for transcriptomic data, pathway enrichment analysis based on phospho-proteomic data directly identified an altered TCR signaling motif. Network analysis of this data set identified drug targets that would promote TCR/CD3-mediated HIV-1 reactivation in the fraction of otherwise TCR/CD3-reactivation inert latently HIV-1 infected T cells, regardless of whether the latency models were based on T cell lines or primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell responsiveness prior to the use of any activating stimulus will likely have to be an element of future HIV-1 cure therapies.

The development of therapies to eliminate the latent HIV-1 reservoir is hampered by our incomplete understanding of the biomolecular mechanism governing HIV-1 latency. To further complicate matters, recent single cell RNA-seq studies reported extensive heterogeneity between latently HIV-1-infected primary T cells, implying that latent HIV-1 infection can persist in greatly differing host cell environments. We here show that transcriptomic heterogeneity is also found between latently infected T cell lines, which allowed us to study the underlying mechanisms of intercell heterogeneity at high signal resolution. Latently infected T cells exhibited a de-differentiated phenotype, characterized by the loss of T cell-specific markers and gene regulation profiles reminiscent of hematopoietic stem cells (HSC). These changes had functional consequences. As reported for stem cells, latently HIV-1 infected T cells efficiently forced lentiviral superinfections into a latent state and favored glycolysis. As a result, metabolic reprogramming or cell re-differentiation destabilized latent infection. Guided by these findings, data-mining of single cell RNA-seq data of latently HIV-1 infected primary T cells from patients revealed the presence of similar dedifferentiation motifs. >20% of the highly detectable genes that were differentially regulated in latently infected cells were associated with hematopoietic lineage development (e.g. HUWE1, IRF4, PRDM1, BATF3, TOX, ID2, IKZF3, CDK6) or were hematopoietic markers (SRGN; hematopoietic proteoglycan core protein). The data add to evidence that the biomolecular phenotype of latently HIV-1 infected cells differs from normal T cells and strategies to address their differential phenotype need to be considered in the design of therapeutic cure interventions. IMPORTANCE HIV-1 persists in a latent reservoir in memory CD4 T cells for the lifetime of a patient. Understanding the biomolecular mechanisms used by the host cells to suppress viral expression will provide essential insights required to develop curative therapeutic interventions. Unfortunately, our current understanding of these control mechanisms is still limited. By studying gene expression profiles, we demonstrated that latently HIV-1-infected T cells have a de-differentiated T cell phenotype. Software-based data integration allowed for the identification of drug targets that would re-differentiate viral host cells and, in extension, destabilize latent HIV-1 infection events. The importance of the presented data lies within the clear demonstration that HIV-1 latency is a host cell phenomenon. As such, therapeutic strategies must first restore proper host cell functionality to accomplish efficient HIV-1 reactivation.

Therapeutic attempts to eliminate the latent HIV-1 reservoir have failed, at least in part due to our incomplete biomolecular understanding of how latent HIV-1 infection is established and maintained. We here address the fundamental question of whether all lentiviruses actually possess a similar capacity to establish latent infections or whether there are differences between the lentiviral lineages driving differential latency establishment that could be exploited to develop improved latency reversal agents.

ABSTRACTAlthough the ability of HIV-1 to reside in a latent state in CD4+ T cells constitutes a critical hurdle to a curative therapy, the biomolecular mechanisms by which latent HIV-1 infection is established and maintained are only partially understood. Ex vivo studies have shown that T cell receptor/CD3 stimulation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, suggesting that parts of the T cell population hosting latent HIV-1 infection events are altered to be TCR/CD3-activation-inert. We provide experimental evidence that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of TCR/CD3 activation-inert latently infected T cells. HIV-1 induced host cell TCR/CD3 inertness is thus a conserved mechanism that contributes to the stability of latent HIV-1 infection. Proteomic and genome-wide RNA-level analysis comparing CD3-responsive and CD3-inert latently HIV-1 infected T cells, followed by software-based integration of the data into protein-protein interaction networks (PINs) suggested two phenomena to govern CD3-inertness: (i) the presence of extensive transcriptomic noise that affected the efficacy of CD3 signaling and (ii) defined changes to specific signaling pathways. Validation experiments demonstrated that compounds known to increase transcriptomic noise further diminished the ability of TCR/CD3 stimulation to trigger HIV-1 reactivation. Conversely, targeting specific central nodes in the generated PINs such as STAT3 improved the ability of TCR/CD3 activation to trigger HIV-1 reactivation in T cell lines and primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell TCR/CD3 responsiveness could enable gradual reservoir depletion without the need for therapeutic activators, driven by cognate antigen recognition.AUTHOR SUMMARYA curative therapy for HIV-1 infection will at least require the eradication of a small pool of CD4+ helper T cells in which the virus can persist in a latent state, even after years of successful antiretroviral therapy. It has been assumed that activation of these viral reservoir T cells will also reactivate the latent virus, which is a prerequisite for the destruction of these cells. Remarkably, this is not the case and following application of even the most potent stimuli that activate normal T cells through their T cell receptor, a large portion of the latent virus pool remains in a dormant state. Herein we demonstrate that a large part of latent HIV-1 infection events reside in T cells that have been rendered activation inert by the actual infection event. We provide a systemwide, biomolecular description of the changes that render latently HIV-1 infected T cells activation inert and using this description, devise pharmacologic interference strategies that render initially activation inert T cells responsive to stimulation. This in turn allows for efficient triggering of HIV-1 reactivation in a large part of the latently HIV-1 infected T cell reservoir.