The role of norovirus capsid flexibility in infection and pathogenesis

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Title: The role of norovirus capsid flexibility in infection and pathogenesis Abstract Virus capsids are metastable structures that transition between a stable form in the environment and an unstable form inside the host. Specific cues, generally thought to occur at or inside the cell, are required to elicit this change. This ability of capsids to be flexible is a fundamental feature of virions that is critical for the success of a virus infection. However, there is a fundamental gap in our understanding how capsid flexibility influences norovirus infection and pathogenesis. Noroviruses are prevalent enteric pathogens that cause significant morbidity and mortality worldwide. However, no directed antiviral strategies are approved for use, in part due to our limited understanding of fundamental aspects of their biology. Therefore, the objective of this application is to investigate the role of capsid dynamics in virus biology using murine norovirus (MNV) as a highly tractable model for studies of norovirus biology. Recent studies from us and others have identified key modes of flexibility in the human and murine norovirus capsid that highlight their dynamic nature. Unlike any other virus structure to date, the norovirus capsid exists in two states outside the cell, an expanded conformation where the protruding (P) domain is raised up off the shell (S) domain, and a contracted conformation, where the P domain rests on top of the S domain. The transition between these two states is mediated by environmental cues, including bile acids, a key constituent in the intestinal lumen, luminal pH and kosmotropic ions, like Ca2+. Multiple antibodies against human norovirus bind to epitopes accessible only in the expanded conformation, while receptor binding occupancy is increased in the contracted conformation. A second layer of flexibility lies within the P domain, in external loops that contain epitopes for neutralizing antibodies. Escape from antibody neutralization and bile acid binding to the capsid influence the positioning of these loops. Published and preliminary findings suggest a model whereby the expanded conformation interfaces with the immune system, while the contracted form is optimized for cell/virus interactions. To investigate this hypothesis, we will pursue the following aims in vitro and in vivo: 1) Determine the importance of the flexible linker between P and S domain mediating contraction of the norovirus capsid on infectivity, and 2) Determine the importance of capsid protein loop flexibility on MNV infectivity. Towards that end, we will test viral mutants with varying levels of flexibility by changing the linker length, and viral mutants lacking the bile acid binding site. These conceptually innovative studies promise to be of high impact, because they will define fundamental features of norovirus capsid dynamics and their role in infection and pathogenesis. Such information is important for norovirus vaccine design.