Ahmad Asrar, Tigabu Bersabeh, Ivanov Andrey, Jerebtsova Marina, Ammosova Tatiana, Ramanathan Palaniappan, Kumari Namita, Brantner Christine A, Pietzsch Colette A, Abdullah Ghadeer, Popratiloff Anastas, Widen Steve, Bukreyev Alexander, Nekhai Sergei
Howard University.
The University of Texas Medical Branch at Galveston.
Res Sq. 2023 Jun 6:rs.3.rs-2963943. doi: 10.21203/rs.3.rs-2963943/v1.
The Ebola virus (EBOV) transcriptional regulation involves host protein phosphatases PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The 1E7-03 compound, which targets PP1, induces VP30 phosphorylation and inhibits EBOV infection. This study aimed to investigate the role of PP1 in EBOV replication. When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was selected. This mutation moderately reduced EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Formation of EBOV capsids, when NP was co-expressed with VP24 and VP35, was impaired with NPE 619K. Treatment with 1E7-03 restored capsid formation by NP E619K mutation, but inhibited capsids formed by WT NP. The dimerization of NP E619K, tested in a split NanoBiT assay, was significantly decreased (~ 15-fold) compared to WT NP. NP E619K bound more efficiently to PP1 (~ 3-fold) but not B56 subunit of PP2A or VP30. Cross-linking and co-immunoprecipitation experiments showed fewer monomers and dimers for NP E619K which were increased with 1E7-03 treatment. NP E619K showed increased co-localization with PP1α compared to WT NP. Mutations of potential PP1 binding sites and NP deletions disrupted its interaction with PP1. Collectively, our findings suggest that PP1 binding to the NP regulates NP dimerization and capsid formation, and that NP E619K mutation, which has the enhanced PP1 binding, disrupts these processes. 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）的转录调控涉及宿主蛋白磷酸酶PP1和PP2A，它们使EBOV聚合酶VP30的转录辅因子去磷酸化。靶向PP1的1E7-03化合物可诱导VP30磷酸化并抑制EBOV感染。本研究旨在探究PP1在EBOV复制中的作用。当用1E7-03持续处理EBOV感染的细胞时，选择了NP E619K突变。该突变适度降低了EBOV微型基因组转录，而用1E7-03处理可使其恢复。当NP与VP24和VP35共表达时，NPE 619K会损害EBOV衣壳的形成。用1E7-03处理可通过NP E619K突变恢复衣壳形成，但会抑制野生型NP形成的衣壳。在分裂纳米生物发光互补分析中测试，与野生型NP相比，NP E619K的二聚化显著降低（约15倍）。NP E619K与PP1的结合效率更高（约3倍），但与PP2A的B56亚基或VP30不结合。交联和免疫共沉淀实验表明，NP E619K的单体和二聚体较少，而1E7-03处理后会增加。与野生型NP相比，NP E619K与PP1α的共定位增加。潜在PP1结合位点的突变和NP缺失破坏了其与PP1的相互作用。总体而言，我们的研究结果表明，PP1与NP的结合调节NP二聚化和衣壳形成，而具有增强PP1结合能力的NP E619K突变会破坏这些过程。我们的结果表明PP1在EBOV复制中具有新作用，其中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。
