STUDIES TOWARD THE IDENTIFICATION OF THE ORIGIN OF ASSEMBLY ON CUCUMBER NECROSIS VIRUS RNA AND ENCAPSIDATION OF HOST RNA by KANKANA GHOSHAL B.Sc., Surendranath College, University of Calcutta, Kolkata, India, 2003 M.Sc., University of Calcutta, Kolkata, India, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Plant Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2015 © Kankana Ghoshal, 2015 Abstract Assembly is one of the major steps in the virus multiplication cycle. Recognition of viral RNA by coat protein (CP) is one means to ensure specific packaging of viral RNA over host RNA for the production of infectious virus particles. Viral RNAs possess specific sequences and/or structures [origin of assembly sequences (OASs)] which serve as high-affinity binding sites for the CP. In this thesis, I aimed to identify the OAS of Cucumber necrosis virus (CNV). Serendipitously, it was found that besides viral RNA, CNV also encapsidates host RNAs albeit to a lower level (~0.1%). Therefore, I extended my research to characterize the host RNAs present in CNV virions and in virus-like particles (VLPs) formed during agro- infiltration with CP. Characterization of encapsidated RNAs showed that both CNV virions and VLPs contained a variety of host RNA species, the most predominant being chloroplast encoded RNAs. Remarkably, certain retrotransposon or retrotransposon-like sequences were among the most efficiently encapsidated nuclear encoded RNAs, indicating that CNV virions may possibly serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution. To my knowledge, this is the first report of a plant virus encapsidating retrotransposon-like sequences and complements the recent findings that Flock house virus, a small icosahedral insect virus, also encapsidates retrotransposons. Interestingly, analysis of the relative encapsidation efficiency of CP mRNA in VLPs was found to be high, indicating that the CNV CP ORF may contain an OAS(s). ii Towards the identification of an OAS in CNV RNA, a 1.2 kb segment encompassing the 3’ terminus of the replicase ORF and 800 nt of the CP ORF was found to stimulate encapsidation of heterologous chimeric viral RNA during co-infection with CNV. However, smaller portions of this region failed to facilitate encapsidation. Interestingly, two chimeric viral RNAs expressing CNV CP were efficiently encapsidated suggesting that the CP ORF may contain an OAS(s). This result raises the possibility that the microenvironment where virus replication and encapsidation occurs may play a role in the specificity of encapsidation, supporting the involvement of multiple factors in the specificity of CNV RNA assembly. iii Preface The research work of this thesis was conducted in Dr. D’Ann Rochon’s lab at the Agriculture and Agri-Food Canada (AAFC), Pacific Agri-Food Research Centre (PARC), Summerland, British Columbia, Canada from January 2010 to August 2015 by the candidate. A list of manuscripts that are either published or in preparation are mentioned below which have resulted from the work of this thesis. The contribution of the candidate is mentioned below. Chapter 1: Literature review The candidate wrote the chapter and Dr. D’Ann Rochon provided editorial support. Chapter 2: Encapsidation of host RNAs by Cucumber necrosis virus coat protein both during agro-infiltration and infection was modified from the manuscript: Ghoshal, K., Theilmann, J., Reade, R., Maghodia. A., and Rochon, D. (2015) Encapsidation of host RNAs by Cucumber necrosis virus coat protein both during agro-infiltration and infection. J. Virol. Accepted manuscript posted online doi:10.1128/JVI.01466-15 The candidate and Dr. D’Ann Rochon designed the research. The candidate conducted agro-infiltration, virus inoculation, extraction of total leaf RNA (TLR), virions, VLPs and virion RNA, Northern blot analyses, Western blot analyses, Next Generation RNA sequencing analyses of VLP-RNAseq and CNV-RNAseq1 and Droplet Digital PCR (ddPCR). Dr. D’Ann Rochon performed the transmission electron microscopy (TEM) and analyzed the CNV- iv RNAseq2 data. Jane Theilmann assisted the candidate in RT-PCR and ddPCR. Ron Reade made pCNVCPpBin(+) construct. Dr. Ajay Maghodia conducted the N-terminal peptide sequencing. The candidate wrote the manuscript and Dr. D’Ann Rochon supervised the work and manuscript preparation and provided editorial support. Chapter 3: Identification and characterization of the OAS on CNV RNA, a version of this chapter along with additional experiments is anticipated to be prepared for publication. Anticipated author list: Ghoshal, K., Reade, R., Theilmann, J. and Rochon, D. The candidate and Dr. D’Ann Rochon designed the research. The candidate conducted cloning, agro-infiltration, virus inoculation, extraction of TLR, virions, VLPs and virion RNA, Northern blot analyses, semi-quantitative RT- PCR and ddPCR. Dr. D’Ann Rochon performed TEM. Ron Reade and Jane Theilmann assisted the candidate in cloning and ddPCR, contributing to approximately 5-10% of the research. The candidate wrote the chapter and Dr. D’Ann Rochon provided editorial support. Chapter 4: General discussion The candidate wrote the chapter and Dr. D’Ann Rochon provided editorial support. v Table of Contents Abstract ....................................................................................................................................... ii Preface ...................................................................................................................................... iv Table of Contents ...................................................................................................................... vi List of Tables ............................................................................................................................. ix List of Figures ............................................................................................................................. x List of Abbreviations ............................................................................................................... xiii Acknowledgments ................................................................................................................... xvi Dedication ............................................................................................................................... xix Chapter 1: Literature review ................................................................................................... 1 1.1 Introduction ....................................................................................................... 1 1.2 Overview of the multiplication cycle of plant positive-sense single-stranded RNA viruses ...................................................................................................... 3 1.3 A brief overview of virus particle structure and assembly mechanisms of viral RNA .......................................................................................................... 6 1.3.1 Monopartite helical viruses: Structure and assembly mechanisms .......... 6 1.3.2 Monopartite icosahedral viruses: Structure and assembly mechanisms .. 7 1.4 Factors involved in assembly of viral RNA .................................................... 10 1.4.1 Electrostatic interactions ........................................................................ 10 1.4.2 Origin of assembly sequence (OAS) ...................................................... 14 1.4.3 Amino acid sequences on CPs that participate in viral RNA interaction ............................................................................................... 16 1.4.4 Replication coupled encapsidation and the microenvironment of assembly .................................................................................................. 17 1.4.5 Physical size constraints ......................................................................... 20 1.5 Detailed discussion of OAS(s) ........................................................................ 20 1.5.1 Helical viruses ........................................................................................ 21 1.5.1.1 Single OAS................................................................................. 21 1.5.1.2 Multiple OASs ........................................................................... 22 1.5.2 Icosahedral viruses ................................................................................. 23 1.5.2.1 Single OAS................................................................................. 23 1.5.2.2 Multiple OASs ........................................................................... 25 1.5.3 Viruses with no apparent OAS ............................................................... 32 1.6 Packaging profile of virions versus virus-like particles (VLPs) ..................... 33 1.7 Cucumber necrosis virus (CNV) ..................................................................... 34 1.7.1 Biology, genome organization and virus particle structure ................... 34 1.7.2 CNV multiplication cycle ...................................................................... 37 1.7.3 Structure and assembly of CNV ............................................................. 40 1.8 Objectives .......................................................................................................