Arenaviruses

Abstract

To my husband, Marc, for his endless encouragement and support iii ACKNOWELDGEMENTS First, I would like to thank my mentor, Dr. Wendy Maury. Without her, none of this would have been possible. I would also like to thank all the people in the Department of Microbiology with whom I have worked over the last four years and my committee members who took the time to meet with me about my work each year.Particularly, I would like to acknowledge Nick Lennemann, Beth Rhein, and Sven Moller-Tank. Sven was always a source of amusement and good advice. Nick taught me essentially everything I needed to know to succeed and helped me start my first project.Lastly, Beth Rhein is a great friend. I cannot imagine my graduate career without her. I would also like to make special note of an undergraduate research assistant who worked tirelessly by my side, Elisabeth Phillips. She regularly exceeded her responsibilities and assisted me with countless experiments needed for my dissertation.Her dedication and work ethic are unmatched.Finally, I would like to thank my friends and family for their love and support. In particular, I thank my husband and best friend Marc. He has always been indispensable. iv ABSTRACTThe family Arenaviridae consists of over 30 members, some of which can infect humans and cause severe hemorrhagic fever. The three arenaviruses studied in this thesis, Machupo virus (MACV), Junín virus (JUNV), and Lassa virus (LASV), are causative agents of Bolivian hemorrhagic fever, Argentine hemorrhagic fever, and Lassa fever, respectively. Epidemics of these diseases can carry high rates of morbidity and mortality, and due to a lack of available countermeasures, all three viruses are considered category A priority pathogens by the CDC.Arenavirus glycoproteins (GPCs) are considered class I viral fusion proteins, but in multiple regards, they are quite unusual for viral envelope proteins. The GP precursor is translated as a polypeptide that is proteolytically processed within the secretory pathway by two sequential cleavage events to produce a tripartite complex (heterotrimers) that assemble into homotrimers to form heterononamers. The three distinct units of the GPC structure are a receptor binding domain, GP1, and fusion domain, GP2, and most peculiarly, a stable signal peptide (SSP) that traverses the membrane twice and associates with the GP2.The glycoprotein complex (GPC) of MACV encodes nine putative sites for Nlinked glycosylation. As N-glycans have been shown to be important for a multitude of factors in glycoprotein biology, we sought to investigate the potential roles of the Nglycans on MACV GPC expression, function, antigenicity, and immunogenicity. To do so, we used MACV GPC-VSVG-eGFP pseudovirions (MACV-VSV) to study the effects of both individual and combinatorial N-glycan losses. Our results demonstrate that loss of N-glycans at sites N178, N370, or N378 resulted in a loss of GPC proteolytic processing, while the accumulation of multiple N-glycans reduced total GPC expression and function. Replacement of the ...

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