Role of Nuclear Interactions of Paramyxoviral Matrix Proteins: Cellular ANP32B in Host Manipulation and Virus Replication

我们最近发现富含酸性亮氨酸的核磷蛋白32家族成员B(ANP32B)是Hendra和Nipah病毒基质蛋白(M)的核靶标。进一步的初步数据表明，副粘病毒家族的另一个成员也能够通过M与ANP32B相互作用。因此，我们得出结论，靶向ANP32B是一种保守机制的一部分，至少在副粘病毒亚家族中是这样。确定这种相互作用在病毒复制和宿主细胞操纵中的详细作用可能导致确定迄今尚不清楚的复制和发病机制。为了阐明ANP32B靶向的作用，我们研究了(I)哪些病毒M蛋白可以与核靶相互作用，(Ii)M蛋白中的哪些序列是特定相互作用所必需的，(Iii)ANP32B如何影响病毒复制，以及(Iv)ANP32B在非常规核输出、基因调控和细胞凋亡抑制中的细胞功能是否受相互作用的M蛋白的调节。宿主细胞的调节活性可能在病毒在感染宿主中的复制中发挥重要作用，因此对了解病毒的毒力和致病机制具有重要意义。在该项目中，已建立的蛋白质相互作用分析将与M蛋白质的系统突变和重组ANP32B结合缺陷病毒突变体的产生相结合。对ANP32B功能的影响将通过核输出、基因调控和在表达质粒的M蛋白存在的情况下以及在病毒感染的细胞中进行凋亡检测来解决。特异性M/ANP32B依赖的分子机制的体外鉴定和结合缺陷病毒突变体的开发将为研究ANP32B在疾病发生和免疫逃避中的作用提供突出的可能性。鉴于这项为期三年的提案的工作计划侧重于分子机制的体外表征，在后续工作中，开发的病毒突变和对受影响途径的了解将使我们能够研究细胞相互作用因子在黑尼帕病毒和其他副粘病毒体内复制和发病中的作用。由于该项目的重点是宿主-细胞界面，这可能对复制和宿主操纵有很大贡献，拟议的工作不仅将有助于更好地从分子上了解病毒复制，还可能为合理干预病毒复制或疾病发展提供新的分子靶点。

We recent identified cellular acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) as a nuclear target for Hendra and Nipah virus matrix protein (M). Further preliminary data show that also another member of the paramyxovirus family is able to interact with ANP32B through M. We therefore conclude that targeting of ANP32B is part of a conserved mechanism, at least within the paramxovirinae subfamily. Identification of the detailed role of this interaction in virus replication and host cell manipulation may lead to identification of a so far unknown mechanism involved in replication and pathogenesis. To clarify the role of ANP32B targeting we here investigate (i) which viral M proteins can interact with the nuclear target, (ii) what sequences in the M proteins are required for specific interaction, (iii) how ANP32B influences virus replication and (iv) whether cellular functions of ANP32B in unconventional nuclear export, gene regulation and apoptosis inhibition are modulated by interacting M proteins. Host cell modulatory activities may play a substantial role in virus replication in infected hosts and thus are of high importance to understand virulence and pathogenesis of viruses. Within the project, established protein interaction assays will be combined with systematic mutagenesis of M proteins and generation of recombinant ANP32B-binding defective virus mutants. Influences on ANP32B functions will be addressed by specific assays for nuclear export, gene regulation and apoptosis detection in the presence of plasmid expressed M proteins and in virus infected cells. In vitro identification of specific M / ANP32B dependent molecular mechanisms and development of binding defective virus mutants will offer the outstanding possibility to investigate the role of ANP32B in disease development and immune escape. Whereas the work program of this three years proposal is focused on in vitro characterization of molecular mechanisms, in continuative work the developed virus mutants and knowledge about affected pathways will allow to investigate the role of the cellular interactor for in vivo replication and pathogenesis of henipa- and other paramyxoviruses. As this project focuses on a host-cell interface that may substantially contribute to replication and host manipulation, the proposed work will not only contribute to a better molecular understanding of virus replication but may also offer novel molecular targets for rational interference with virus replication or disease development.