Modulation of host cell innate immune proteins by viral non-coding RNAs Open Access

Vachon, Virginia Katherine (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/n296wz73b?locale=en
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Abstract

Precise control of protein synthesis is essential for regulation of normal cellular processes and responses to cell stress. In addition, the innate immune response exploits varied strategies of translational control in order to limit viral protein synthesis and thus replication. The double-stranded RNA-activated protein kinase (PKR) and 2'-5'-oligoadenylate synthetases (OAS) sense cytosolic double-stranded RNA, a molecular hallmark of virus infection, to initiate general shut down of protein synthesis. The central importance of PKR and OAS is highlighted by numerous diverse strategies employed by a range of viruses to thwart their effects. Among them, the Adenovirus non-coding (nc)RNA transcript, VA RNAI, accumulates to high levels in later stages of infection, and is critical for efficient adenoviral replication. VA RNAI is well recognized for its inhibition of dsRNA-activated protein kinase (PKR)-mediated shut-down of general translation but, counterintuitively, VA RNAI activates rather than inhibits OAS1. The fundamental basis for control of these proteins by VA RNAI and other viral or cellular non-coding RNAs, including the range of RNA sequences and structures capable of regulating their activity remains underexplored.

Here, VA RNAI is used as a model to further our understanding of RNA-mediated regulation of PKR and OAS1. Following a review of the current state of knowledge surrounding the multifaceted roles of VA RNAI, a description is provided of methods developed for solution-based probing of VA RNAI and other short RNA structures. These methods are then applied to a study on the role of VA RNAI structure in PKR inhibition. Finally, using VA RNAI as a starting point, discovery and characterization of a general, novel motif for optimal activation of OAS1 is presented.

Defining the molecular signatures which govern the activities of PKR and OAS1 is essential to understanding how these proteins maximize their protective role against a broad range of pathogens while accurately discriminating between foreign and self molecules, avoiding inadvertent activation. Detailed knowledge of specific pathogen-associated molecular patterns recognized by proteins of the innate immune system is an essential foundation for development of therapeutic strategies that either directly target viral pathogens or enhance the host cell response to them.

Table of Contents

TABLE OF CONTENTS

ABSTRACT...................................................................................................................... iv ACKNOWLEDGEMENTS..................................................................................................... vi CHAPTER 1:.................................................................................................................... 1 Introduction.................................................................................................................... 1 BACKGROUND................................................................................................................. 2 RESEARCH GOALS........................................................................................................... 14 REFERENCES.................................................................................................................. 17 CHAPTER 2:.................................................................................................................... 31

Adenovirus VA RNA: An essential pro-viral non-coding RNA................................................... 31

ABSTRACT...................................................................................................................... 32 INTRODUCTION.............................................................................................................. 33 Expression, Localization, and Structure of AdV VA RNAs....................................................... 35 Cellular Localization of VA RNAI................................................................................... 39 VA RNA Structure......................................................................................................... 41 VA RNAs Usurp Multiple Cellular Functions.......................................................................... 46

VA RNA interference with RNAi machinery........................................................................... 46

Competition for nuclear export by Exportin 5 (Exp 5)........................................................... 48

Dicer Saturation.............................................................................................................. 48

mivaRNA incorporation into RISC: a mechanism of viral control of host cell gene expression? .....50

VA RNAI-mediated inhibition of PKR.................................................................................... 54

VA RNAI regulation of 2'-5' oligoadenylate synthetase-1 (OAS1)............................................. 61

Pro-viral roles of Apical Stem-Central Domain fragment from Dicer-processing of VA RNAI .........65

VA RNAI induces type-I interferon late in infection................................................................. 68 Considerations for Ad vectors............................................................................................. 69 CONCLUSIONS................................................................................................................. 70 ACKNOWLEDGEMENTS...................................................................................................... 71 REFERENCES................................................................................................................... 71 CHAPTER 3:..................................................................................................................... 91

Plasmid template design and in vitro transcription of short RNAs within a 'structure cassette' for

structure probing experiments.............................................................................................91

ABSTRACT........................................................................................................................ 92 INTRODUCTION................................................................................................................ 93 MATERIALS...................................................................................................................... 97

Generating a structure cassette plasmid containing a target RNA sequence............................... 97

Cloning Method 1: PCR...................................................................................................... 98

Cloning Method 2: Direct ligation of chemically synthesized DNA oligonucleotides.......................99

Analysis of transcribed RNA................................................................................................ 99 METHODS....................................................................................................................... 100

Generating structure cassette plasmids with new target RNAs................................................ 100

Cloning Method 1: PCR...................................................................................................... 102

Cloning Method 2: Direct ligation of chemically synthesized DNA oligonucleotides.......................103

Analysis and preparation of transcribed RNA for probing experiments........................................104

NOTES............................................................................................................................. 106 ACKNOWLEDGEMENTS...................................................................................................... 110 REFERENCES..................................................................................................................... 110 CHAPTER 4:.......................................................................................................................112

Dissection of the adenoviral VA RNAI Central Domain structure reveals minimal requirements for

RNA-mediated inhibition of PKR ............................................................................................112

ABSTRACT......................................................................................................................... 113 INTRODUCTION................................................................................................................. 113 EXPERIMENTAL PROCEDURES.............................................................................................. 116 RESULTS AND DISCUSSION................................................................................................. 121 REFERENCES......................................................................................................................146 ACKNOWLEDGEMENTS........................................................................................................ 151 FUNDING...........................................................................................................................151 CHAPTER 5:...................................................................................................................... 152

A novel RNA molecular signature for activation of 2'-5' oligoadenylate synthetase-1.....................152

ABSTRACT......................................................................................................................... 153 INTRODUCTION................................................................................................................. 153 MATERIAL AND METHODS................................................................................................... 155 RESULTS........................................................................................................................... 159 DISCUSSION..................................................................................................................... 173 ACKNOWLEDGEMENT.......................................................................................................... 177 FUNDING.......................................................................................................................... 177

SUPPLEMENTAL FIGURES.................................................................................................... 178

REFERENCES..................................................................................................................... 183 CHAPTER 6:...................................................................................................................... 189 Conclusions...................................................................................................................... 189

VA RNAI INHIBITION OF PKR: OPEN QUESTIONS................................................................... 190

DICER PROCESSING OF VA RNAI......................................................................................... 194

FUNCTIONAL IMPLICATIONS OF THE 3'-ssPy MOTIF............................................................... 196

ADDITIONAL POTENTIAL PROVIRAL ROLES FOR VA RNAI........................................................ 202

THE FUNCTIONS OF EBV EBER-1 REMAIN UNCLEAR............................................................... 203

CONCLUSION................................................................................................................... 204

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