The long term goal of this research is to understand the mechanism by which enteroviruses such as poliovirus (PV) and Coxsackievirus (CVB3) control cellular and viral translation in infected cells and in turn, discern how translation and gene expression are regulated normally. Translation regulation mechanisms now encompass translation silencing (e.g. microRNAs) and dynamic assembly/disassembly of RNA granules, stress granules (SG) and P-bodies (PB) that contain translationally-silenced mRNPs. These structures assist cell homeostasis during stress and serve as temporary storage/triage sites for mRNPs, and in the case of PBs, sites for mRNA decay. We discovered that PV and CVB3 destroys-disperses both SGs and PBs, the former by cleavage of G3BP1, a key factor that nucleates formation of stress granules. Our emerging evidence suggests stress responses are linked to innate immune responses at several levels to form an integrated stress/innate immune response. In this funding period we discovered that SGs are stress-activated platforms to signal innate immunity and that G3BP1 mediates activation of PKR and NF-kB. We have also discovered that G3BP1 assembly of SGs is mediated by reversible arginine methylation on G3BP1 and have linked methylation to functional innate immunity output of SGs. In this proposal we will elucidate the role of protein arginine methylation in innate immune activation as we have now found recruitment of a key methyl- reader protein TDRD3 and several innate immune factors to SGs is dependent on methylation state of G3BP1. We have found the methyl-reader TDRD3 is antiviral, Aim 2 will elucidate the role of TDRD3 in recruitment and activation of innate immune factors in the absence of G3BP1 and the impact of its cleavage by virus protease.
Aim 3 will determine molecular mechanisms of activation of both PKR that is non-methylation dependent and NF-kB by G3BP1. This proposal is innovative since the both the (i) role SGs as a signaling platforms in innate immunity and (ii) the role of protein methylation in innate immune activation are novel. The proposed work is significant since it is relevant to a broad range of DNA and RNA viruses that promote SG formation and it promises to uncover unprecedented insights into novel protein-mRNP interactions that link stress signaling to innate immune activation. This will open new conceptual avenues for antiviral development.
This proposal will determine how cytoplasmic RNA granules, particularly stress granules which are conserved from plants to yeast to humans, activate and regulate innate immune functions that repress virus reproduction using arginine methylation marks on proteins to initiate signaling. In humans dysfunction of RNA granules and their components is linked to neurodegenerative disease, cancer and virus infection. The proposed studies will enhance our understanding of the biology and regulation of these stress activated membraneless organelles in cells and provide the basis for development of future tools to regulate their function in human disease and infections.
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