ABSTRACT

Flaviviruses such as the dengue (DENV) and the Zika virus (ZIKV) are important human pathogens causing around 100 million symptomatic infections each year. During infection, small subgenomic flavivirus RNAs (sfRNAs) are formed inside the infected host cell as a result of incomplete degradation of the viral RNA genome by cellular exoribonuclease XRN1. Although the full extent of sfRNA functions is to be revealed, these non-coding RNAs are key virulence factors and their detrimental effects on multiple cellular processes seem to consistently involve molecular interactions with RNA-binding proteins (RBPs). Discovery of such sfRNA-binding host-factors has followed established biochemical pull-down approaches skewed towards highly abundant proteins hampering proteome-wide coverage. Yeast three-hybrid (Y3H) systems represent an attractive alternative approach. To facilitate proteome-wide screens for RBP, we revisited and improved existing RNA-Y3H methodology by (1) implementing full-length ORF libraries in combination with (2) efficient yeast mating to increase screening depth and sensitivity, and (3) stringent negative controls to eliminate over-representation of non-specific RNA-binders. These improvements were validated employing the well-characterized interaction between DDX6 (DEAD-box helicase 6) and sfRNA of DENV as paradigm. Our advanced Y3H system was used to screen for human proteins binding to DENV and ZIKV sfRNA, resulting in a list of 69 putative sfRNA-binders, including several previously reported as well as numerous novel RBP host factors. Our methodology requiring no sophisticated infrastructure or analytic pipeline may be employed for the discovery of meaningful RNA–protein interactions at large scale in other fields.

摘要

登革热 (DENV) 和寨卡病毒 (ZIKV) 等黄病毒是重要的人类病原体，每年导致约 1 亿例有症状的感染。在感染期间，由于病毒 RNA 基因组被细胞外切核糖核酸酶 XRN1 不完全降解，小亚基因组黄病毒 RNA (sfRNA) 在受感染的宿主细胞内形成。尽管 sfRNA 功能的全部范围有待揭示，但这些非编码 RNA 是关键的毒力因子，它们对多个细胞过程的不利影响似乎始终涉及与 RNA 结合蛋白 (RBP) 的分子相互作用。这种 sfRNA 结合宿主因子的发现遵循了既定的生化下拉方法，偏向于阻碍蛋白质组范围覆盖的高丰度蛋白质。酵母三杂交 (Y3H) 系统代表了一种有吸引力的替代方法。为了促进 RBP 的蛋白质组范围筛选，我们通过 (1) 实施全长 ORF 文库结合 (2) 有效酵母交配以增加筛选深度和灵敏度，以及 (3) 严格阴性控制以消除非特异性 RNA 结合剂的过度表达。这些改进采用 DDX6（DEAD-box 解旋酶 6）和 DENV 的 sfRNA 之间的充分表征的相互作用作为范例进行了验证。我们先进的 Y3H 系统用于筛选与 DENV 和 ZIKV sfRNA 结合的人类蛋白质，产生了 69 种推定的 sfRNA 结合剂的列表，包括先前报道的几种以及许多新型 RBP 宿主因子。