The Reoviridae family of segmented dsRNA viruses includes many members that are of considerable health and economic importance. Additionally, mammalian orthoreoviruses (MRV) are oncolytic viruses that selectively kill tumor cells. Many members of this virus family induce the phosphorylation of translation initiation factor eIF2?, which leads to inhibition of mRNA translation. MRV mRNAs escape cellular translation shutoff. Inhibition of protein synthesis also leads to sequestration of cellular mRNAs and translation initiation factors into cytoplasmic structures termed stress granules (SGs). Recent data suggests that MRV induces SGs during viral entry. As viral infection proceeds, even though eIF2? remains phosphorylated, SGs are disrupted in a manner dependent on protein synthesis. SG disruption also correlates with the formation of virally encoded structures called virus factories (VFs). MRV prevents sodium arsenite (SA) induction of SGs downstream of eIF2? phosphorylation, and strains of MRV that differ in their ability to shutoff host cell translation are all able to translate high levels of mRNA in the presence of SA. This suggests MRV-induced host cell translation shutoff and MRV escape from translational shutoff are independent of each other, and additionally, that escape from shutoff is independent of inhibition of eIF2? phosphorylation. These findings support a hypothesis that SG disruption plays an important role in MRV infection and escape from cellular shutoff of protein synthesis. Experiments in this proposal will test this hypothesis by accomplishing two specific goals. The mechanism of MRV-induced SG disruption will be elucidated by defining the relationship between VFs and SGs, and by determining if VF formation is necessary for SG disruption. Additional experiments will identify MRV proteins required for SG disruption, and determine if MRV infection alters cellular pathways necessary for SG formation. Additionally the mechanism(s) used for MRV translation in the host cell shutoff environment will be investigated by separating the impact of eIF2? phosphorylation and SG formation on MRV translation, and identifying viral proteins and mRNA sequences that are necessary for MRV mRNA translation in the presence of phosphorylated eIF2?. Elucidation of the mechanisms behind this complex virus-host cell interaction will increase our understanding of the basic mechanisms used by these viruses to overcome host cell defenses, and may lead to the development of strategies to either interdict the pathogens in this family using targeted anti-virals, or alternatively, to enhance the oncolytic properties of MRV. PUBLIC HEALTH RELEVANCE: Mammalian orthoreoviruses are members of a family of viruses that includes important human pathogens, and are also oncolytic viruses that selectively kill tumor cells alone or synergistically when combined with radiation or other chemotherapeutic treatments. This proposal is designed to elucidate the mechanisms used by these viruses to escape an important aspect of the host cell defense system that was recently found to play a role in tumor resistance to chemotherapy and radiation. Understanding this complex virus-host cell interaction may lead to the development of targeted anti-virals against the pathogens in this family, or allow educated manipulation of the virus to enhance its individual or synergistic oncolytic properties.