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Structural Integrity Is Essential for the Replication of the Virusoid RNA of Lucerne Transient Streak Sobemovirus

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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 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 ........................................................................................................ 26 10.3 Extraction of viral RNA ................................................................................................. 27 11 Infectivity assays of cDNA clones........................................................................................ 28 11.1 Coinoculation of TRosV with (+) and (-) RNA transcripts............................................ 28 11.2 Coinoculation of TRosV with dsDNA ........................................................................... 28 11.3 Coinoculation of TRosV with mutants........................................................................... 29 11.4 Total RNA extraction from B. rapa leaves .................................................................... 29 12 Reverse transcription, PCR, and cloning .............................................................................. 30 12.1 Reverse transcription (RT) ............................................................................................. 30 12.2 Polymerase chain reaction (PCR)................................................................................... 30 12.3 Cloning ........................................................................................................................... 31 Chapter 4 Results .......................................................................................................................... 33 13 Infectivity Assays of vLTSV as RNA or dsDNA................................................................. 33 13.1 RNA transcripts.............................................................................................................. 33 13.2 Double-stranded DNA.................................................................................................... 35 14 Structure-function analysis ................................................................................................... 37 14.1 Infectivity of large
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