A genome-wide analysis of the spatio-temporal distribution of herpesviral proteins in the course of infection

疱疹病毒生命周期的关键环节，包括DNA复制、基因转录和RNA输出、衣壳形态发生和出口，都发生在宿主核中，需要大量的疱疹病毒蛋白在宿主胞浆和细胞核之间运输。因此，病毒蛋白在细胞核和细胞质之间的时空协调分布对病毒复制至关重要。进入的疱疹病毒衣壳与核孔复合体(NPC)对接，并将病毒DNA释放到核内部，是核输入的一种变体，也是病毒感染的另一个关键步骤。一般来说，蛋白质和一些RNA在细胞质和细胞核之间的运输是通过与Importin(和)家族的运输因子直接或间接结合而介导的，并沿着小GTP酶Ran的梯度发生。到目前为止，大量病毒蛋白的核质转运机制尚不清楚，那些介导衣壳对接的病毒蛋白还没有被鉴定出来。在这个由两个在核质转运(S.Bailer)和高通量传染病筛查技术(J.Haas)方面拥有高度专业知识的团队合作的项目中，我们将应用一种新的方法，通过并行执行大规模酵母双杂交(Y2H)筛查和体外生化分析，全面分析疱疹病毒蛋白与真核细胞Importin(和)家族所有已知运输因子之间的相互作用。这些运输因子的新型病毒载体将得到功能验证，并系统地确定病毒蛋白及其核运输信号的亚细胞定位。然后，生物信息学工具将收到的数据结合在一起，创建疱疹病毒感染期间病毒蛋白质和衣壳的时空分布的动态模型。这一分析不仅将大大增加我们对疱疹病毒生物学的了解，揭示病毒治疗的潜在新靶点，而且还将改进对细胞核运输货物的新共识序列的电子预测。

Crucial aspects of the herpesviral life cycle including DNA replication, gene transcription and RNA export, capsid morphogenesis, and egress occur in the host nucleus and require that numerous herpesviral proteins are transported between the host cytosol and the nucleus. The coordinated spatio-temporal distribution of viral proteins between nucleus and cytoplasm is thus vital for viral replication. The docking of incoming herpesviral capsids to the nuclear pore complex (NPC) and the release of viral DNA into the nuclear interior represents a variant of nuclear import and another critical step of viral infection. In general, transport of proteins and some RNAs between cytoplasm and nucleus is mediated by a direct or indirect binding to transport factors of the importin ( and (-families and occurs along a gradient of the small GTPase Ran. So far the mechanism of nucleo-cytoplasmic transport is unknown for a large number of viral proteins and those viral proteins mediating capsid docking have not been identified. In this collaborative project between two groups with high expertise in nucleocytoplasmic transport (S. Bailer) and high-throughput-based screening technologies in infectious diseases (J. Haas), we will apply a novel approach to comprehensively analyse the interactions between herpesviral proteins and all known transport factors of the eukaryotic importin ( and (-families by performing large-scale yeast-two-hybrid (Y2H) screens and in vitro biochemical assays in parallel. The novel viral cargoes of these transport factors will be functionally verified and the subcellular localization of viral proteins and their nuclear transport signals determined systematically. The data received will then be combined by bioinformatical tools to create a dynamic model of the spatio-temporal distribution of viral proteins and capsids during herpesviral infection. This analysis will not only considerably increase our understanding of herpesviral biology and reveal potential novel targets for viral treatment, but also improve the in silico prediction of novel consensus sequences for cellular nuclear transport cargoes.