Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2010 Characterization of mammalian orthoreovirus (MRV) induced stress granules (SGs) and implications of eIF2α phosphorylation on viral translation Qingsong Qin Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Veterinary Preventive Medicine, Epidemiology, and Public Health Commons Recommended Citation Qin, Qingsong, "Characterization of mammalian orthoreovirus (MRV) induced stress granules (SGs) and implications of eIF2α phosphorylation on viral translation" (2010). Graduate Theses and Dissertations. 11731. https://lib.dr.iastate.edu/etd/11731 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Characterization of mammalian orthoreovirus (MRV) induced stress granules (SGs) and implications of eIF2 phosphorylation on viral translation by Qingsong Qin A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Veterinary Microbiology Program of Study Committee: Cathy L Miller, Major Professor Qijing Zhang W. Allen Miller Michael J. Wannemuehler Mark Ackermann Iowa State University Ames, Iowa 2010 Copyright © Qingsong Qin, 2010. All rights reserved. ii TABLE OF CONTENTS ABSTRACT ............................................................................................................................. iv CHAPTER 1. GENERAL INTRODUCTION ......................................................................... 1 Introduction ........................................................................................................................... 1 Dissertation organization....................................................................................................... 3 CHAPTER 2. A REVIEW OF MAMMALIAN ORTHOREOVIRUS PROPERTIES, VIRAL INFECTION, CELLULAR STRESS RESPONSE, AND VIRAL ONCOLYSIS ................... 5 Introduction and significance ................................................................................................ 5 Mammalian orthoreovirus properties .................................................................................... 6 Components of viral particles ............................................................................................... 7 MRV infection, the cellular stress response, and shutoff of host protein synthesis ............ 15 Reovirus-induced apoptosis ................................................................................................ 20 Reovirus oncolysis .............................................................................................................. 22 References ........................................................................................................................... 24 CHAPTER 3. MAMMALIAN ORTHOREOVIRUS PARTICLES INDUCE AND ARE RECRUITED INTO STRESS GRANULES AT EARLY TIMES POST INFECTION ....... 43 Abstract ............................................................................................................................... 43 Introduction ......................................................................................................................... 44 Materials and methods ........................................................................................................ 47 Results ................................................................................................................................. 51 Discussion ........................................................................................................................... 59 Acknowledements ............................................................................................................... 65 References ........................................................................................................................... 65 CHAPTER 4. MAMMALIAN ORTHOREOVIRUS ESCAPE FROM HOST TRANSLATIONAL SHUTOFF CORRELATES WITH STRESS GRANULE DISRUPTION AND IS INDEPENDENT OF eIF2 PHOSPHORYLATION AND PKR ... 85 Abstract ............................................................................................................................... 85 Introduction ......................................................................................................................... 86 Materials and methods ........................................................................................................ 90 Results ................................................................................................................................. 93 Discussion ......................................................................................................................... 104 Acknowledgements ........................................................................................................... 109 iii References ......................................................................................................................... 109 CHAPTER 5. ESTABLISHMENT OF A 10 PLASMID-BASED REVERSE GENETICS SYSTEM AND INDUCIBLE CELL LINES FOR FUTURE STUDY OF MRV TRANSLATION................................................................................................................... 132 Abstract ............................................................................................................................. 132 Introduction ....................................................................................................................... 133 Materials and methods ...................................................................................................... 136 Results ............................................................................................................................... 140 Discussion ......................................................................................................................... 143 Acknowledgements ........................................................................................................... 149 References ......................................................................................................................... 150 CHAPTER 6. GENERAL CONCLUSIONS ........................................................................ 166 ACKNOWLEDGEMENTS .................................................................................................. 168 iv ABSTRACT Mammalian orthoreoviruses (MRV) are non-fusogenic, nonenveloped, icosahedral, RNA viruses, containing a 10-segmented double-stranded RNA genome, belonging to the family Reoviridae. Infection with many mammalian orthoreovirus (MRV) strains results in shutoff of host, but not viral, protein synthesis via protein kinase R (PKR) activation and phosphorylation of translation initiation factor eIF2 . When cells are under stressful environments, such as heat shock, oxidative stress, nutritional starvation, and viral infection, several kinases (PKR, PERK, HRI, or GCN) are activated, which phosphorylate eIF2 ser), resulting in the formation of stress granules (SGs), discrete areas in the cytoplasm where cellular mRNAs are held in a translationally inactive state. We examined MRV-infected cells to characterize SG formation in response to MRV infection. We found SGs formed at early times following infection (2-6 h p.i.) in a manner dependent on phosphorylation of eIF2 . MRV induced SG formation in all four eIF2 kinase knockout cell lines, suggesting at least two kinases are involved in induction of SGs. Inhibitors of MRV disassembly prevented MRV-induced SG formation, indicating that viral uncoating is a required step for SG formation. Inactivation of MRV virions by ultraviolet (UV) light, or treatment of MRV-infected cells with the translational inhibitor, puromycin, did not prevent SG formation, suggesting that viral transcription and translation are not required for SG formation. Viral cores were found to colocalize with SGs, however, cores from UV- inactivated virions did not associate with SGs, suggesting viral core particles are recruited into SGs in a process that requires the synthesis of viral mRNA. These results demonstrate that MRV particles induce SGs in a step following viral disassembly but preceding viral mRNA transcription, and that core particles are themselves recruited to SGs, suggesting the v cellular stress response may play an inhibitory role in viral translation. As infection proceeds, MRV disrupts SGs despite sustained levels of phosphorylated eIF2 , and further, interferes with the induction of SGs by other stress inducers. MRV interference with SG formation occurs downstream of eIF2α phosphorylation suggesting the virus uncouples the cellular stress signaling machinery from SG formation. We additionally examined mRNA translation in the presence of SGs induced by eIF2α phosphorylation dependent and independent mechanisms. We found that irrespective of eIF2α phosphorylation status, the presence of SGs in cells correlated with inhibition of viral and cellular translation. In contrast, MRV disruption of SGs correlated with release of viral mRNAs from translational inhibition, even in the presence of phosphorylated