Murine Norovirus 1 Productively Infects Murine Macrophages in a Dynamin II-, Cholesterol-, and USP14 Dependent Mechanism but is Independent of Endosome Acidification, Clathrin, Caveolin, Flotillin, GRAF 1, and Phagocytosis.

Abstract

Viruses are obligate intracellular parasites that require their hosts for all steps of the viral life cycle. The mechanism of how an inert particle of protein, lipid, and nucleic acid usurps the machinery of the host to enter its host is an critical first step to elucidating the pathogenesis of a virus. The mechanism by which Murine Norovirus (MNV), a nonenveloped postive strand RNA virus, productively infects murine macrophages and dendritic cells was the topic of my thesis work. I have demonstrated that MNV enters cells in a cholesterol-, and dynamin II- dependent mechanism. After entering into the permissive cell, I observed that the virus does not require acidification of the endosome as a trigger to begin infection. To demonstrate the requirements MNV-1 entry, I performed experiments with pharmacological inhibitors, dominant negative constructs, and siRNA knockdown. Once the viral genome has been released into the host cytoplasm, it trafficks to the site of viral replication. For positive strand RNA viruses, this site of replication requires the recruitment of host derived membrane. For MNV-1, the requirement of membrane derived from the endoplasmic reticulum (ER) has been previously demonstrated. We serendipitiously discovered a small inhibitor of MNV-1 infection which required activation of the unfolded protein response (UPR) through inhibition of host deubiquitinases, specifically USP14. The UPR is a cellular response to the accumulation of unfolded protein in the endoplasmic reticulum, and activation of the UPR by various stimulants, including virus infection, has been suggested. Once the UPR is activated the synthesis and recruitment of ER membranes could be impaired. The impairment of ER membrane recruitment to the viral replication factories is one mechanism to explain the inhibition of MNV-1 replication observed during treatment with the small molecule. We also demonstrated that La Crosse virus, encephalomyocarditis virus, and Sindbis virus infections are inhibited by induction of the UPR. These results suggest that the UPR could be an important target for the development of antiviral therapies. Clearly, there is further investigation required to determine the requirements of norovirus entry and infection, but my research has laid a framework for further investigation.