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    Interaction of mouse norovirus (MNV) with the cellular immune response of host cells
    Fritzlar, Svenja ( 2017)
    Human noroviruses (HuNoV) cause the majority of non-bacterial gastroenteritis cases worldwide and generate an economic burden of 60 billion USD every year. Noroviruses are highly infectious and predominantly cause issues in closed environments such as cruise ships, hospitals and nursing homes. Due to the lack of a tissue culture or small animal model, HuNoV research has been impaired and so far no drug treatment or vaccine is available. Despite recent advances in the field and the successful replication of HuNoV in B cells and human intestinal organoids, models of HuNoV replication in vitro still remain to be established. Fortunately, murine norovirus (MNV) was discovered in 2003 and has since been used as a model system to investigate NoV infections. In this study we show that MNV infection reduces the surface expression of MHC class I proteins. The reduction in MHC class I levels on the cell surface is based on reduced intracellular levels of the protein. We reveal that MHC class I transcription is not reduced during MNV infection, implying that either MHC class I translation is affected or MHC class I proteins are degraded during MNV infections. We were able to partially rescue the surface expression of MHC class I proteins on MNV infected cells with MG132, a proteasome inhibitor. These findings indicate that MNV interferes with the MHC class I pathway in either directly degrading the protein or targeting it for the degradation pathway within the cell. Furthermore, we identified MNV NS3 as the viral protein which is essential and sufficient for the MHC class I surface reduction when separately expressed in cells. Additionally, we investigated the effect of MNV on cytokine secretion. The secretion of the cytokines IFNβ and TNFα is significantly reduced in MNV infected cells, which is not due to a down regulation of cytokine mRNA transcription. Analysis of the intracellular expression of cytokines and host cell translation in general, revealed a continuous decrease in global host cell translation in MNV infected cells. The translational shutdown seems to be induced by the dsRNA-sensitive regulator PKR. PKR becomes phosphorylated and phosphorylates the translation initiation factor eIF2α, impeding host cell translation. Whilst the translation of host proteins is stalled, viral proteins are still able to be translated due to a cap-independent mechanism. Furthermore, we interrogated the interaction of MNV with the microtubules and the microtubule-associated protein GEF-H1. We discovered an interaction of GEF-H1 with the viral protein MNV NS3, which leads to changes in the expression levels and location of GEF-H1 within the cell and prevents the formation of GEF-H1 induced microtubule fibres. This indicates a potential interference of MNV NS3 with GEF-H1, which has been proposed to play a major role in the immune detection of viral replication. Despite various approaches to identify a similar role of GEF-H1 during MNV infection, we have so far not been able to support the proposed function of the protein. Considering the multiple roles of GEFs like GEF-H1, it is possible that MNV and specifically NS3 acts on a different GEF-H1-regulated pathway during MNV infection.