The E3 ligase RAD18-mediated ubiquitination of henipavirus matrix protein promotes its nuclear-cytoplasmic trafficking and viral egress

The nuclear-cytoplasmic trafficking of matrix proteins (M) is essential for henipavirus budding, with M protein ubiquitination playing a pivotal role in this dynamic process. Despite its importance, the intricacies of the M ubiquitination cascade have remained elusive. In this study, we elucidate a novel mechanism by which Nipah virus (NiV), a highly pathogenic henipavirus, utilizes a ubiquitination complex involving the E2 ubiquitin-conjugating enzyme RAD6A and the E3 ubiquitin ligase RAD18 to ubiquitinate the virus's M protein, thereby facilitating its nuclear-cytoplasmic trafficking. We demonstrate that RAD18 interacts with RAD6A, enabling the latter to supply ubiquitins for the RAD18-mediated transfer of ubiquitin to M through RAD18-M interactions. Specifically, M is ubiquitinated by the RAD6A-RAD18 complex at lysine (K) 258 through a K63-linked ubiquitination, a modification crucial for M's function. This ubiquitination drives M's relocation to the cytoplasm, directing it to plasma membranes for effective viral egress. Conversely, disrupting the RAD6A-RAD18-M axis, mutating RAD18′s E3 ligase activity, or inhibiting RAD6A activity with TZ9 (a RAD6-ubiquitin thioester formation inhibitor) impairs M ubiquitination, resulting in defective nuclear export and budding of NiV. Significantly, live NiV and Hendra virus infection is attenuated in RAD18 knockout cells or in cells treated with TZ9, highlighting the critical physiological role of RAD6A-RAD18-mediated M ubiquitination in the henipavirus life cycle. Our findings not only reveal how NiV manipulates a nucleus-localized ubiquitination complex to promote virus's M protein ubiquitination and nuclear export, but also suggest that the small molecule inhibitor TZ9 could serve as a potential therapeutic against henipavirus infection.

基质蛋白（matrix proteins，简称M）的核质运输对于亨尼帕病毒（henipavirus）出芽至关重要，M蛋白泛素化在该动态过程中发挥关键作用。尽管其重要性不言而喻，但M蛋白泛素化级联反应的复杂机制迄今仍未明确。本研究阐明了高致病性亨尼帕病毒——尼帕病毒（Nipah virus，NiV）的全新机制：其通过包含E2泛素结合酶（E2 ubiquitin-conjugating enzyme）RAD6A与E3泛素连接酶（E3 ubiquitin ligase）RAD18的泛素化复合物对病毒M蛋白进行泛素化，进而促进其核质运输。

我们证实RAD18可与RAD6A相互作用，使后者能够通过RAD18与M蛋白的结合，为RAD18介导的泛素向M蛋白的转移提供泛素分子。具体而言，M蛋白在赖氨酸（lysine，K）258位点被RAD6A-RAD18复合物以K63位连接的泛素化方式修饰，这一修饰对M蛋白的功能至关重要。该泛素化修饰驱动M蛋白向细胞质移位，并引导其抵达细胞膜以实现高效的病毒出芽释放。

反之，破坏RAD6A-RAD18-M调控轴、突变RAD18的E3连接酶活性，或使用TZ9（一种RAD6-泛素硫酯形成抑制剂，RAD6-ubiquitin thioester formation inhibitor）抑制RAD6A活性，均会损害M蛋白的泛素化，导致尼帕病毒的核输出与出芽过程出现缺陷。值得注意的是，在RAD18基因敲除细胞或经TZ9处理的细胞中，活尼帕病毒与亨德拉病毒（Hendra virus）的感染能力均被减弱，这凸显了RAD6A-RAD18介导的M蛋白泛素化在亨尼帕病毒生命周期中的关键生理作用。

本研究的发现不仅揭示了尼帕病毒如何利用定位于细胞核的泛素化复合物促进病毒M蛋白泛素化与核输出，同时也提示小分子抑制剂TZ9可作为对抗亨尼帕病毒感染的潜在治疗手段。