Structural Integrity is Essential for the Replication of the Virusoid RNA of Lucerne Transient Streak Sobemovirus by Kayvan Mirhadi A thesis submitted in conformity with the requirements for the degree of Master of Science Cell and Systems Biology University of Toronto © Copyright by Kayvan Mirhadi 2010 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l’édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-72912-0 Our file Notre référence ISBN: 978-0-494-72912-0 NOTICE: AVIS: The author has granted a non- L’auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l’Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L’auteur conserve la propriété du droit d’auteur ownership and moral rights in this et des droits moraux qui protège cette thèse. Ni thesis. Neither the thesis nor la thèse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent être imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author’s permission. In compliance with the Canadian Conformément à la loi canadienne sur la Privacy Act some supporting forms protection de la vie privée, quelques may have been removed from this formulaires secondaires ont été enlevés de thesis. cette thèse. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n’y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Structural Integrity is Essential for the Replication of the Virusoid RNA of Lucerne Transient Streak Sobemovirus Kayvan Mirhadi Master of Science Cell and Systems Biology University of Toronto 2010 Abstract Lucerne transient streak sobemovirus (LTSV) supports the replication of a small, 322-nucleotide, untranslated virusoid (vLTSV) that has an extensively base-paired, viroid-like structure. Since vLTSV does not code for its own proteins or share sequence homology with its helper virus (LTSV), it is presumed that it uses structural motifs to signal the helper virus (and host) machinery for its replication. In order to elucidate these structural domains, insertion-deletion mutations were introduced to disrupt the secondary structure. Infectivity assays of these mutants showed that they were all lethal, except a 9-nucleotide, palindromic insertion, which preserved the overall rod-like structure of the virusoid. Sequence analysis of cDNA clones prepared from progeny virusoid RNA revealed that the palindromic sequence was replicated up to twelve days of infection but discarded afterwards. Results indicate that vLTSV has an optimum size and secondary structure for replication and packaging within the LTSV helper virus. ii Acknowledgements I would like to thank Professor Mounir G. AbouHaidar, who was my supervisor and mentor, for making everything possible. He has given me much support and encouragement and most importantly an opportunity to experience research first hand. Many thanks goes to my supervisory committee members, Professor Maurice Ringuette and Professor Keiko Yoshioka for their continuous help and support. I would not have reached this level without their guidance and helpful remarks. Thanks goes to my current and former colleagues: Taqueer, Vidya, Amanda, and Huda. A special thanks goes to Taqueer for helping my out with this project as well as the Begomovirus side project. You are truly an example of hard work and determination. I especially want to thank my parents and bigger brother for their love, support, and friendship. They gave me confidence to know that I can accomplish anything and they were always supportive of my goals in life. And, of course, a especial thanks to my beautiful Niloofar, for her love, patience, understanding, and helping me out through thick and thin. iii Table of Contents Abstract……………………………………………………………………………………………ii Acknowledgements........................................................................................................................iii Table of Contents........................................................................................................................... iv List of Figures ............................................................................................................................... vii List of Tables ...............................................................................................................................viii List of Abbreviations ..................................................................................................................... ix Chapter 1 Literature Review........................................................................................................... 1 1 Viroids and Viroid-like Satellite RNAs (Virusoids)................................................................. 1 1.1 General features and organization...................................................................................... 1 1.2 Viroid and virusoid replication .......................................................................................... 4 2 Structure-Function Relationship ............................................................................................... 9 2.1 Functional domains of viroids............................................................................................ 9 2.2 Functional domains of satellite RNAs ............................................................................. 11 Chapter 2 Introduction .................................................................................................................. 12 3 Lucerne Transient Streak virus ............................................................................................... 12 4 The LTSV Virusoid ................................................................................................................ 13 4.1 Satellite-like nature and symptom induction.................................................................... 13 4.2 Helper and host specificity............................................................................................... 13 4.3 Sequence and structure..................................................................................................... 14 4.4 Replication and ribozyme activity.................................................................................... 15 5 Full-length infectious cDNA clones ....................................................................................... 16 6 Research proposal ................................................................................................................... 18 6.1 Overview of previous work.............................................................................................. 18 6.2 Current objectives ............................................................................................................ 18 6.3 Hypothesis........................................................................................................................ 19 Chapter 3 Materials and Methods ................................................................................................. 20 iv 7 General Molecular Techniques............................................................................................... 20 7.1 Plasmid DNA isolation from E. coli (miniprep) .............................................................. 20 7.2 Heat shock transformation of E. coli................................................................................ 21 7.3 Glycerol stock preparation ............................................................................................... 21 7.4 Phenol-chloroform extraction of DNA/RNA................................................................... 22 8 Sub-cloning of 322I8 to generate multimers .......................................................................... 23 8.1 Restriction digest and agarose gel electrophoresis........................................................... 23 8.2 Ligation ............................................................................................................................ 23 8.3 Screening of colonies ....................................................................................................... 23 9 In vitro runoff transcription of cDNA clones ......................................................................... 25 10 Purification of viruses........................................................................................................... 26 10.1 Lucerne transient streak virus......................................................................................... 26 10.2 Turnip rosette virus .......................................................................................................