Dissertation/Thesis Abstract

Functional Domains of Reovirus Protein μ2 that Determine the Interferon Response and Reovirus Replication
by Irvin, Susan C., Ph.D., North Carolina State University, 2011, 145; 3497187
Abstract (Summary)

The innate immune system provides a critical first line of protection against invading pathogens. In particular, the type I interferon (IFN) response enables the host to establish an antiviral state upon infection. Reovirus infection in vitro and in vivo provides an opportunity to investigate mechanisms underlying the IFN response as well as its impact on disease. We have focused on viral replication and damage in cardiac cells which together determine myocarditis in a mouse model. A non-myocarditic reovirus can induce myocarditis in mice when the IFN response is ablated either by deletion of genes that are required for induction of IFN or by treatment with anti-IFN antibody. Thus reovirus is an excellent tool for to identifying viral determinants of the IFN response and myocarditis.

Previously, we identified a reovirus strain-specific repressor of IFN signaling: the M1 gene-encoded protein μ2. Here, we demonstrate through the use of recombinant viruses that amino acid 208 of the μ2 protein is both required and sufficient for repression of IFN signaling. A virus encoding a proline at μ2 amino acid 208, such as strain type 1 Lang (T1L), represses IFN signaling while a virus encoding a serine at μ2 amino acid 208, such as type 3 Dearing (T3D), does not repress IFN signaling. Furthermore, in primary cardiac myocyte cultures, .2 amino acid 208 modulates reovirus induction of IFN, determines reovirus replication, and influences cytopathic effect after multiple cycles of replication. Previously, we found that reovirus induces significantly higher levels of IFN in cardiac myocytes than in cardiac fibroblasts. Accordingly while μ2 amino acid 208 modulates induction of IFN in cardiac fibroblasts, the impact of IFN on reovirus replication is less dramatic than in cardiac myocytes. Given that the IFN response limits viral spread and cytopathic effect in primary cardiac myocyte cultures, we next investigated the effect of μ2 amino acid 208 on reovirus-induced myocarditis in a mouse model. We determined that the T1L M1 gene is sufficient for reovirus-induced myocarditis and that μ2 amino acid 208 modulates myocarditis, likely through repression of IFN signaling.

Next, we investigated the role of immunoreceptor tyrosine-based activation motifs (ITAMs) in reovirus proteins. ITAMs are signaling domains found in the cytoplasmic tails of receptors that mediate immune cell activation, and in the cytoplasmic tails of surface glycoproteins of some enveloped viruses. Here, ITAMs were identified in three proteins, σ2, μ2, and λ2, which are constituents of the reovirus inner core and outer capsid. These are the first ITAMs identified in a non-enveloped virus. ITAM-mediated cell signaling requires the phosphorylation of two tyrosine residues within the motif. Reverse genetics was used to engineer mutant T3D reoviruses in which the two critical tyrosines in ITAMs of σ2, μ2, and λ2 were replaced with phenylalanine to inhibit ITAM phosphorylation. Although the λ2 mutant virus was not viable, replication kinetics and final yields of the σ2 and μ2 ITAM mutant viruses in L929 cells and in primary cardiac myocyte cultures demonstrated the σ2 and μ2 ITAMs are not required for reovirus replication. Importantly, the μ2 ITAM regulates the activation of NF-κB and influences the induction of interferon-β (IFN-β) in both L929 cells and primary cardiac myocyte cultures. Moreover, the consequences of these μ2 ITAM effects are cell type-specific. In L929 cells, the μ2 ITAM proved to be advantageous for viral fitness. In contrast, in primary cardiac myocyte cultures where the IFN response is critical for antiviral protection and NF-κB is not required for apoptosis, the μ2 ITAM diminishes viral fitness. Results here suggest the μ2 ITAM has a cell type-specific role in viral spread, likely reflecting the cell type-specific roles of NF-κB and IFN-β.

Indexing (document details)
Advisor: Sherry, Barbara
School: North Carolina State University
School Location: United States -- North Carolina
Source: DAI-B 73/05, Dissertation Abstracts International
Subjects: Molecular biology, Physiology, Virology
Keywords: Cardiac myocyte, Heart, Interferon, Myocarditis, Reovirus protein, Reovirus replication
Publication Number: 3497187
ISBN: 978-1-267-17673-8
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