BackgroundHIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency.ResultsTo quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts (“read-through,” initiated, 5′ elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival.ConclusionsHIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript.

中文翻译：

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潜伏性和生产性感染细胞系模型中 HIV 和细胞转录谱的异质性：对 HIV 潜伏期的影响

背景 HIV 感染的细胞系被广泛用于研究潜在的 HIV 感染，这被认为是 HIV 治愈的主要障碍。我们假设这些细胞系彼此不同，并且与来自 HIV 感染个体的细胞在潜伏期的潜在机制不同。结果为了量化 HIV 表达在 HIV 转录的不同阶段被阻断的程度，我们采用了最近描述的面板使用 RT-ddPCR 检测方法测量 7 种 HIV 转录物的水平（“通读”、启动、5' 延长、中间转录/未剪接 [Pol]、远端转录 [Nef]、多聚腺苷酸化和多切片 [Tat- Rev]）在大量潜伏感染（U1、ACH-2、J-Lat）和大量感染（8E5，激活的 J-Lat）细胞系中。为了评估单细胞变异并研究与 HIV 转录块相关的细胞基因，我们开发了一种新型多重 qPCR 面板，并量化了潜伏感染和高效感染细胞系中 7 个 HIV 靶标和 89 个细胞转录本的单细胞水平。大量细胞 HIV 转录谱在细胞系和来自 ART 抑制个体的细胞之间存在显着差异。与来自 ART 抑制个体的细胞相比，潜伏细胞系显示出较低水平的 HIV 转录起始和较高水平的多聚腺苷酸化和剪接。ACH-2 和 J-Lat 细胞表现出不同形式的转录干扰，而 U1 细胞表现出对延伸的阻滞。单细胞研究揭示了 HIV 转录本、T 细胞表型标志物、抗病毒因子、和与潜伏期有关的基因。多剪接 HIV Tat-Rev 的表达与参与激活、组织保留、T 细胞转录和细胞凋亡/存活的细胞基因的表达有关。控制潜伏性 HIV 感染的机制。在衡量特定细胞系是否适合未来 HIV 研究时，必须考虑病毒和细胞基因表达的这些差异。同时，一些特征在细胞系之间是共享的，例如抗病毒防御基因的低表达以及生产性感染与存活相关基因之间的关系。这些特征可能有助于 HIV 在体内的潜伏期或持久性，