CRM1出核转运通路在杆状病毒AcMNPV诱导的病毒蛋白核聚集现象中的作用与分子机制

As a nucleopolyhedrovirus, Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) replication is accompanied by nuclear accumulation of a variety of viral and cellular proteins, which is required for nucleocapsid assembly in the nucleus. Except for a limited number of viral proteins, of which nuclear accumulation mechanisms are previously identified, how most viral proteins relocate to the nucleus during AcMNPV infection remains obscure...Chromosomal maintenance 1 (CRM1) is the most important exportin in eukaryotic cells, and mediates the nuclear export of protein bearing leucine-rich nuclear export sequence (NES). Bioinformatics assay showed that many viral proteins contain putative NESes, and are supposed to be exported from the nucleus through the CRM1-dependent pathway. This finding suggested that AcMNPV may suppress the CRM1-dependent nuclear export, which leads to NES-bearing proteins accumulating in the nucleus, and eventually participating in nucleocapsid assembly process...Our published data demonstrated that the CRM1-dependent nuclear export is inhibited during AcMNPV infection, and viral late gene product Ac34 is responsible for the inhibition (PLoS Pathog. 2016 Nov 1). In this project, we will continue to investigate the impact of Ac34 on virus-induced protein nuclear accumulation and the molecular mechanism of how Ac34 suppressing CRM1 nuclear export. Since AcMNPV is the only virus that specifically inhibiting CRM1, unveiling its inhibition mechanism and significance will shed light on how virus interferes and harnesses host material transport mechinery.

苜蓿银纹夜蛾核型多角体病毒(AcMNPV)在复制过程中需要将众多来自病毒和宿主的蛋白聚集于胞核内，用于核壳体装配等病毒复制过程。除部分病毒蛋白可通过自身编码核定位信号等方式入核，仍有大量病毒蛋白发生核聚集的机制未知。.CRM1是最重要的蛋白出核转运受体之一。我们前期发现AcMNPV可以通过Ac34阻断CRM1通路，从而导致病毒复制所需的宿主蛋白在核内滞留、聚集(PLoS Pathog. 2016 Nov 1)。由于生物信息学预测发现大量AcMNPV蛋白含有潜在的CRM1靶序列，提示我们从出核转运受阻这一全新角度去探索病毒蛋白发生核聚集的原因。我们将利用既有的AcMNPV ORF文库来识别因CRM1出核转运受阻而发生核聚集的病毒蛋白，并探索Ac34阻断CRM1通路的分子机制。鉴于AcMNPV是迄今发现的唯一一种特异性阻断CRM1通路的病毒，该研究必将深化我们对病毒调控细胞物质运输机器的认识。

苜蓿银纹夜蛾核型多角体病毒（AcMNPV）是典型的大DNA病毒。病毒基因组编码超过100个以上的蛋白产物。由于AcMNPV需要在宿主细胞核中完成核壳体的装配，因此需要将大量病毒结构蛋白由胞质转移到核内进行装配。在病毒蛋白核转运过程中，除了被广泛认识的importin介导的核输入作用以外，我们前期发现AcMNPV编码的非结构蛋白Ac34可以通过抑制CRM1介导的核输出通路引起携带有核输出信号（NES）的蛋白滞留在核中。通过对AcMNPV所有病毒蛋白进行生物信息学分析，发现许多病毒蛋白都携带有潜在的NES，提示Ac34-CRM1在病毒蛋白核转移过程中发挥了重要作用。为了识别依赖该通路发生核转移的病毒蛋白，我们利用先前构建的AcMNPV ORF表达文库进行筛选。结果发现在所有140个病毒蛋白中，有37个蛋白主要定位于胞质，43个蛋白主要定位于胞核，60个蛋白呈现全细胞分布。依赖Ac34-CRM1发生核转运的病毒蛋白有Ac102和E26。..为了探讨Ac34抑制CRM1功能的分子机制，我们从三个方面进行了探索。首先，我们发现Ac34对CRM1的蛋白质稳定性并无下调作用；其实，Ac34也没有与含有NES的底物竞争性结合CRM1，即与目前已知的Leptomycin B等CRM1小分子抑制剂作用机制不同；最后我们发现Ac34可以显著增强CRM1与Ran的相互作用，提示Ac34有可能通过调控CRM1出核相关分子来实现对蛋白质出核转运的抑制作用。进一步的分子机制，还有待于深入探索。