The Cardiac Response to Reovirus Infection

Abstract

LI, LIANNA. The Cardiac Response to Reovirus Infection. (Under the direction of Dr. Barbara Sherry).

Viral myocarditis is a common disease in humans. Interferon-β (IFN-β) has been identified as critical for protection against viral myocarditis in mouse models, and IFN-α or -β treatment is beneficial in the treatment of human viral myocarditis. IFN-β expression and its antiviral effects are cell-type specific in murine cardiac myocytes and fibroblasts. However, expression and function of individual IFN-α subtypes in cardiac cells has not previously been investigated. Therefore, IFN-α subtype expression and antiviral effects were studied in reovirus-infected murine primary cardiac myocyte and cardiac fibroblast cultures. In order to quantify the thirteen highly conserved IFN-α subtype, a quantitative Real-Time PCR assay was developed. Results demonstrated that IFN-α induction by reovirus T3D in cardiac cells is both subtype- and cell type-specific, and that some individual IFN-α subtypes are likely important in the antiviral cardiac response. In brief, reovirus T3D induced five IFN-α subtypes in primary cultures of cardiac myocytes and fibroblasts: IFN-α1, -α2, -α4, -α5, and -α8/6. The levels of IFN-α expression were both higher and spanned a greater range in cardiac myocytes than in fibroblasts. Viral induction of IFN-α1, -α2, -α5, and -α8/6 required IFN-α/β signaling in both cell types, while induction of IFN-β and -α4 was more dependent on IFN signaling in myocytes than fibroblasts. Murine IFN-α1, -α2, -α4, or -α5 treatment induced IRF7 and ISG56 in both cardiac cell types, however induction was always greater in cardiac fibroblasts than in cardiac myocytes. Finally, each IFN-α subtype inhibited reovirus T3D replication in both cell types, but protection was subtype-specific. To discover novel proteins or protein post-translational modifications involved in the IFN pathway or displaying antiviral effects against viral myocarditis, a proteomics tool, two-dimensional difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF-TOF, was used to investigate the reovirus-induced proteome changes in murine primary cardiac myocyte cultures. Results demonstrated that the 2D-DIGE technique is quantitative and reproducible. Whole proteome changes based on differentially expressed proteins were clustered according to viral pathogenic phenotypes and induction of IFN. One hundred and twenty-four differentially expressed proteins were identified, including those involved in calcium signaling, ERK/ MAPK signaling, protein ubiquitination, mitochondrial dysfunction, oxidative stress, amino acid metabolism, and other pathways. Interestingly, 2D-DIGE results and additional studies demonstrated that heat shock protein Hsp25 is modulated differentially by myocarditic and non-myocarditic reoviruses, and suggested that it may play a role in the cardiac antiviral response. This is the eighth virus family found to modulate Hsp25 or its human homolog, Hsp27, suggesting that Hsp25/27 participation in the antiviral response may be widespread. However, results here provide the first evidence for a virus-induced decrease in Hsp25/27, and suggest that viruses may have evolved a mechanism to subvert this protective response, as they have for IFN.