Integrated Single-cell Multiomic Analysis of HIV Latency Reversal Reveals Novel Regulators of Viral Reactivation

Abstract

Despite the success of antiretroviral therapy, human immunodeficiency virus (HIV) cannot be cured because of a reservoir of latently infected cells that evades therapy. To understand the mechanisms of HIV latency, we employed an integrated single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) approach to simultaneously profile the transcriptomic and epigenomic characteristics of ~125,000 latently infected primary CD4+ T cells after reactivation using three different latency reversing agents. Differentially expressed genes and differentially accessible motifs were used to examine transcriptional pathways and transcription factor (TF) activities across the cell population. We identified cellular transcripts and TFs whose expression/activity was correlated with viral reactivation and demonstrated that a machine learning model trained on these data was 75%–79% accurate at predicting viral reactivation. Finally, we validated the role of two candidate HIV-regulating factors, FOXP1 and GATA3, in viral transcription. These data demonstrate the power of integrated multimodal single-cell analysis to uncover novel relationships between host cell factors and HIV latency.

1. Introduction

The formation of a latently infected reservoir in CD4+ T cells is a key barrier to an human immunodeficiency virus (HIV) cure [1,2]. The reservoir is highly stable and long lived, and is not eliminated by current antiretroviral therapy [3]. Additionally, clonal expansion in vivo counteracts gradual erosion of infected cells [4–8]. A key mechanism in the formation of this reservoir is the phenomenon of viral latency, in which HIV transcription is reversibly silenced post integration, allowing it to evade the host immune defenses. Sporadic reactivation of these cells generates “blips” of viremia during therapy [9] and seeds rapid rebound upon interruption of therapy [10,11]. Current cure strategies for HIV involve transiently inducing viral gene expression in latent proviruses using latency reversing agents (LRAs), followed by immune clearance of the reactivated cells. However, this approach has thus far only achieved limited success [12–16]. Reactivation of latently infected cells with existing LRAs is inefficient, with typically ~10% of replication-competent proviruses being reactivated in patient-derived cells, even with “potent” LRAs [17]. For the LRA/clearance approach to be successful, broad reactivation of the reservoir will be required. This inefficiency of latency reversal with LRAs is likely due to a combination of stochastic processes that regulate viral gene expression, the existence of multiple layers of repression to HIV gene expression, and the heterogeneity in both the cellular environment and proviral integration sites in infected cells. Fully defining the nature of these repressive mechanisms is urgently needed to allow the development of broader acting LRAs or approaches with a combination of different LRAs.

Prior work indicates that silencing of HIV in CD4+ T cells involves the combined effects of low levels of positive transcription factors (TFs), such as nuclear factor kappa B (NF-κB) and activating protein-1 (AP-1) [18–20], active repression by cellular factors (e.g., NELF and DSIF) [21,22], sequestration of the elongation complex P-TEFb (cyclinT1/CDK9) [23], and histone modifications that create a repressive heterochromatin environment around the virus promoter [23–27]. Nevertheless, the regulation of HIV gene expression remains incompletely understood, and additional mechanisms likely exist that will need to be fully characterized.