Lentiviral Nef Proteins Differentially Govern the Establishment of Viral Latency.

Despite the clinical importance of latent human immunodeficiency virus type 1 (HIV-1) infection, our understanding of the biomolecular processes involved in HIV-1 latency control is still limited. This study was designed to address whether interactions between viral proteins, specifically HIV Nef, and the host cell could affect latency establishment. The study was driven by three reported observations. First, early reports suggested that human immunodeficiency virus type 2 (HIV-2) infection in patients produces a lower viral RNA/DNA ratio than HIV-1 infection, potentially indicating an increased propensity of HIV-2 to produce latent infection. Second, Nef, an early viral gene product, has been shown to alter the activation state of infected cells in a lentiviral lineage-dependent manner. Third, it has been demonstrated that the ability of HIV-1 to establish latent infection is a function of the activation state of the host cell at the time of infection. Based on these observations, we reasoned that HIV-2 Nef may have the ability to promote latency establishment. We demonstrate that HIV-1 latency establishment in T cell lines and primary T cells is indeed differentially modulated by Nef proteins. In the context of an HIV-1 backbone, HIV-1 Nef promoted active HIV-1 infection, while HIV-2 Nef strongly promoted latency establishment. Given that Nef represents the only difference in these HIV-1 vectors and is known to interact with numerous cellular factors, these data add support to the idea that latency establishment is a host cell-virus interaction phenomenon, but they also suggest that the HIV-1 lineage may have evolved mechanisms to counteract host cell suppression. IMPORTANCE 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. Research investigating the viral RNA/DNA ratio in HIV-1 and HIV-2 patients could suggest that HIV-2 indeed has a much higher propensity to establish latent infections, a trait that we found, at least in part, to be attributable to the HIV-2 Nef protein. Reported Nef-mediated effects on host cell activation thus also affect latency establishment, and HIV-1 vectors that carry different lentiviral nef genes should become key tools to develop a better understanding of the biomolecular basis of HIV-1 latency establishment.

Host T Cell Dedifferentiation Effects Drive HIV-1 Latency Stability.

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 sequencing (scRNA-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 show here 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 dedifferentiated 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 redifferentiation 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. More than 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, and 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 that of 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 dedifferentiated T cell phenotype. Software-based data integration allowed the identification of drug targets that would redifferentiate viral host cells and, by 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.