Our overall goal is to identify novel components of the host innate antiviral response and novel mechanisms for viral subversion of this response to impact disease. Cardiac myocytes are essential and non-replenishable, thus the heart is exceptionally dependent on this first-line defense. Indeed, we have shown that the cardiac interferon (IFN) response is unique, and that in reovirus-induced murine myocarditis the IFN response differs between virus strains and is critical for protection. We continue to use this powerful tool-kit of viruses to probe the innate response in a highly vulnerable organ, the heart, and have made two new discoveries. First, we found that reovirus inhibits IFN signaling by an entirely novel mechanism. Specifically, reovirus induces unusual nuclear accumulation of transcription factor IRF9, likely reflecting concomitant interference with IRF9 function in induction of IFN-stimulated genes. This virus strain specific-effect is determined by a single amino acid in reovirus protein ?2. We hypothesize that reovirus protein ?2 modulates IRF9 structure / function to inhibit IFN signaling, and that ?2 repression of IFN signaling in cardiac cells is critical for myocarditis.
In Specific Aim 1, we will determine the mechanism for this modulation and its impact on myocarditis and other disease. Results will provide significant new insights into functional domains and protein binding partners of participants in IFN signaling. Second, using a proteomic discovery approach, we identified a new IFN- independent protective response which can be subverted by virus. We found that Heat Shock Protein-25 (Hsp25) is phosphorylated (non-myocarditic reoviruses) or decreased (myocarditic reovirus) in infected cardiac myocytes, and that this is cell type-specific and IFN-independent. Hsp25 is modulated by many viruses and it is protective against stress, particularly in the heart. However, an Hsp25 decrease has never been reported for any stimulus in any cell type. Inhibition of Hsp25 by a highly myocarditic reovirus, but no other stimulus, suggests that Hsp25 is protective and can be subverted by viruses. We hypothesize that phosphorylated Hsp25 plays a cell type-specific protective role during infection, and that viruses can subvert this innate response.
In Specific Aim 2, we will determine the protective effects of Hsp25, the mechanisms by which viruses modulate it, and the impact on virus tropism and disease. Results will define a completely new protective response against viral infection. In sum, by studying both a component of the IFN response (IRF9) and an IFN-independent protective factor (Hsp25), we gain a more complete picture of virus modulation of cell factors in disease. By studying cells critically dependent on innate antiviral responses, we uncover effectors that may be present in many cell types but not readily detected. Finally, the remarkably strong correlation between viral effects in cardiac myocyte cultures and murine myocarditis provides an outstanding platform to test hypotheses generated in vitro for their validity in vivo. The broader impact is to increase the catalog of protective host factors that can be sabotaged by viruses and manipulated for therapeutic intervention.
The host's immediate innate response to viral infection can be a critical determinant of disease outcome. We have identified a new mechanism by which viruses can repress that innate response, and a new component of the innate response that may be important for protection against disease. We will investigate both topics in this proposal.
