Profound phenotypic and epigenetic heterogeneity of the HIV-1-infected CD4+ T cell reservoir

Abstract

Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.

Single-cell multiomic understanding of HIV-1 reservoir at epigenetic, transcriptional, and protein levels

“…When Tn5 binds to HIV-1 DNA, these HIV-1 DNA+ cells can be bioinformatically identified. This study identified host transcription factor activity (derived from chromatin accessibility) in addition to surface protein expression of HIV-1 DNA+ cells [56 ▪▪ ]. Of note, the majority of these HIV-1 DNA+ cells are also likely to be defective.…”

“…Wu et al [56 ▪▪ ] in the Betts group applied ASAP-seq to identify HIV-1 DNA and host chromatin accessibility (by ATAC-seq), and surface protein expression. Vahedi group first proposed the concept of using transposase to identify cells harboring lentiviral DNA, using lentiviral transduced CAR-T cells as an example [57 ▪▪ ].…”

“…Yet, defining the rare HIV-1-infected cells has to be stringent and rigorous to prevent false positive discovery of cell types as a result of sequencing artifacts. By mapping ATAC-seq [56 ▪▪ ] or RNA-seq reads [25 ▪▪ ] to the HXB2 reference (for which the clinical and mechanistic implications of HIV-1 mutations are annotated), the identification of HIV-1 RNA transcripts in single cells acts as a surrogate for transcriptionally active HIV-1-infected cells [17 ▪▪ ,25 ▪▪ ]. Increasing the breadth of reference genomes by mapping transcriptomic reads to autologous HIV-1 sequences in addition to HXB2 further increases mapping rate by approximately 20% and increases detection of spliced HIV-1 RNA [25 ▪▪ ].…”

“…In previous studies of HIV-1+ individuals under suppressive ART, nearly 0.13% memory CD4 + T cells harbor HIV-1 DNA as captured by ATAC-seq [56 ▪▪ ] and nearly 0.02% CD4 + T cells harbor HIV-1 RNA as captured by RNA-seq [25 ▪▪ ]. Both HIV-1 DNA and RNA sequences are sparse: the nearly 9719 bp of HIV-1 provirus only account for 0.00016% of the 6 x 10 9 bp of diploid human genome and may not be captured by Tn5 transposase in ATAC-seq.…”

“…For example, as enlisted by Liu et al [17 ▪▪ ], some of HIV-1 sequences deposited in the public database contain human genome, which may lead to false discovery of HIV-1-infected cells. Second, although HIV-1 integration sites can sometimes be captured by identifying the junction of HIV-1 DNA and human genome [56 ▪▪ ] or the junction of HIV-1-host chimeric RNA through aberrant splicing [17 ▪▪ ], sequencing artifacts have to be identified and removed. There cannot be any additional sequences between the human genome and the HIV-1 genome, as these additional sequences are artifacts generated during the PCR amplification during library preparation, as described by Sherrill-Mix et al in the Bushman group [101].…”