Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America.
Molecular Biology and Virology Laboratory, Faculty of Medicine and Health Sciences, Corporación Universitaria Empresarial Alexander von Humboldt, Armenia, Colombia.
PLoS Biol. 2024 Feb 29;22(2):e3002544. doi: 10.1371/journal.pbio.3002544. eCollection 2024 Feb.
Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.
埃博拉病毒（EBOV）属于高度致病病毒家族，可导致人类严重出血热。EBOV 的复制需要病毒聚合酶复合物的活性，该复合物包括辅助因子和干扰素拮抗剂 VP35。我们之前的研究表明，VP35 的共价泛素化通过调节与聚合酶复合物的相互作用来促进病毒复制。此外，VP35 还可以与泛素（Ub）非共价相互作用；然而，这种相互作用的功能尚不清楚。在这里，我们报告 VP35 与游离（无锚）K63 连接的多 Ub 链相互作用。异位表达已知可降解无锚多 Ub 链的肽酶 T（USP5），可减少 VP35 与 Ub 的结合，并与微基因组测定中的聚合酶活性降低相关。我们使用计算方法构建了 VP35-Ub 非共价相互作用的模型，鉴定了 VP35-Ub 相互作用的表面，并测试了突变以验证该界面。对接模拟确定了可阻断 VP35-Ub 相互作用从而降低病毒聚合酶活性的化学化合物。这些化合物的处理降低了细胞内和体内感染性 EBOV 的复制，在小鼠模型中也有很好的效果。总之，我们发现了无锚多 Ub 在调节埃博拉病毒聚合酶功能中的新作用，并发现了具有潜在抗埃博拉病毒活性的化合物。
