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 extend to dynamic assembly/disassembly of RNA granules, called 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 have discovered that G3BP, a key factor that nucleates formation of stress granules, is cleaved in PV-infected cells by virus 3C protease, resulting in loss of SG in cells. Experiments are proposed to discern interactions of G3BP with initiation factors in mRNP complexes that regulate translation and how G3BP cleavage abrogates this function. Experiments will also define alternate mechanism(s) employed by Coxsackievirus to block SG formation and the minimal viral proteins required. We have also shown that G3BP expression assembles SGs that induce antiviral states, and block PV replication.
The second aim will elucidate the mechanism of this potent antiviral activity and discover if it stems from sequestration of factors required b the virus or if SG assembly itself signals JNK or other pathways that activate innate immunity. Our emerging evidence suggests stress responses are linked to innate immune responses at several levels to form an integrated stress/innate immune response. We have also determined that PV disperses PB in cells and in the process, degrades/cleaves three critical components of the mRNA decay pathway, Xrn1, Dcp1a and Pan3. We will elucidate the mechanism of PB dispersal and determine if loss of these factors during infection stabilizes viral RNA. Overall, proposed studies are significant since the virus is attacking the translation/mRNP regulatory apparatus at a novel level of RNA granules, which are poorly understood but play key roles in maintaining cell homeostasis and stress survival. SG and perhaps PBs can strongly inhibit virus replication and new insights from this study may lead to novel antiviral strategies.
This work will provide new information how human enteroviruses interfere with cellular gene expression regulation by blocking the assembly of mRNA granules known as stress granules and P-bodies. Formation of stress granules is strongly antiviral in cells;we will discern how these structures block virus replication. This wil enhance understanding of regulation of stress in uninfected cells and virus infected cells and may provide bases for new therapeutic approaches for fighting these diseases.
|Reineke, Lucas C; Tsai, Wei-Chih; Jain, Antrix et al. (2017) Casein Kinase 2 Is Linked to Stress Granule Dynamics through Phosphorylation of the Stress Granule Nucleating Protein G3BP1. Mol Cell Biol 37:|
|Tsai, Wei-Chih; Reineke, Lucas C; Jain, Antrix et al. (2017) Histone arginine demethylase JMJD6 is linked to stress granule assembly through demethylation of the stress granule nucleating protein G3BP1. J Biol Chem :|
|Tsai, Wei-Chih; Gayatri, Sitaram; Reineke, Lucas C et al. (2016) Arginine Demethylation of G3BP1 Promotes Stress Granule Assembly. J Biol Chem 291:22671-22685|
|Lloyd, Richard E (2016) Enterovirus Control of Translation and RNA Granule Stress Responses. Viruses 8:93|
|Reineke, Lucas C; Lloyd, Richard E (2015) The stress granule protein G3BP1 recruits protein kinase R to promote multiple innate immune antiviral responses. J Virol 89:2575-89|
|Reineke, Lucas C; Kedersha, Nancy; Langereis, Martijn A et al. (2015) Stress granules regulate double-stranded RNA-dependent protein kinase activation through a complex containing G3BP1 and Caprin1. MBio 6:e02486|
|Lloyd, Richard E (2015) Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses. Virology 479-480:457-74|
|Dougherty, Jonathan D; Tsai, Wei-Chih; Lloyd, Richard E (2015) Multiple Poliovirus Proteins Repress Cytoplasmic RNA Granules. Viruses 7:6127-40|
|Dougherty, Jonathan D; Reineke, Lucas C; Lloyd, Richard E (2014) mRNA decapping enzyme 1a (Dcp1a)-induced translational arrest through protein kinase R (PKR) activation requires the N-terminal enabled vasodilator-stimulated protein homology 1 (EVH1) domain. J Biol Chem 289:3936-49|
|Feng, Qian; Langereis, Martijn A; Lork, Marie et al. (2014) Enterovirus 2Apro targets MDA5 and MAVS in infected cells. J Virol 88:3369-78|
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