A single-amino-acid polymorphism in reovirus protein µ2 determines repression of interferon signaling and modulates myocarditis.

Myocarditis is indicated as the second leading cause of sudden death in young adults. Reovirus induces myocarditis in neonatal mice, providing a tractable model system for investigation of this important disease. Alpha/beta-interferon (IFN-α/ß) treatment improves cardiac function and inhibits viral replication in patients with chronic myocarditis, and the host IFN-α/ß response is a determinant of reovirus strain-specific differences in induction of myocarditis. Virus-induced IFN-ß stimulates a signaling cascade that establishes an antiviral state and further induces IFN-α/ß through an amplification loop. Reovirus strain-specific differences in induction of and sensitivity to IFN-α/ß are associated with the viral M1, L2, and S2 genes. The reovirus M1 gene-encoded µ2 protein is a strain-specific repressor of IFN-ß signaling, providing one possible mechanism for the variation in resistance to IFN and induction of myocarditis between different reovirus strains. We report here that µ2 amino acid 208 determines repression of IFN-ß signaling and modulates reovirus induction of IFN-ß in cardiac myocytes. Moreover, µ2 amino acid 208 determines reovirus replication, both in initially infected cardiac myocytes and after viral spread, by regulating the IFN-ß response. Amino acid 208 of µ2 also influences the cytopathic effect in cardiac myocytes after spread. Finally, µ2 amino acid 208 modulates myocarditis in neonatal mice. Thus, repression of IFN-ß signaling mediated by reovirus µ2 amino acid 208 is a determinant of the IFN-ß response, viral replication and damage in cardiac myocytes, and myocarditis. These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-ß response and, consequently, damage to the heart.

Reovirus mu2 protein inhibits interferon signaling through a novel mechanism involving nuclear accumulation of interferon regulatory factor 9.

The secreted cytokine alpha/beta interferon (IFN-alpha/beta) binds its receptor to activate the Jak-STAT signal transduction pathway, leading to formation of the heterotrimeric IFN-stimulated gene factor 3 (ISGF3) transcription complex for induction of IFN-stimulated genes (ISGs) and establishment of an antiviral state. Many viruses have evolved countermeasures to inhibit the IFN pathway, thereby subverting the innate antiviral response. Here, we demonstrate that the mildly myocarditic reovirus type 1 Lang (T1L), but not the nonmyocarditic reovirus type 3 Dearing, represses IFN induction of a subset of ISGs and that this repressor function segregates with the T1L M1 gene. Concordantly, the T1L M1 gene product, mu2, dramatically inhibits IFN-beta-induced reporter gene expression. Surprisingly, T1L infection does not degrade components of the ISGF3 complex or interfere with STAT1 or STAT2 nuclear translocation as has been observed for other viruses. Instead, infection with T1L or reassortant or recombinant viruses containing the T1L M1 gene results in accumulation of interferon regulatory factor 9 (IRF9) in the nucleus. This effect has not been previously described for any virus and suggests that mu2 modulates IRF9 interactions with STATs for both ISGF3 function and nuclear export. The M1 gene is a determinant of virus strain-specific differences in the IFN response, which are linked to virus strain-specific differences in induction of murine myocarditis. We find that virus-induced myocarditis is associated with repression of IFN function, providing new insights into the pathophysiology of this disease. Together, these data provide the first report of an increase in IRF9 nuclear accumulation associated with viral subversion of the IFN response and couple virus strain-specific differences in IFN antagonism to the pathogenesis of viral myocarditis.

Basal expression levels of IFNAR and Jak-STAT components are determinants of cell-type-specific differences in cardiac antiviral responses.

Viral myocarditis is an important human disease, and reovirus-induced murine myocarditis provides an excellent model system for study. Cardiac myocytes, like neurons in the central nervous system, are not replenished, yet there is no cardiac protective equivalent to the blood-brain barrier. Thus, cardiac myocytes may have evolved a unique antiviral response relative to readily replenished cell types, such as cardiac fibroblasts. Our previous comparisons of these two cell types revealed a conundrum: reovirus T3D induces more beta-interferon (IFN-beta) mRNA in cardiac myocytes, yet there is a greater induction of IFN-stimulated genes (ISGs) in cardiac fibroblasts. Here, we investigated possible underlying molecular determinants. We found that greater basal expression of IFN-beta in cardiac myocytes results in greater basal activated nuclear STAT1 and STAT2 and greater basal ISG mRNA expression and provides greater basal antiviral protection relative to cardiac fibroblasts. Conversely, cardiac fibroblasts express greater basal IFN-alpha/beta receptor 1 (IFNAR1) and greater basal cytoplasmic Jak1, Tyk2, STAT2, and IRF9, leading to a greater increase in reovirus T3D- or IFN-induced nuclear activated STAT1 and STAT2 and greater induction of ISGs for a greater IFN-induced antiviral protection relative to cardiac myocytes. Our results suggest that high basal IFN-beta expression in cardiac myocytes prearms this vulnerable, nonreplenishable cell type, while high basal expression of IFNAR1 and latent Jak-STAT components in adjacent cardiac fibroblasts renders these cells more responsive to IFN and prevents them from inadvertently serving as a reservoir for viral replication and spread to cardiac myocytes. These studies provide the first indication of an integrated network of cell-type-specific innate immune components for organ protection.

Retinoic acid-inducible gene-I and interferon-beta promoter stimulator-1 augment proapoptotic responses following mammalian reovirus infection via interferon regulatory factor-3.

During viral infection, cells initiate antiviral responses to contain replication and inhibit virus spread. One protective mechanism involves activation of transcription factors interferon regulatory factor-3 (IRF-3) and NF-kappaB, resulting in secretion of the antiviral cytokine, interferon-beta. Another is induction of apoptosis, killing the host cell before virus disseminates. Mammalian reovirus induces both interferon-beta and apoptosis, raising the possibility that both pathways are initiated by a common cellular sensor. We show here that reovirus activates IRF-3 with kinetics that parallel the activation of NF-kappaB, a known mediator of reovirus-induced apoptosis. Activation of IRF-3 requires functional retinoic acid inducible gene-I and interferon-beta promoter stimulator-1, but these intracellular sensors are dispensable for activation of NF-kappaB. Interferon-beta promoter stimulator-1 and IRF-3 are required for efficient apoptosis following reovirus infection, suggesting a common mechanism of antiviral cytokine induction and activation of the cell death response.