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

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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 The West lab has solved the high-resolution X-ray structures of variant EBNA1 DBDs (unpublished).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.