Transcriptomic HIV-1 reservoir profiling reveals a role for mitochondrial functionality in HIV-1 latency

Identifying cellular mechanisms maintaining HIV-1 latency in the viral reservoir is crucial for devising effective cure strategies. Here we developed a flow cytometry-fluorescent in situ hybridization (flow-FISH) approach using a combination of probes that detects abortive and elongated HIV-1 transcripts for ex vivo isolation and characterization of viral reservoir cells in peripheral blood from people with HIV-1. Following the isolation of three distinct cell populations from CD4+ T cells (i.e. cells harboring transcriptionally latent HIV-1, cells harboring transcriptionally active HIV-1, or uninfected cells), we determined their transcriptomic profile by RNA sequencing (RNAseq). Supervised gene expression analysis identified several differentially expressed mitochondrial genes in infected cell populations compared to uninfected cells, but also in latently infected compared to productively infected CD4+ T cells. Our transcriptomic profiling data shows an association between diminished mitochondrial functioning and the transcriptional activity of the viral reservoir. These findings underline the relevance of metabolic regulation in HIV-1 infection, and support the development of strategies modulating immunometabolism to target viral latency. To investigate the mechanisms underlying HIV-1 latency in the viral reservoir, we designed probes targeting abortive TAR and elongated Gag HIV-1 transcripts for flow-FISH detection and isolation of transcriptionally latent or active HIV-1-infected CD4+ T cells. Using this method, we isolated transcriptionally latent HIV-1-infected cells (TAR+Gag-), transcriptionally active HIV-1-infected cells (TAR+Gag+), and uninfected cells (TAR-Gag-) from the CD4+ T cell population in peripheral blood mononuclear cells (PBMCs) from people with HIV-1 (N=5) by cell sorting. We then performed 3’ RNAseq and gene expression profiling analysis to assess the transcriptomic profiles of the three isolated populations.

鉴定维持病毒储存库中HIV-1潜伏性的细胞机制，对于制定有效的HIV-1治愈策略至关重要。本研究开发了一种流式细胞术-荧光原位杂交（flow cytometry-fluorescent in situ hybridization，简称flow-FISH）方法，该方法通过组合多种探针检测流产型与延长型HIV-1转录本，可用于从HIV-1感染者外周血中体外分离并鉴定病毒储存库细胞。我们从CD4+ T细胞中分离出三类不同的细胞群，即携带转录潜伏性HIV-1的细胞、携带转录活跃性HIV-1的细胞以及未感染细胞，随后通过RNA测序（RNA sequencing，简称RNAseq）分析其转录组谱特征。有监督的基因表达分析显示，与未感染细胞相比，感染细胞群中存在多个差异表达的线粒体基因；与产性感染的CD4+ T细胞相比，潜伏感染细胞中亦是如此。本研究的转录组谱分析数据表明，线粒体功能减弱与病毒储存库的转录活性存在显著关联。这些发现凸显了代谢调控在HIV-1感染中的重要意义，并为开发靶向调节免疫代谢以干预病毒潜伏的治疗策略提供了支持。为探究病毒储存库中HIV-1潜伏的潜在机制，我们设计了靶向流产型TAR与延长型Gag HIV-1转录本的探针，用于flow-FISH检测及分离转录潜伏或活跃的HIV-1感染CD4+ T细胞。利用该方法，我们通过细胞分选从5名HIV-1感染者的外周血单个核细胞（peripheral blood mononuclear cells，简称PBMCs）的CD4+ T细胞群中，分离出转录潜伏性HIV-1感染细胞（TAR+Gag-）、转录活跃性HIV-1感染细胞（TAR+Gag+）以及未感染细胞（TAR-Gag-）。随后我们开展了3'端RNA测序及基因表达谱分析，以评估这三类分离细胞群的转录组特征。