Characterization of the Cricket Paralysis Virus 3C Protease And
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CHARACTERIZATION OF THE CRICKET PARALYSIS VIRUS 3C PROTEASE AND ITS SUBSTRATE SPECIFICITY by Ruhi Nichalle Brito B.Sc., Trent University 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2018 © Ruhi Nichalle Brito, 2018 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: Characterization of the Cricket Paralysis Virus 3C Protease and its Substrate Specificity submitted by Ruhi Nichalle Brito in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology Examining Committee: Dr. Eric Jan, Department of Biochemistry and Molecular Biology Supervisor Dr. Helene Sanfacon, Department of Botany Supervisory Committee Member Dr. Dieter Bromme, Department of Biochemistry and Molecular Biology Supervisory Committee Member Dr. Thibault Mayor, Department of Biochemistry and Molecular Biology Additional Examiner Additional Supervisory Committee Members: Dr. Chris Overall, Department of Biochemistry and Molecular Biology Supervisory Committee Member Supervisory Committee Member ii Abstract Many positive-sense single-stranded RNA (+ssRNA) viruses encode an open reading frame that is translated as a polyprotein. This viral polyprotein is subsequently cleaved by its virally encoded protease or in some instances with the aid of host proteases. It has been well established that +ssRNA viruses, such as poliovirus encode protease(s) that can cleave and target host protein substrates in order to facilitate viral infection. The Dicistrovirade family, are +ssRNA viruses that primarily infect arthropods such as honey bees, shrimp, and crickets and can have an impact on agriculture and the economy. Dicistroviruses encode a cysteine protease, 3C, that is responsible for the cleavage of its own polyprotein. To date little is known about dicistrovirus protease structure, catalytic efficiency, cleavage site specificity and substrate specificity. Cricket paralysis virus (CrPV), a dicistrovirus, has been well characterized within its family. CrPV has been characterized for its translation mechanism as well as a few of its encoded proteins such as 1A, thus making it a good model to study. Given that other +ssRNA viral 3C proteases, such as poliovirus, cleave host substrates during infection, it could be thought that the CrPV 3C protease cleaves target host proteins during infection. In order to better understand the fundamental processes that are regulated during infection, CrPV was chosen as a model. In this thesis CrPV 3C protease was purified to address two aims. 1) Purify and verify activity of CrPV 3C protease and 2) Determine cleavage site specificity of CrPV 3C protease. This will help give a better understanding of the catalytic efficiency and target substrate specificity of the purified protease. iii Lay Summary Viruses use resources available to them from the host they infect. This is because the virus is small and does not contain all the essential components for it to survive by itself. One way for the virus to trick the host into helping the virus, is by stopping regular functions in the host. This in turn limits the resources in the host. The way that the virus stops these functions is by this enzyme known as a protease. This protease cut host proteins, making them unusable to the host. Unfortunately, we do not know what these host proteins are and how some of these virus enzymes act. To understand this, we isolated a type of viral protease and then determined what it could possibly cut. iv Preface All experiments were conducted by me. The fluorescent peptides were designed with the help of Dr. Eric Jan and made by Biomatik. The construction of the peptide libraries for Aim 2 were made with the help of Dr. Nestor Solis, as well as the analysis of the mass spectrometry data. The phylogenetic tree of +ssRNA virus cysteine proteases was made with the help of Dr. Marli Vlok. All experiments were designed by my supervisor Dr. Eric Jan and myself. I finished the Biological Safety Training Course [Certificate ID: 2016-qajCN] , Chemical Safety course [Certificate ID: 2018-Xa7BQ], and Radionuclide Safety and Methodology course [Certificate ID: 2017-Rc7NX] provided by Risk Management Services as required for this research. v Table of Contents Abstract ................................................................................................................................... iii Lay Summary ........................................................................................................................... iv Preface ....................................................................................................................................... v Table of Contents ..................................................................................................................... vi List of Tables ............................................................................................................................ ix List of Figures ........................................................................................................................... x List of Symbols ........................................................................................................................ xii List of Abbreviations .............................................................................................................xiii Acknowledgements ................................................................................................................. xv Dedication ............................................................................................................................. xvii CHAPTER 1: INTRODUCTION ...................................................................................................... 1 1.1 General overview of RNA viruses ........................................................................... 1 1.1.1 Positive-sense single stranded RNA viruses ......................................................... 1 1.1.2 Viral life cycle ..................................................................................................... 5 1.2 Host substrates cleaved during +ssRNA viral infection ............................................ 8 1.2.1 Host translation shutoff ........................................................................................ 8 1.2.2 Host transcription shutoff ..................................................................................... 9 1.2.3 Immune response and stress granules ................................................................... 9 1.3 Proteases ............................................................................................................... 10 1.3.1 Protease families ................................................................................................ 10 vi 1.3.2 Cysteine proteases ............................................................................................. 12 1.3.3 Protease Kinetics ............................................................................................... 16 1.4 Dicistrovirus .......................................................................................................... 17 1.4.1 Classification and genome organization ............................................................. 17 1.4.2 Cricket Paralysis Virus ...................................................................................... 21 1.4.3 Dicistrovirus 3C protease and their cleavage specificity ..................................... 21 1.5 Approaches to identify candidate substrates ........................................................... 24 1.5.1 Classical and new approaches of identification................................................... 24 1.6 Thesis approach ..................................................................................................... 29 CHAPTER 2: MATERIALS AND METHODS ................................................................................. 31 2.1 Generation of plasmid GST-tagged CrPV 3C ......................................................... 31 2.2 Optimization of expression of CrPV 3C ................................................................. 31 2.3 GST CrPV 3C Purification and cleavage conditions............................................... 32 2.4 Determination of GST CrPV 3C stability ............................................................... 33 2.5 Determination of protease activity by Fluorescence quenching assay ..................... 33 2.6 In-vitro translation reaction .................................................................................... 34 2.7 Proteome Identification of Cleavage Site ............................................................... 35 CHAPTER 3: OPTIMIZATION, PURIFICATION AND KINETICS OF 3C PROTEASE .......................... 37 3.1 Background ........................................................................................................... 37 3.2 Results ................................................................................................................... 37 3.2.1 Expression of CrPV 3C ...................................................................................... 37 3.2.2 Purification of CrPV 3C and cleavage