Differential Remodeling of Multivesicular Bodies by Human Cytomegalovirus (HCMV) in Endothelial Cells and Fibroblasts
AdvisorWilson, Jean M.
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PublisherThe University of Arizona.
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EmbargoRelease after 01/01/2021
AbstractHuman cytomegalovirus (HCMV) is a prevalent and opportunistic pathogen. HCMV has many modes of cellular entry and egress, depending upon the cell type. How the virus uses host cell machinery to exit different cell types is poorly understood. Previous studies found that viral tegument proteins affect the multivesicular body (MVB) formation and viral maturation differently in fibroblasts and endothelial cells, providing a possible mechanism for the distinct cellular tropisms. We hypothesize that HCMV infection induces cell-type specific remodeling of the MVBs in endothelial cells (EC) and fibroblasts to facilitate viral egress. To define the nature of infection-induced MVBs, we infected cells with HCMV that had been engineered to express a fusion protein containing the tegument protein pp150 (gene UL32) fused in frame with green fluorescent protein (GFP). Four days after infection, cells were fixed and labeled with antibodies against subcellular compartment markers and imaged using confocal and super-resolution microscopy. In fibroblasts, we find that the UL32/GFP- positive vesicles colocalize with CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the coat protein, clathrin. In endothelial cells, UL32/GFP- positive vesicles colocalized with clathrin, GM130 (cis-Golgi marker), LAMP1 (lysosomal marker), and Rab27a (associated with exocytosis of lysosome related organelles), but not with classical late endosome (LE) marker, CD63 and LBPA. These findings suggest that virus containing MVBs in fibroblasts are derived from the endocytic pathway whereas those in endothelial cells are derived from the early biosynthetic pathway. We propose that fibroblasts use the classical MVB/exosome pathway to facilitate viral egress whereas endothelial cells exploit a less characterized early Golgi/LAMP1 associated secretory granule pathway for egress.
Degree ProgramGraduate College
Cellular & Molecular Medicine