Single-cell understanding of HIV persistence
Approach: Single-cell genomics, HIV virology, immunology, bioinformatics, and translational approaches using clinical samples
Goal: Understanding HIV persistence to guide the development of HIV cure strategies
Despite effective antiretroviral therapy (ART), HIV persists in the latent reservoir as the major barrier to cure. Although ART effectively suppresses plasma viral load to clinically undetectable levels, once ART is interrupted, high levels of viremia will inevitably occur. Therefore, all 37 million people living with HIV need to take lifelong ART. This is because HIV hides in the memory CD4+ T cells and evades immune clearance. First, HIV stably integrates into chromosomes of infected cells. ART cannot remove these integrated proviruses. Second, HIV infects the long-lived memory CD4+ T cells and persists for decades. Third, HIV enters latency and becomes transcriptionally silent when memory CD4+ T cells return to a quiescent state. Latently infected cells do not express viral antigens and cannot be recognized and cleared by the immune system. Fourth, HIV-infected cells undergo clonal expansion through antigen stimulation, homeostatic cytokine-mediated proliferation, and integration-site driven proliferation. This means that HIV-infected cells not only survive but also increase over time. Finally, despite ART, millions of HIV-infected cells already established the latent reservoir within 3 days of infection. Since ART does not inhibit HIV LTR promoter activity, these infected cells express viral antigens through stochastic activation and continue to induce immune activation and immune exhaustion. The exhausted and dysfunctional immune effector cells cannot effectively eliminate HIV-infected cells. A cure is desperately needed. However, many questions remain unsolved: how does virally suppressed HIV infection induce chronic immune exhaustion? How do HIV-infected cells survive, proliferate, and persist over time? How can we specifically target HIV-infected CD4+ T cells without harming uninfected cells?
Our goal is to understand how HIV induces immune dysfunction over different stages of HIV infection over time, both at the systemic level characterizing all CD4+ T cells and the single-cell level in HIV-infected cells. We will focus on CD4+ T cells, the central orchestrator of adaptive immunity and the major target of HIV infection. We hypothesize that despite the heterogeneity of HIV-infected cells, HIV drives a distinct cellular program that promotes persistence. We propose that identifying and targeting HIV-driven immune dysregulation leads to cure. The challenge to reaching this goal is the heterogeneity, rarity, and lack of marker of HIV-infected cells. Our approach is to develop cutting-edge single-cell methods, to apply these methods to challenging clinical samples, and to validate with CRISPR-mediated up- and down-regulation of candidate cellular pathways.
Single-cell transcriptional landscapes reveal HIV-1–driven aberrant host gene transcription as a potential therapeutic target
We developed HIV-1 SortSeq which captures the rare HIV-1-infected cells for single-cell RNAseq.
First authors: our postdocs Runxla Liu (HIV-1 SortSeq) and Yang-Hui Jimmy Yeh (HIV-1-driven aberrant cancer gene expression landscape) and Johns Hopkins PhD student Ales Varabyou (HIV-host chimeric RNA bioinformatic capture), with help from our PhD student Jack Collora (single-cell transcriptome analysis and CRISPRa/i system) and Kristen Albrecht (HIV-1 gRNA construction).
Understanding HIV-1–host interactions can identify the cellular environment supporting HIV-1 reactivation and mechanisms of clonal expansion. We developed HIV-1 SortSeq to isolate rare HIV-1–infected cells from virally suppressed, HIV-1–infected individuals upon early latency reversal. Single-cell transcriptome analysis of HIV-1 SortSeq+ cells revealed enrichment of nonsense-mediated RNA decay and viral transcription pathways. HIV-1 SortSeq+ cells up-regulated cellular factors that can support HIV-1 transcription (IMPDH1 and JAK1) or promote cellular survival (IL2 and IKBKB). HIV-1–host RNA landscape analysis at the integration site revealed that HIV-1 drives high aberrant host gene transcription downstream, but not upstream, of the integration site through HIV-1–to–host aberrant splicing, in which HIV-1 RNA splices into the host RNA and aberrantly drives host RNA transcription. HIV-1–induced aberrant transcription was driven by the HIV-1 promoter as shown by CRISPR-dCas9–mediated HIV-1–specific activation and could be suppressed by CRISPR-dCas9–mediated inhibition of HIV-1 5′ long terminal repeat. Overall, we identified cellular factors supporting HIV-1 reactivation and HIV-1–driven aberrant host gene transcription as potential therapeutic targets to disrupt HIV-1 persistence.
Video abstract: https://youtu.be/QGnn6IspsrA
News summary: https://news.yale.edu/2020/05/13/yale-researchers-discover-how-hiv-hides-treatment
Targeting RNA processing and T cell activation halts HIV-1-driven aberrant host-transcription
Using a drug screen and transcriptome analysis, we identified FDA-approved drugs that can suppress HIV-driven aberrant host gene transcription. Published on June 24, 2020 at Journal of Clinical Investigation (as in-press preview).
Our postdoc Jimmy did the experiments using blood samples from HIV-infected individuals and performed all bioinformatic analysis.
Despite effective antiretroviral therapy, HIV-1-infected cells continue to produce viral antigens and induce chronic immune exhaustion. We propose to identify HIV-1-suppressing agents which can inhibit HIV-1 reactivation and reduce HIV-1-induced immune activation. Using a novel dual reporter system and a high-throughput drug screen, we identified FDA-approved drugs which can suppress HIV-1 reactivation in both cell line models and CD4+ T cells from virally suppressed, HIV-1-infected individuals. We identified 11 cellular pathways required for HIV-1 reactivation as druggable targets. Using differential expression analysis, gene set enrichment analysis and exon-intron landscape analysis, we examined the impact of drug treatment on the cellular environment at a genome-wide level. We identified a new function of a JAK inhibitor filgotinib which suppresses HIV-1 splicing. First, filgotinib preferentially suppresses spliced HIV-1 RNA transcription. Second, filgotinib suppresses HIV-1-driven aberrant cancer-related gene expression at the integration site. Third, we found that filgotinib suppresses HIV-1 transcription by inhibiting T cell activation and by modulating RNA splicing. Finally, we found that filgotinib treatment reduces the proliferation of HIV-1-infected cells. Overall, the combination of a drug screen and transcriptome analysis provides systemic understanding of cellular targets required for HIV-1 reactivation and drug candidates that may reduce HIV-1-related immune activation.
Single-cell immune profiling reveals mechanisms of HIV persistence and HIV-induced immune dysfunction
R01/NIAID: Role of clonal expansion in HIV-1 persistence (Ho)
*Awarded within 1 year after starting the Ho Lab.
R61/NIDA: Evaluating the role of opioid medication assisted therapies in HIV-1 Persistence for persons living with HIV and opioid use disorders (Springer/Ho/Kluger)
*Awarded within 1 year after starting the Ho Lab.
Gilead HIV Scholar: Single-cell characterization of HIV-1-infected cells (Ho)
CHEETAH/NIH Pilot Award: Identification of cellular factors critical for HIV-1 silencing (Ho)
Yale Top Scholar (Ho)
Rudolf J. Anderson Endowed Postdoctoral Fellowship (Ho)
R21/NIAID: High-throughput measurement and transcriptome analysis of the latent reservoir using HIV-1 RNA flow-FISH (Ho)
*Awarded within 15 months after PhD thesis defense.
Johns Hopkins CFAR Developmental Award: Identification of cellular factors necessary for latent HIV-1 reactivation (Ho)
W. W. Smith AIDS Research Grant: Expansion dynamics and functional analysis of the HIV-1 latent reservoir (Ho)
Gilead HIV Research Grant: Measuring the latent reservoir for HIV (Siliciano/Ho/Quinn)
HHMI International Student Research Fellowship (Ho)