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HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5' untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5'UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5' rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH. Author summary: People living with HIV establish a persistent reservoir which includes latently infected cells that fuel viral rebound upon treatment interruption. However, the majority of HIV-1 genomes in these persistently infected cells are defective. Whether these defective HIV genomes are expressed and whether they contribute to HIV associated diseases including accelerated aging, neurodegenerative symptoms, and cardiovascular diseases are still outstanding questions. In this paper, we demonstrate that acute infection of macrophages and resting T cells is biased towards generating defective viruses which are expressed by DNA regulatory elements in the HIV genome. These studies describe an alternative mechanism for chronic expression of HIV genomes. [ABSTRACT FROM AUTHOR]

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 (scRNAseq) 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. [ABSTRACT FROM AUTHOR]