An ITAM in a Nonenveloped Virus Regulates Activation of NF-κB, Induction of Beta Interferon, and Viral Spread

Abstract

Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains located within the cytoplasmic tails of many transmembrane receptors and associated adaptor proteins that mediate immune cell activation. ITAMs also have been identified in the cytoplasmic tails of some enveloped virus glycoproteins. Here, we identified ITAM sequences in three mammalian reovirus proteins: 2, 2, and 2. We demonstrate for the first time that 2 is phosphorylated, contains a functional ITAM, and activates NF-B. Specifically, 2 and NS recruit the ITAM-signaling intermediate Syk to cytoplasmic viral factories and this recruitment requires the 2 ITAM. Moreover, both the 2 ITAM and Syk are required for maximal 2 activation of NF-B. A mutant virus lacking the 2 ITAM activates NF-B less efficiently and induces lower levels of the downstream antiviral cytokine beta interferon (IFN-␤) than does wild-type virus despite similar replication. Notably, the consequences of these 2 ITAM effects are cell type specific. In fibroblasts where NF-B is required for reovirus-induced apoptosis, the 2 ITAM is advantageous for viral spread and enhances viral fitness. Conversely, in cardiac myocytes where the IFN response is critical for antiviral protection and NF-B is not required for apoptosis, the 2 ITAM stimulates cellular defense mechanisms and diminishes viral fitness. Together, these results suggest that the cell type-specific effect of the 2 ITAM on viral spread reflects the cell type-specific effects of NF-B and IFN-␤. This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host.

A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2

“…9). Posttranslational modifications of 2, including acetylation (33) and phosphorylation (34), have been reported, and yet the impact(s) of these modifications on 2 function has not been fully elucidated. Future studies will address the roles of SRSF2 and posttranslational modifications on 2 subcellular localization.…”

“…A plasmid expressing C-terminus hemagglutinin (HA)-tagged T1L 2 in a pCAGGs backbone was described previously (34). The C-terminus HA-tagged T3D 2 was generated from pCAGGs-M1-T3D (6) using a similar strategy and the primer 5=-CCCGGGTCAGCTAGCGT AATCTGGAACATCGTATGGGTACGCCAAGTCAGATCGGAAAGCTAGTC-3=.…”

NF-κB activation is cell type-specific in the heart

“…To determine whether cardiac myocytes are relatively recalcitrant to virus-induced NF-κB nuclear translocation, cultures were infected with reovirus type 3 Dearing (T3D), a serotype known to activate NF-κB in different cell types (20, 34-36) (Figure 7). While there were more mock-treated cardiac fibroblasts with nuclear NF-κB than in previous experiments (Figures 5 and 6), likely reflecting the stress induced by two media changes, the number of cells with nuclear NF-κB was still much lower in cardiac fibroblast cultures than in identically-treated cardiac myocyte cultures.…”

“…Finally, reovirus-induced myocarditis is the result of a direct viral cytopathic effect to cardiac myocytes (59), which is largely apoptotic (57). Reovirus-induced apoptosis requires activation of NF-κB in most cell types (20, 34-36, 60-62) but not in cardiac myocytes (20, 36). Thus reovirus-induced myocarditis does not require activation of NF-κB in cardiac myocytes, consistent with evidence here that cardiac myocytes are relatively recalcitrant to NF-κB activation (Figures 1 and 6-8).…”

“…T3D was chosen as a strong inducer of NF-κB activity (20, 34-36, 60, 61). Unless stated otherwise, cardiac cultures plated in 8-well chamber slides were inoculated with reovirus T3D at 50 plaque forming units (PFU) per cell and were fixed at the indicated times post-infection.…”