Cellular Determinants of Reovirus Entry and Infection

Efficient entry is a critical determinant of reovirus oncolysis. While a model for the entry of reovirus virions has been described, the method by which partially uncoated intermediate subviral particles (ISVPs) enter host cells remains unknown. Biochemical studies have suggested that ISVPs can directly penetrate the plasma membrane since these particles can cause membrane damage and hemolysis at high multiplicities of infection. We used a combination of techniques to show that these particles can use endocytosis to enter and productively infect host cells. We found that uptake of reovirus virions and ISVPs was inhibited in cells treated with a small molecule inhibitor of dynasore. Analysis of reovirus replication in cells treated with genistein and in cells expressing dominant-negative caveolin-1 revealed that ISVPs use dynamin-dependent caveolar endocytosis to enter host cells. These studies also showed that reovirus virions were able to take advantage of caveolar endocytosis to infect cells. Because many viruses have recently been found to enter cells through dynamin-dependent and -independent endocytic pathways, we also assessed the role of cholesterol in reovirus infection. The growth of reovirus virions, but not ISVPs, was inhibited in cells treated with methyl-β-cyclodextrin, which extracts membrane cholesterol. We also wanted to learn whether reovirus entry had cell type-specific differences. Analysis of reovirus infection in a rat embryo fibroblast (CREF) and Ras-transformed CREF cell line suggested that reovirus particles lose the ability to use clathrin-mediated endocytosis in CREF cells following Ras transformation. Reovirus can also infect cells that lack caveolae. To assess whether reovirus used different endocytic pathways to infect cells devoid of caveolae, we analyzed reovirus entry and infection in polarized Caco-2 cells. While reovirus infection of these cells was dynamin-independent, particle internalization occurred through both dynamin-dependent and -independent pathways. Visualization of particle uptake showed that particles internalized through dynamin-dependent endocytosis were transcytosed to the basolateral surface. These findings have demonstrated that reovirus can use a diverse set of endocytic pathways to enter and infect cells. The data presented in this thesis show that reovirus entry is a complex process that can vary in different cell types. In addition, it has revealed that particles internalized into various endocytic pathways are trafficked to different destinations in polarized epithelial cells.