Department of Chemistry, University of Wisconsin-Madison College of Letters and Sciences, Madison, Wisconsin 53706, United States.
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin 53705, United States.
J Proteome Res. 2024 Sep 6;23(9):4128-4138. doi: 10.1021/acs.jproteome.4c00505. Epub 2024 Jul 30.
A critical part of the hepatitis B virus (HBV) life cycle is the packaging of the pregenomic RNA (pgRNA) into nucleocapsids. While this process is known to involve several viral elements, much less is known about the identities and roles of host proteins in this process. To better understand the role of host proteins, we isolated pgRNA and characterized its protein interactome in cells expressing either packaging-competent or packaging-incompetent HBV genomes. We identified over 250 host proteins preferentially associated with pgRNA from the packaging-competent version of the virus. These included proteins already known to support capsid formation, enhance viral gene expression, catalyze nucleocapsid dephosphorylation, and bind to the viral genome, demonstrating the ability of the approach to effectively reveal functionally significant host-virus interactors. Three of these host proteins, AURKA, YTHDF2, and ATR, were selected for follow-up analysis. RNA immunoprecipitation qPCR (RIP-qPCR) confirmed pgRNA-protein association in cells, and siRNA knockdown of the proteins showed decreased encapsidation efficiency. This study provides a template for the use of comparative RNA-protein interactome analysis in conjunction with virus engineering to reveal functionally significant host-virus interactions.
乙型肝炎病毒（HBV）生命周期的一个关键部分是将前基因组 RNA（pgRNA）包装到核衣壳中。虽然已知这个过程涉及几个病毒元件，但宿主蛋白在这个过程中的身份和作用知之甚少。为了更好地了解宿主蛋白的作用，我们在表达包装能力或包装能力丧失的 HBV 基因组的细胞中分离了 pgRNA，并对其蛋白质相互作用组进行了表征。我们从包装能力丧失的病毒版本中鉴定出了 250 多种与 pgRNA 优先相关的宿主蛋白。这些蛋白包括已经被证实支持衣壳形成、增强病毒基因表达、催化核衣壳去磷酸化和与病毒基因组结合的蛋白，这表明该方法能够有效地揭示具有功能意义的宿主-病毒相互作用体。我们选择了其中的三种宿主蛋白 AURKA、YTHDF2 和 ATR 进行后续分析。RNA 免疫沉淀 qPCR（RIP-qPCR）在细胞中证实了 pgRNA-蛋白的关联，并且这些蛋白的 siRNA 敲低显示出封装效率降低。这项研究为使用比较 RNA-蛋白质相互作用组分析结合病毒工程来揭示具有功能意义的宿主-病毒相互作用提供了模板。
