HIV-1 expression is heterogeneous among clones of CD4+ T cells carrying authentic intact latent proviruses

Antiretroviral therapy suppresses HIV-1 infection but is not curative because it fails to eliminate a reservoir of intact latent proviruses that reside primarily in CD4+ T cells. This compartment is composed of rare T cells that predominantly express memory and effector memory markers. The lack of precise understanding of the latent compartment has made it challenging to develop curative strategies for HIV-1 infection. Here we report on the properties of CD4+ T cells clones carrying intact latent proviruses, expanded in vitro from single cells obtained from the reservoir of people living with HIV-1. The latent proviruses in the clones were integrated into ZNF genes, non-genic satellite and centromeric regions, frequently associated with latency. Notably, the transcriptome of the cultured clones resembled their cells of origin. Despite their descent from single cells, only a fraction of the cells ranging from 0.4-14% expressed relatively low levels of HIV-1 that did not measurably alter host gene transcriptome. Latency reversing agents (LRAs) variably increased the number and amount of expression per cell, but the effects were modest and clone and LRA specific. The results suggest that pharmacologic and immunologic approaches to clear the reservoir should be optimized to accommodate intra- and inter-clonal diversity. Overall design: CD4+ T cells harboring intact latent proviruses were obtained from single cells from 7 ART-suppressed individuals and expanded in vitro. These expanded clones were analyzed at baseline (resting) and after 24 hours of stimulation (activated) with anti-CD3/CD28 + IL-2. We performed 5' single-cell RNA sequencing (scRNA-seq) and T-cell receptor (scV(D)J-seq) using the 10X Genomics platform to characterize proviral expression and the host cell transcriptome.

抗逆转录病毒治疗（Antiretroviral therapy）可抑制HIV-1感染，但无法实现治愈，因其无法清除主要藏于CD4+ T细胞中的完整潜伏前病毒库。该病毒库由罕见的T细胞组成，此类细胞主要表达记忆性与效应记忆性标志物。由于对潜伏库缺乏精准认知，开发HIV-1感染的治愈策略极具挑战。本研究报道了携带完整潜伏前病毒的CD4+ T细胞克隆的特性，这些克隆从HIV-1感染者病毒库中的单个细胞体外扩增获得。克隆中的潜伏前病毒整合至ZNF基因、非基因卫星序列及着丝粒区域，这类整合位点常与病毒潜伏状态相关。值得注意的是，培养的克隆的转录组与其起源细胞高度相似。尽管所有克隆均源自单个细胞，但仅0.4%~14%的细胞表达水平较低的HIV-1，且未显著改变宿主基因的转录组。潜伏期逆转剂（Latency reversing agents, LRAs）可不同程度提升每细胞的HIV-1表达量与表达细胞数，但效应较为温和，且具有克隆和LRA特异性。研究结果提示，清除病毒库的药物与免疫学策略应进行优化，以适应克隆内与克隆间的异质性。

总体实验设计：从7名经抗逆转录病毒治疗实现病毒抑制的感染者的单个细胞中获取携带完整潜伏前病毒的CD4+ T细胞，并进行体外扩增。分别在基线（静息状态）以及经抗CD3/CD28+IL-2刺激24小时（活化状态）后对这些扩增后的克隆进行分析。采用10X Genomics平台开展5'端单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与T细胞受体测序（T-cell receptor, scV(D)J-seq），以表征前病毒表达情况与宿主细胞转录组。