In this proposal we seek to understand the underlying mechanisms by which reoviruses induce apoptosis in cells and how this process is regulated to optimize viral replication in cultured cells and in vivo. Apoptosis is an evolutionarily conserved mechanism by which cells are removed during development and normal tissue homeostasis is maintained. Under pathologic conditions, suppression of apoptosis can lead to cancer and autoimmunity or conversely induction of apoptosis can lead to tissue injury. Many pathogenic human viruses, e.g., dengue virus, influenza virus, and West Nile virus, cause disease by inducing apoptosis. Some viruses induce apoptosis late in the infectious cycle, which allows the virus to spread within the host. Little is known about how viruses regulate the initiation and timing of apoptosis induction. Mammalian reoviruses are important models for identification of general pathogenic mechanisms by which viruses cause disease. Reoviruses cause pathologic damage (myocarditis and encephalitis) by inducing apoptosis. In addition, these viruses preferentially target transformed cell lines and are showing increasing promise as oncolytic agents because of their safety in humans and activity against a broad range of tumor types (they are currently being tested in several phase II/III clinical trials). Importantly, the primary mechanism of tumor cell killing is by virus-induced apoptosis. Here we will use the reovirus model to dissect the process of virus-induced apoptosis and its regulation by focusing on the viral structural protein, ?1, which functions to permeabilize cell membranes during virus entry and induces apoptosis later in the infectious cycle.
Two specific aims are proposed. In the first, mechanisms by which outer-capsid protein ?1 induces apoptosis will be determined. These experiments will test the hypothesis that a C-terminal fragment of ?1 is a viroporin that can directly permeabilize cellular membranes, including the plasma membrane, allowing leakage of molecules into the cytosol that activate cellular stress pathways, inducing apoptosis. In the second, post-translational modifications of ?1 that regulate the induction of apoptosis, cytotoxicity, and pathogenesis will be defined. These experiments will test the hypothesis that proteolytic cleavage and ubiquitination of free ?1 in the cytosol of infected cells regulates virus release, cytotoxicity, and pathogenicity. Collectively, these studies will enhance a basic understanding of virus-induced cell killing and may foster development of improved reovirus vectors for oncolytic applications and lead to new antiviral therapeutics that target apoptotic pathways.
The proposed studies will investigate a novel uninvestigated area of Reovirus biology that is applicable to understanding how human pathogenic viruses induce apoptosis in cells and to the development of reovirus as a therapeutic agent for cancer therapy in humans. This is relevant to public health because of the recognized importance of apoptosis in viral pathogenesis and because of the clinical promise of viral oncolytic therapy.
|Desmet, Emily A; Anguish, Lynne J; Parker, John S L (2014) Virus-mediated compartmentalization of the host translational machinery. MBio 5:e01463-14|