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Title page for ETD etd-08012014-145043


Type of Document Dissertation
Author Freeman, Megan Culler
URN etd-08012014-145043
Title Cell Biology of Coronavirus Replication
Degree PhD
Department Microbiology and Immunology
Advisory Committee
Advisor Name Title
John Williams Committee Chair
Alissa Weaver Committee Member
Earl Ruley Committee Member
Jim Goldenring Committee Member
Mark Denison Committee Member
Keywords
  • cell to cell spread
  • macropinocytosis
  • fluorescent reporter viruses
  • MHV
  • MERS-CoV
  • SARS-CoV
  • Coronaviruses
  • virology
  • viral replication
Date of Defense 2014-07-16
Availability unrestricted
Abstract
Coronaviruses (CoVs) are positive-strand RNA viruses that induce modifications to host-cell cytoplasmic membranes during formation of replication complexes. While important for viral replication, the dynamics of this process remain poorly understood. Existing reagents largely limited the study of these processes to fixed times, so I developed reporter viruses containing green fluorescent protein or firefly luciferase as a fusion with nonstructural protein (nsp) 2 or 3 to quantitate replication complex formation and virus replication in real time. These viruses replicated with kinetics similar to WT, demonstrating that the CoV genome has the flexibility to accommodate foreign gene addition, despite being the largest replicating RNA molecule. Use of these viruses in live-cell imaging experiments revealed continuous plasma membrane ruffling, vesicle internalization, and production of extended filopodia during CoV infection. Using several complementary techniques, I demonstrate that these membrane rearrangements are due to continuous macropinocytosis induced late during SARS-CoV and MHV infection. Additionally, I discovered that the presence of fusogenic spike on the cell surface was required for macropinocytosis induction, and that this was dependent upon EGFR activation. Ultimately, these results are the first to demonstrate the use of macropinocytosis independent from virus entry, and suggest that CoVs could exploit macropinocytosis to facilitate cell-to-cell spread during infection. During this work, a new CoV, Middle East Respiratory Syndrome (MERS) CoV, emerged in Saudi Arabia. Due to our interest in replication complex formation and membrane rearrangements, we developed a panel of antibodies to recognize MERS-CoV nsps. Several of these antibodies were highly specific for MERS-CoV nsps, allowing us to investigate the spatial and temporal formation of replication complexes in relation to cellular markers. This work is the first to visualize formation of these complexes during MERS-CoV infection and provides new reagents critical for future studies. Overall, my work has used novel reporter viruses to demonstrate the flexibility of the CoV genome, quantitate replication complex formation during infection, and provide early measurement of viral translation. Furthermore, I identified a novel function for viral exploitation of macropinocytosis and defined new virus-host interactions required for efficient CoV replication through the potential interaction of spike and EGFR.
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