Structural Study of Tulane Virus and Its Host Cell Factors and Applications in Cryo-EM
thesisposted on 2021-12-03, 03:26 authored by Chen SunChen Sun
Currently, human norovirus is the leading cause of acute gastroenteritis and accounts for most cases of foodborne illnesses in the United States each year. Due to its tissue culture inefficiency, studies of human norovirus have been crippled for more than forty years.Tulane virus (TV) stands out as a suitable surrogate of human norovirus given its high amino acid identity with human norovirus and its well-established cell culture system. It was first isolated from rhesus macaques (Macaca mulatta) in 2008 and identified as a novel Calicivirusrepresenting a new genus, Recovirus genus (Farkas et al., 2008). However, there are still unanswered questions about its infectious cycle and the essential factors for its infection. In this study, we have obtained a TV variant (the 9-6-17 strain) that has lost the binding ability to the B-type histo-blood group antigen (HBGA), which was proposed to be the receptor of both TV and human norovirus. In the first chapter, we outline how the sequence analysis,structural biology studies, and mutagenesis studies of the 9-6-17 TV strain have shed light on the interaction with its host cell receptor. To investigate the key residues for HBGA binding, we established the full-length infectious clone of the 9-6-17 TV strain. We present a highly selective transformation of serine 367, located in the predicted HBGA binding site, into a lysine residu e. Our results advance the understanding of genetic changes in TV required for adaptation to cell culture environments. Cryo-EM is an awarding winning technique that has been the greatest scientific breakthrough in recent years. It was awarded the Nobel Prize in Chemistry in 2017. Despite the technological advances of the direct electron detector and image processing software, several major roadblocks remain for high-resolution structure determination with cryo-EM. In the later chapters, we explored the most efficient way of using VPP to enhance image contrast, how to tackle the airwater interface problem by encapsulating target protein, how to reach a higher resolution by refining high order parameters, and the helical indexing problem in real space. These technical advances would benefit the whole cryo-EM community by providing convenient tools or insights for future directions.