Ahmad Asrar, Tigabu Bersabeh, Ivanov Andrey, Jerebtsova Marina, Ammosova Tatiana, Ramanathan Palaniappan, Kumari Namita, Brantner Christine A, Pietzsch Colette A, Simhadri Jyothirmai, Abdullah Ghadeer, Uversky Vladmir N, Paromov Victor, Popratiloff Anastas, Widen Steve, Bukreyev Alexander, Nekhai Sergei
Center for Sickle Cell Disease, Howard University, Washington, District of Columbia, USA.
Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, Texas, USA.
J Biol Chem. 2025 Apr 25;301(6):108541. doi: 10.1016/j.jbc.2025.108541.
Ebola virus (EBOV) replication is regulated by the host protein phosphatases, PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The PP1-targeting compound 1E7-03 induces VP30 phosphorylation and inhibits EBOV infection. Here, we investigate the broader role of PP1 in EBOV replication and transcription, including its interaction with nucleoprotein (NP). When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was found and selected for further analysis. The NP E619K mutation moderately reduced the EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Proteomics, immunoprecipitation, dimerization, split NanoBit, and colocalization analyses indicated that NP interacts with PP1 and that NP E619K mutations enhanced this binding. Treatment with 1E7-03 dissociated PP1-NP complex, but enhanced NP dimerization, which was more pronounced for NP E619K mutant. Mutation and deletion analyses pointed to several potential PP1-binding sites in NP that were located in the moderately disordered NP regions. When NP was co-expressed with VP24 and VP35, formation of EBOV capsids was impaired with NP E619K mutation. Treatment with 1E7-03 restored the capsid formation by the NP E619K mutant but inhibited capsids formed by WT NP. Our findings suggest that PP1 binds to NP and that this binding might regulate NP dimerization and capsid formation. Collectively, our results point to a new role for PP1 in EBOV replication, in which NP binding to PP1 may facilitate viral transcription by delaying capsid formation and EBOV replication.
埃博拉病毒（EBOV）的复制受宿主蛋白磷酸酶PP1和PP2A调控，这两种酶使EBOV聚合酶VP30的转录辅因子去磷酸化。靶向PP1的化合物1E7-03可诱导VP30磷酸化并抑制EBOV感染。在此，我们研究PP1在EBOV复制和转录中的更广泛作用，包括其与核蛋白（NP）的相互作用。当用1E7-03持续处理EBOV感染的细胞时，发现并选择了NP E619K突变进行进一步分析。NP E619K突变适度降低了EBOV微型基因组转录，而用1E7-03处理可使其恢复。蛋白质组学、免疫沉淀、二聚化、分裂纳米荧光互补分析以及共定位分析表明，NP与PP1相互作用，且NP E619K突变增强了这种结合。用1E7-03处理可使PP1-NP复合物解离，但增强了NP二聚化，这在NP E619K突变体中更为明显。突变和缺失分析指出NP中几个潜在的PP1结合位点位于中度无序的NP区域。当NP与VP24和VP35共表达时，NP E619K突变会损害EBOV衣壳的形成。用1E7-03处理可恢复NP E619K突变体的衣壳形成，但抑制野生型NP形成的衣壳。我们的研究结果表明，PP1与NP结合，这种结合可能调节NP二聚化和衣壳形成。总体而言，我们的结果指出了PP1在EBOV复制中的新作用，即NP与PP1的结合可能通过延迟衣壳形成和EBOV复制来促进病毒转录。
