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Authors: Jean Beltran, Pierre Michel
Advisors: Cristea, Ileana M
Contributors: Molecular Biology Department
Keywords: cytomegalovirus
Subjects: Virology
Molecular biology
Issue Date: 2018
Publisher: Princeton, NJ : Princeton University
Abstract: The cell is a complex system that is organized into multiple subcellular compartments known as organelles. Viruses, obligate intracellular pathogens, exploit organelle functions to facilitate viral replication and spread. However, our understanding of the interplay between organelles and virus replication remains limited. I performed a global investigation of organelles during viral infection through the integration of proteomics, computational tools, microscopy, and molecular virology. This study was conducted in the context of Human Cytomegalovirus (HCMV) infection, an important human pathogen. I found that HCMV causes a major rearrangement of organelle morphology. Therefore, I designed a proteomic approach to present the first comprehensive analysis of organelles in both time and space throughout an infected cell. This analysis assigned HCMV proteins to specific organelles, highlighting potential functions in organelle modulation to viral proteins previously uncharacterized. Lysosomes were found to form two subpopulations with distinct protein content late in infection. Moreover, numerous proteins translocated between organelles throughout the infection process. One key finding was that a molecular motor involved in movement of organelles across the cell, known as MYO18A, is targeted to viral-loaded vesicles and is required for efficient virus production. This study revealed an increase in the abundance of peroxisome proteins, which I further investigated by a targeted proteomics approach. I determined that infection causes a temporal regulation of peroxisome proteins, increasing proteins involved in peroxisome biogenesis and lipid metabolism. Moreover, microscopy shows a substantial increase of peroxisome numbers. To investigate the mechanism that underlies this infection-induced increase in peroxisome biogenesis, I integrated microscopy and mathematic modeling to infer the rates of processes controlling peroxisome numbers. This demonstrated that peroxisome fission dominates. By generating a series of CRISPR-mediated knockouts, we demonstrate that peroxisomes are required for efficient viral replication and that the elongated morphology is beneficial to viral replication. Finally, I demonstrate that the infection-induced peroxisome biogenesis increases a peroxisome-derived lipid known as plasmalogen. Probing the different stages of viral replication, I show that plasmalogens are required at the step of cytoplasmic viral assembly. Overall, this study shows that HCMV hijacks peroxisome structure, function, and numbers, increasing plasmalogen abundance used during viral assembly.
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog:
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Molecular Biology

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