Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.

病毒通过劫持宿主的泛素机制来抵消或重定向细胞过程来促进感染。腺病毒编码两种早期蛋白，E1B55K 和 E4orf6，它们共同选择细胞泛素连接酶复合物来克服宿主防御并促进病毒产生。缺乏 E1B55K 或 E4orf6 的腺病毒突变体在病毒 RNA 加工和蛋白质生产方面表现出缺陷，但先前鉴定的重定向连接酶的底物并不能解释这些表型。在这里，我们使用定量蛋白质组学方法来鉴定 E1B55K/E4orf6 介导的泛素化底物，以促进 RNA 加工。虽然目前已知的所有 E1B55K 和 E4orf6 细胞底物都会被蛋白酶体降解，但我们发现 RNA 结合蛋白是高置信度底物，其总体丰度并未减少。我们重点关注两种 RNA 结合蛋白 RALY 和 hnRNP-C，我们确认它们被泛素化而不会降解。在 E1B55K 缺失病毒感染期间，RALY 和 hnRNP-C 的敲低增加了病毒 RNA 剪接水平、蛋白质丰度和后代产量。此外，感染 E1B55K 缺失病毒会导致 hnRNP-C 与病毒 RNA 的相互作用增加，并减弱病毒 RNA 加工。这些数据表明，病毒介导的 RALY 和 hnRNP-C 泛素化缓解了对病毒 RNA 加工的限制，并揭示了非降解性泛素化在病毒感染期间操纵细胞过程中的意外作用。