使用整合位点特异性 qPCR 追踪 HIV-1 感染的细胞克隆。

Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR.

机构信息

Department of Medicine, University of Pittsburgh, 3550 Terrace Street, Scaife Hall-818, Pittsburgh, PA 15261, USA.

Cancer Research Technology Program, Leidos Biomedical Research, Inc., 8560 Progress Drive, ATRF, Room C3004, Frederick, MD 21701, USA.

出版信息

Viruses. 2021 Jun 25;13(7):1235. doi: 10.3390/v13071235.

Abstract

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed . The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies ( = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

摘要

为了治愈 HIV-1 感染,需要更好地定量 HIV-1 储存库,特别是那些携带具有复制能力(完整)前病毒的细胞克隆,这些细胞克隆被称为。目前常用的数字液滴 PCR 检测方法无法区分特定的复制子,因此复制子的动力学尚未明确界定。跟踪复制子的主要挑战是携带特定完整前病毒的细胞相对较少。迄今为止,检测和准确量化复制子需要深入整合位点测序。在这里,我们描述了一种简化的工作流程,使用整合位点特异性 qPCR(IS-qPCR)来确定单个复制子中整合的前病毒的频率。我们设计了 IS-qPCR 来确定来自三个供体样本中携带缺陷前病毒的复制子和细胞克隆的频率。将 IS-qPCR 的结果与深度整合位点测序数据进行比较表明,这两种方法对克隆频率的估计结果一致(=0.838)。IS-qPCR 是一种很有价值的工具,可以应用于多个样本和细胞类型,以随时间测量单个复制子的动力学和旨在消除它们的治疗效果。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6b38/8310066/0cef920bbbb3/viruses-13-01235-g001.jpg

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