Using apathogenic and mammalian-ubiquitous reoviruses to safely discover genetic and phenotypic changes in viruses that promote adaptation to new host species and distinct niches within a host.

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Viruses are continuously entering new hosts, and whether they thrive depends on their ability to complete all steps of replication while subduing anti-viral host responses. It is important to understand how genetic and phenotypic changes promote virus adaptation to new niches. Mammalian orthoreovirus (MRV) is a perfect virus to study adaptation since it infects a wide assortment of mammals without causing disease. MRV naturally infects intestinal epithelia; yet most of our understanding of MRV comes from viruses adapted for 70 years to non-enteric cells such as the L929 tumorigenic mouse fibroblasts. Using 4 "wild" MRVs from primary effluent, we discovered inconsistencies in knowledge from lab-adapted MRVs. For example, MRV are said to undergo the essential proteolytic capsid uncoating step within cells, mediated by lysosomal proteases. We discovered that wild MRVs poorly uncoat intracellularly and exhibit deficiencies in other yet-to-be identified replication steps in non-enteric cell cultures. After 5 passages on L929 cells and isolation of single clonogenic sub-isolates, polymorphisms in the reovirus outercapsid protein s3 enable intracellular uncoating. This proposal seeks to further apply MRV as a model system to better understand virus adaptation to cells from distinct tissues and mammalian sources. Aim 1 will establish the true molecular features of MRV. We will evaluate all major steps of MRV replication for lab-adapted versus wild MRVs, and in L929 cells versus normal polarized intestinal epithelial cells from porcine, bovine and rat origins. Given MRV naturally encounters digestive proteases during enteric infection, we will also establish the true kinetics of extracellular MRV uncoating and sensitivity to proteases from distinct mammals. Given that even lab-adapted MRV strains differ substantially in their ability to induce host IFN and NfkB signaling pathways, we will also establish the true host response to wild MRVs in the context of different host cell cultures. Finally, we will derive a mathematical model for replication of MRVs in intestinal cells that can be applied to characterize MRV fitness between hosts and virus variants. AIM 2 will discover the path(s) to adaptation of MRVs. Using the clonogenic sub-isolates of wild MRVs that adapted to uncoat intracellularly, we will now determine which polymorphisms in s3 enable intracellular uncoating, whether different wild MRVs take the same or different paths of adaptation to cell culture, whether the s3 polymorphisms were present in the wild quasispecies or arose in passage, what relationship s3 polymorphisms have on other s3 functions such as cell signaling, and what polymorphisms in other MRV genes arise to promote non-uncoating steps of adaptation. We will also passage wild MRVs on distinct intestinal cell cultures and apply our clonogenic isolation approach to discover what polymorphisms arise during adaptation to intestinal cultures from distinct mammalian species.