Ebola virus VP24 alters hnRNP C nuclear import: implications for replication

描述（由申请人提供）：人类感染埃博拉病毒（EBOV）通常导致严重的出血热，在高达90%的感染病例中是致命的。因此，了解埃博拉病毒发病机制的分子机制对于开发成功的治疗方法至关重要。先前的研究表明，EBOV VP24通过结合核丝蛋白a （KPNA）家族的特定成员来阻止STAT-1的核积累，从而阻止I型和II型干扰素（IFN）信号传导。VP24阻断IFN信号传导的能力是EBOV抑制宿主对感染反应的一个例子。本提案的目的是测试VP24与KPNAs的结合是否会阻止其他细胞蛋白的结合和随后的核易位。我们推测VP24阻断了额外细胞蛋白的细胞核输入，并将这些蛋白重新分配到细胞质中，从而促进EBOV的复制。为此，我们证明了异质核糖核蛋白C1/C2复合物（hnRNP C1/C2）与KPNA家族成员相互作用，并且在VP24的存在下，hnRNP C1/C2与KPNA1的结合减弱。VP24抑制hnRNP C1/C2与KPNA1的结合是非常有趣的，因为hnRNP CI/ C2在其他RNA病毒的复制周期中发挥了重要作用。因此，本研究的第一个目的是全面表征VP24、NPI-I KPNA家族成员和hnRNP C1/C2之间的相互作用，并确定VP24/KPNA相互作用是否足以阻止hnRNP CI/ C2核积累。由于hnRNP C1/C2在其他系统中促进病毒RNA和蛋白质的合成，第二个目的将测试hnRNP C1/C2是否与EBOV RNA相互作用并影响病毒复制和/或蛋白质翻译。本提案的第三个目标侧重于开发检测方法来筛选抗VP24 IFN拮抗剂的小分子抑制剂。识别小分子抑制剂，削弱EBOV介导的IFN拮抗作用（和可能的复制），将对治疗EBOV非常有用。公共卫生相关性：EBOV是一种极其致命的微生物，虽然感染罕见，但可能发生来自自然或非自然来源的其他疫情。本提案的目的是进一步确定EBOV VP24如何调节正常的细胞核输入，并鉴定抑制VP24功能的小化合物！这些研究获得的数据可以加强对EBOV发病机制的分子机制的认识，并确定有效的药物来抑制EBOV的复制。

DESCRIPTION (provided by applicant): Ebola virus (EBOV) infection of humans often results in severe hemorrhagic fever which is lethal in up to 90% of infected cases. Therefore, understanding the molecular mechanisms of Ebola virus pathogenesis is essential for development of successful therapeutics. Previous work has demonstrated that EBOV VP24 prevents type I and II Interferon (IFN) signaling by binding to the specific members of the karyopherin a (KPNA) family of nuclear import proteins to prevent nuclear accumulation of STAT-1. The ability of VP24 to block IFN signaling is one example of how EBOV can suppress the host response to infection. The goal of this proposal is to test if VP24 binding to KPNAs prevents binding and subsequent nuclear translocation of additional cellular proteins. We hypothesize VP24 blocks the nuclear import of additional cellular proteins and redistributes these proteins to the cytoplasm to promote EBOV replication. To this end, we have demonstrated that the heterogeneous ribonuclear protein C1/C2 complex (hnRNP C1/C2) interacts with members of the KPNA family, and that hnRNP C1/C2 binding to KPNA1 is diminished in the presence of VP24. VP24 inhibition of hnRNP C1/C2 binding to KPNA1 is of great interest since hnRNP CI/ C2 has been shown to play an important role in the replication cycle of other RNA viruses. Therefore, the first aim of this proposal is to fully characterize the interactions between VP24, the NPI-I KPNA family members and hnRNP C1/C2 and determine if VP24/KPNA interactions are sufficient to prevent hnRNP CI/ C2 nuclear accumulation. Since hnRNP C1/C2 facilitates both viral RNA and protein synthesis in other systems, the second aim will test if hnRNP C1/C2 interacts with EBOV RNA and impacts viral replication and/or protein translation. The third aim of this proposal focuses on developing assays to screen for small molecule inhibitors against VP24 IFN antagonism. Identifying small molecular inhibitors that impair EBOV mediated IFN antagonism (and likely replication) would be very useful for treating EBOV. PUBLIC HEALTH RELEVANCE: EBOV is an extremely lethal microbe and while infection is rare, it is possible that additional outbreaks either from a natural or unnatural source could occur. The aims presented in this proposal further define how EBOV VP24 modulates the normal cellular nuclear import and identify small compounds to inhibit VP24 function! Data obtained from these studies could strengthen the fields' understanding of the molecular mechanism of EBOV pathogenesis as well as identify effective drugs to inhibit EBOV replication.