Department of Chemistry, University of Wisconsin, Madison, United States.
McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin, Madison, United States.
Elife. 2021 Feb 25;10:e62470. doi: 10.7554/eLife.62470.
HIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs, each playing distinct roles in viral replication. Elucidating their host protein 'interactomes' is crucial to understanding virus-host interplay. Here, we present HyPR-MS for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes, including preferential association of regulators of RNA stability with US and PS transcripts and, unexpectedly, mitochondria-linked proteins with US transcripts. Remarkably, >80 of these factors screened by siRNA knockdown impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MS for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.
HIV-1 产生未剪接 (US)、部分剪接 (PS) 和完全剪接 (CS) 类 RNA，每一类在病毒复制中都发挥着独特的作用。阐明它们的宿主蛋白“相互作用组”对于理解病毒-宿主相互作用至关重要。在这里，我们提出了 HyPR-MS 用于从感染的 CD4+ T 细胞的单个群体中分离 US、PS 和 CS 转录本，并进行体内蛋白质相互作用组的质谱鉴定。分析显示，212 种蛋白与独特的 RNA 类有差异关联，包括 RNA 稳定性调节剂与 US 和 PS 转录本的优先关联，以及出乎意料的是与 US 转录本的线粒体相关蛋白的关联。值得注意的是，通过 siRNA 敲低筛选出的 >80 个因素会影响 HIV-1 基因表达。荧光显微镜证实其中的几个因素与 HIV-1 US RNA 共定位，并在感染过程中表现出丰度和/或定位的变化。这项研究验证了 HyPR-MS 用于发现病毒剪接变体蛋白相互作用组的方法，并提供了一个前所未有的因素和途径资源，这些因素和途径可能对 HIV-1 复制很重要。
