Jorge F Guerrero, Laraine L Zimdars, James W Bruce, Jordan T Becker, Edward L Evans, Soroosh Torabi, Rob Striker, Scott M Berry, Nathan M Sherer
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引用次数: 0
Abstract
Human immunodeficiency virus type 1 (HIV-1) genome diversification is a key determinant of viral evolution and the pathogenesis of HIV/AIDS. Antiretroviral therapy is non-curative, and in the context of monitoring the latent reservoir, precision tools are needed to detect and enumerate HIV-1 genomes as well as to assess their heterogeneity, replication potential, and predict responses to therapy. Current sequencing-based methodologies are often unable to confirm intact genomes and most cell-based reporters provide limited information pertaining to viral fitness. In this study, we describe dual reporter sensor cells (DRSCs), an imaging-based reporter system designed to detect HIV-1 infection and measure several independent attributes of the virus in a single-cell high-content assay. We show that the DRSC assay can be used to measure infection, viral gene activation kinetics, and quantify viral circumvention of host antiviral responses. Using the DRSCs, we confirmed markedly different functional heterogeneity for vif alleles derived from diverse HIV-1 strains and subtypes affecting both rates of APOBEC3G degradation and the cell cycle. Furthermore, the assay allowed for the delineation of virus co-receptor preference (X4- vs R5-tropism) and visualization of virion assembly. Overall, our study illustrates proof-of-principle for a multivariate imaging-based cell-based system capable of detecting HIV-1 and studying viral genetic variability with greater data richness relative to prior available modalities.
Importance: Human immunodeficiency virus type 1 (HIV-1) is highly heterogeneous and constantly mutating. These changes drive immune evasion and can cause treatment efforts to fail. Here, we describe the "dual reporter sensor cell" (DRSC) assay; a novel imaging-based approach that allows for the detection of HIV-1 infection coupled with a multivariate definition of several independent phenotypic aspects of viral genome activity in a single integrated assay. We validate the DRSC system by studying lab-adapted and patient isolate-derived versions of the viral Vif accessory protein, confirming marked differences in the capacity of diverse vif alleles to mediate downregulation of antiviral APOBEC3G proteins and dysregulate the cell cycle.
人类免疫缺陷病毒1型(HIV-1)基因组多样化是病毒进化和HIV/AIDS发病机制的关键决定因素。抗逆转录病毒治疗是不可治愈的,在监测潜伏病毒库的背景下,需要精确的工具来检测和枚举HIV-1基因组,以及评估它们的异质性、复制潜力和预测对治疗的反应。目前基于测序的方法通常无法确认完整的基因组,而且大多数基于细胞的报告者提供的关于病毒适应性的信息有限。在这项研究中,我们描述了双报告传感细胞(DRSCs),这是一种基于成像的报告系统,旨在检测HIV-1感染,并在单细胞高含量试验中测量病毒的几个独立属性。我们表明,DRSC试验可用于测量感染,病毒基因激活动力学,并量化病毒规避宿主抗病毒反应。使用DRSCs,我们证实了来自不同HIV-1毒株和亚型的vif等位基因的显著不同的功能异质性,影响APOBEC3G降解率和细胞周期。此外,该分析允许描绘病毒共受体偏好(X4- vs r5 -趋向性)和可视化病毒粒子组装。总的来说,我们的研究证明了一种基于细胞的多变量成像系统的原理,该系统能够检测HIV-1并研究病毒遗传变异性,与先前可用的模式相比,数据更丰富。重要性:人类免疫缺陷病毒1型(HIV-1)是高度异质和不断突变的。这些变化会导致免疫逃避,并可能导致治疗失败。在这里,我们描述了“双报告传感器细胞”(DRSC)测定;这是一种新的基于成像的方法,可以检测HIV-1感染,并在一次综合分析中对病毒基因组活性的几个独立表型方面进行多变量定义。我们通过研究实验室适应的病毒Vif辅助蛋白和患者分离衍生的病毒Vif辅助蛋白来验证DRSC系统,证实了不同Vif等位基因介导抗病毒APOBEC3G蛋白下调和细胞周期失调的能力存在显著差异。
期刊介绍:
Journal of Virology (JVI) explores the nature of the viruses of animals, archaea, bacteria, fungi, plants, and protozoa. We welcome papers on virion structure and assembly, viral genome replication and regulation of gene expression, genetic diversity and evolution, virus-cell interactions, cellular responses to infection, transformation and oncogenesis, gene delivery, viral pathogenesis and immunity, and vaccines and antiviral agents.