Browsing by Author "Barbara Sherry, Committee Chair"

Filter results by typing the first few letters
Now showing 1 - 3 of 3
  • No Thumbnail Available
    Cardiac Cell Type-Specific Differences in the Interferon (IFN) Response, and Reovirus Repression of IFN
    (2008-08-08) Zurney, Jennifer Michelle; Barbara Sherry, Committee Chair; Michael Sikes, Committee Member; Ian T.D. Petty, Committee Member; Robert C. Smart, Committee Member
  • No Thumbnail Available
    The Cardiac Response to Reovirus Infection
    (2009-08-03) Li, Lianna; Barbara Sherry, Committee Chair; Frank Scholle, Committee Member; James L. Stephenson, Jr., Committee Member; Jonathan M. Horowitz, Committee Member
    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.
  • No Thumbnail Available
    PKR, Myocarditis and the Cardiac Response to Reovirus Infection
    (2004-06-20) Stewart, Michael Jude; Scott Laster, Committee Member; Tim Petty, Committee Member; Fred Fuller, Committee Member; Barbara Sherry, Committee Chair
    Viral myocarditis is an important human disease associated with many viruses. Mechanistically, cardiac damage associated with viral myocarditis can be immune mediated and/or the result of direct cytopathic effect. Reovirus induced myocarditis is not immune mediated, and thus, provides an excellent model for the study of direct cytopathic effect in the heart. Previous work has demonstrated that reovirus myocarditic potential reflects induction and/or sensitivity to interferon (IFN). Specifically, nonmyocarditic reoviruses induce greater IFN-β and/or are more sensitive to the antiviral effects IFN than are myocarditic reoviruses. Importantly, IFN mediates its antiviral effects through the induction of interferon-stimulated genes (ISGs); ISGs function as the antiviral effector proteins that block replication in the host cell. The work presented in this dissertation further defines the cardiac response to reovirus infection. In chapter 2, we examined the double-stranded activated protein kinase PKR: the role of PKR in cardiac IFN induction and protection against reovirus-induced myocarditis. We demonstrate that PKR is critical to the robust induction of IFN-β in primary cardiac myocyte cultures (PCMCs). Additionally, we show that nonmyocarditic reoviruses become myocarditic in PKR-null mice, even though reovirus growth in PKR-null hearts is similar to that in wild type mouse hearts. Finally, we demonstrate that relative to wild type mice, reoviruses induce significantly greater morbidity in PKR-null mice. In the following chapter, we compare the IFN response in PCMCs: a non-replenished, critical cell type, to that in primary cardiac fibroblast cultures (PCFCs): a readily replenished cardiac cell type. By quantitative real-time PCR, we find that PCMCs express IFN-β and the ISGs IRF-7 and 561 constitutively at higher levels than PCFCs. We also identify constitutive IFN as a primary means of constitutive ISG expression. Additionally, we demonstrate that PCFCs, more so than PCMCs are more dependent on IFN-mediated protection against reovirus infection. The regulation of IFN-β expression is achieved primarily through the actions of Interferon Regulatory Factors (IRFs). In Appendix 1, we show contributions to a publication demonstrating that IRF-1 is dispensable for IFN-β induction in PCMCs, and yet critical for defense against reovirus-induced myocarditis in the mouse. Finally, a summary of this work is provided.