The loud minority: Transcriptionally active HIV-1-infected cells survive, proliferate, and persist

Abstract

The shock-and-kill strategy reactivates HIV-1 latent reservoir for immune clearance. Einkauf et al. found that some HIV-1-infected cells that persist and proliferate have transcriptionally active HIV-1 in permissive chromatin. Silent proviruses in repressive chromatin resist reactivation. Understanding HIV-1-chromatin interactions and how transcriptionally active HIV-1-infected cells survive is a pressing need.

Despite the success of antiretroviral therapy (ART), which effectively blocks new rounds of infections, HIV-1 persists as proviruses integrated into human genome in infected cells. Although most HIV-1-infected cells harboring intact HIV-1 proviruses die of viral cytopathic effects or immune clearance, some turn into a transcriptionally inactive latent state and evade viral cytopathic effects and immune clearance. This latent reservoir persists lifelong in people living with HIV-1 and is the major barrier to cure. On the other hand, ART does not suppress HIV-1 promoter function or transcription. Infected cells harboring defective HIV-1 proviruses remain transcriptionally active and induce chronic immune activation (Pollack et al., 2017). In addition to the intactness of HIV-1 proviral genome, HIV-1 integration site is critical for HIV-1 persistence. HIV-1 integration into cancer genes may drive aberrant transcription of cancer genes and promote clonal expansion of infected cells (Liu et al., 2020; Maldarelli et al., 2014; Wagner et al., 2014), while integration into transcriptionally silent regions may promote immune evasion and persistence (Jiang et al., 2020). Altogether, HIV-1 genome intactness, HIV-1 transcription, and HIV-1-host genome interaction shape cell survival, clonal expansion dynamics, and immune surveillance of infected cells.

An important HIV-1 cure strategy is the shock-and-kill strategy, which involves reactivating latent HIV-1 proviruses, inducing viral protein expression, and exposing latently infected cells for immune clearance. Understanding HIV-1 reactivation is essential for an effective shock-and-kill strategy. However, studying reactivation from the rare and heterogeneous HIV-1-infected cells has been extremely challenging: <0.1% of CD4⁺ T cells harbor defective HIV-1 proviruses, and even fewer (<0.01%) harbor intact HIV-1 proviruses. Recently, researchers developed methods to map the HIV-1 integration site and proviral genome intactness at the same time via matched integration site and proviral sequencing (MIP-seq) (Einkauf et al., 2019) or combined HIV-1 sequence and integration site analysis (Patro et al., 2019). In this issue of Cell, Einkauf et al. push our capabilities further, allowing researchers to examine HIV-1 reactivation simultaneously with integration site and proviral genome of the same provirus (Einkauf et al., 2022). This tour de force and innovative method, called PRIP-seq, answers many pressing questions for mechanisms of HIV-1 persistence and therapeutic strategies.

Does host epigenetic regulation at the integration site affect HIV-1 persistence, or does HIV-1 harness host epigenetic regulation to promote persistence? Generally, actively transcribed genes have more transcription factor binding, higher chromatin accessibility, low CpG methylation, abundant active chromatin modifications, and increased interactions with chromatin loops (Figure 1B). Epigenetic regulators can affect HIV-1 transcriptional activity in vitro, evidenced by the fact that HIV-1 expression is strongest near enhancers (Chen et al., 2017) and that HIV-1 integration sites are enriched in super-enhancer regions (Lucic et al., 2019). The impact of HIV-1-host chromatin interactions in vivo remains unknown. Einkauf et al. profiled the epigenetic landscape on multiple axes, including chromatin conformation (by HiC), chromatin accessibility (by assay for transposase-accessible chromatin sequencing [ATAC-seq]), active versus repressive chromatin marks (by chromatin immunoprecipitation sequencing [ChIP-seq]), DNA CpG methylation profiles (by bisulfite sequencing), and gene expression (by RNA-seq), using CD4⁺ T cells from HIV-1-infected individuals or existing databases. The authors identified the chromatin locations in which HIV-1 is integrated and examined the corresponding epigenetic landscape and transcription profiles in primarily uninfected CD4⁺ T cells. The results are striking (Figure 1C).

Figure 1. The impact of chromatin environment on HIV-1 expression, persistence, and clonal expansion dynamics.

(A) PRIP-seq: parallel HIV-1 RNA, integration site, and proviral DNA analysis. Cells are plated at limiting dilution such that there is only one infected cell in a group of cells. HIV-1 RNA is separated from genomic DNA using HIV-1-specific biotinylated probes, reverse transcribed with the same probes, amplified, and measured by droplet digital PCR. Genomic DNA from cells in each well is amplified using phi29-mediated multiple displacement whole-genome amplification using universal primers and then used for HIV-1 integration site sequencing and HIV-1 proviral genome sequencing in separate aliquots.

(B) The chromatin environment that determines gene expression is orchestrated by multiple levels of epigenetic regulators. The chromatin environment can be examined by chromatin conformation (by HiC), chromatin accessibility (by ATAC-seq), active versus repressive chromatin marks (by ChIP-seq), and DNA CpG methylation profiles (by bisulfite sequencing).

(C) Host determinants of HIV-1 transcription and persistence in vivo. Despite ART, a substantial proportion (>25%) of HIV-1-infected cells harboring intact HIV-1 are transcriptionally active. The level of HIV-1 transcription is associated with the chromatin environment. Most transcriptionally active HIV-1-infected cells are eliminated by the immune selection pressure. However, some transcriptionally active HIV-1-infected cells harboring intact HIV-1 that reside in permissive chromatin can survive, persist, and proliferate despite long-term ART. Silent HIV-1 proviruses that reside in repressive chromatin resist latency reversal. Latency reversing agents may increase HIV-1 expression from already transcriptionally active HIV-1-infected cells but may not reactivate transcriptionally silent HIV-1-infected cells.

First, the chromatin environment determines HIV-1 proviral transcription. Transcriptionally active HIV-1 proviruses are enriched in permissive chromatin and are associated with closer proximity to frequently interacting chromatin regions (FIRE), higher chromatin accessibility, active histone marks, lower CpG methylation, and higher levels of host gene expression. In contrast, transcriptionally silent HIV-1 proviruses mainly reside in repressive chromatin environments.

Second, HIV-1 proviruses respond differently to latency reversal depending on the integration site. Upon ex vivo maximum T cell activation by phorbol myristate acetate (PMA)/ionomycin, HIV-1 proviruses that are transcriptionally active in vivo respond robustly to latency reversal and express higher levels of HIV-1 RNA. However, HIV-1 proviruses that are transcriptionally silent in vivo, especially those in the repressive chromatin environment, cannot be reactivated despite maximum T cell activation. New ways to reactivate HIV-1 proviruses in epigenetically repressed chromatin environment need to be considered in the shock-and-kill strategy.

Third, many more HIV-1-infected cells are transcriptionally active in vivo than previously thought. Around one-third defective proviruses during viremia (33.6%, 377/1123) and after long-term ART (31.6%, 269/852) are transcriptionally active, reflecting the fact that ART does not suppress HIV-1 transcription. Around half (56.8%, 25/44) intact HIV-1 proviruses during viremia and one-fourth (26.7%, 27/101) intact HIV-1 proviruses under long-term ART are transcriptionally active. This reflects productive infections during viremia and the immune selection pressure that eliminates HIV-1-infected cells harboring intact and transcriptionally active HIV-1 genome under long-term ART. However, despite ART, a substantial proportion of infected cells harbor intact HIV-1 proviruses residing in permissive chromatin environments, allowing them to remain transcriptionally active. Surprisingly, these cells survive viral cytopathic effects, evade immune clearance, and undergo clonal expansion over time. Mechanisms promoting long-term persistence of these transcriptionally active HIV-1 RNA⁺ cells require further investigation.

The ultimate question of whether host epigenetic regulation of HIV-1 expression or HIV-1-driven aberrant host gene expresssion is more functionally important remains to be answered. This is because the HIV-1 integration site was not directly epigenetically profiled, but rather the epigenetics were determined by correlating with datasets from primarily uninfected cells. HIV-1 insertion into the human genome brings in additional transcriptional factor binding sites, drives aberrant host gene transcription, and can change the local epigenetic regulators and chromatin environment. Methods that can examine the epigenetic regulators directly at the HIV-1 integration sites are required, although such methods remain technically challenging and unavailable.

Overall, the unprecedented singlegenome study by Einkauf et al. revolutionizes our understanding of the HIV-1 reservoir in vivo and identified the impact of the chromatin environment on HIV-1 transcription and persistence. The study also identified transcriptionally active, clonally expanding, HIV-1-infected cells–the loud minority–as a key target for a cure. Understanding how cells harboring transcriptionally active HIV-1 proviruses evade host elimination likely require advanced single-cell multi-omics studies to decipher the interactions between the heterogeneous HIV-1 integration sites and the diverse memory CD4⁺ T cell transcriptional programs.