Viral variants: assessing the impact of natural strain variation on the structure and function of virus replication and transcription factors

流行的伽马疱疹病毒Epstein-Barr病毒有两个主要毒株(类型1和类型2)，它们只在驱动致病性的两个关键转录因子(EBNA2和EBNA3C)的序列上存在差异。缺乏关于这些转录因子结构的信息，阻碍了对这种变异的功能影响的理解。West实验室最近展示了EBNA2序列变异如何影响与宿主细胞转录抑制因子的相互作用(Ponnusamy等人，2019年)。重要的EBV复制因子EBNA1最近被发现。这种变异既存在于基本DNA结合域(DBD)内部，也存在于其外部。West实验室已经解决了不同EBNA1 DBD的高分辨率X射线结构(未发表)。EBNA1 N末端的额外变异与DBD变异有关，但没有EBNA1的全长结构来理解这些共同相关变化的结构或功能基础。这个项目将：1.使用蛋白质模型来深入了解类型1和类型2的变异对EBNA2和EBNA3C结构和功能的影响，并使用合理的突变和建立的生化和基于细胞的功能分析来检验预测。2.利用EBNA1 DBD变异X射线结构和蛋白质模拟揭示DBD中共伴生氨基酸变异的结构基础。二聚化和DNA结合分析将被用来检验预测。3.利用蛋白质模型预测EBNA1的全长结构，以了解EBNA1 N末端和DBD共同变化的结构基础。预测将在功能分析中得到验证。影响：增加对病毒变异与病毒学和疫苗学许多方面有关的功能影响的了解，以及关于复制和转录因子功能的结构基础的关键信息。

The prevalent gamma-herpesvirus Epstein-Barr Virus has two main strains (type1 and type2) that vary only in the sequence of two key transcription factors (TFs) that drive pathogenicity (EBNA2 and EBNA3C). Understanding the functional impact of this variation is hampered by a lack of information on the structure of these TFs. The West lab recently showed how EBNA2 sequence variation impacts interactions with host cell transcription repressors (Ponnusamy et al, 2019).New variation in the essential EBV replication factor, EBNA1, was recently identified. This variation lies both within and outside of the essential DNA-binding domain (DBD). The West lab has solved the high-resolution X-ray structures of variant EBNA1 DBDs (unpublished). Additional variation in the N-terminus of EBNA1 co-segregates with DBD variation, but there is no full length structure of EBNA1 to understand the structural or functional basis of these co-associated changes. This project will: 1. Use protein modelling to gain insights into the impact of type 1 and type 2 variation on the structure and function of EBNA2 and EBNA3C and to test predictions using rational mutagenesis and established biochemical and cell-based functional assays. 2. Use variant EBNA1 DBD X-ray structures and protein modelling to uncover the structural basis for co-associated amino acid variation in the DBD. Dimerisation and DNA-binding assays will be used to test predictions. 3. Use protein modelling to predict the full-length structure of EBNA1 to understand the structural basis of co-associated changes in the EBNA1 N-terminus and DBD. Predictions will be tested in functional assays. Impact: increased understanding of the functional impact of viral variation relevant to many aspects of virology and vaccinology and key information on the structural basis of replication and transcription factor function.