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Role of P33 in Tombusvirus Replication

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Role of P33 in Tombusvirus Replication University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2009 ROLE OF P33 IN TOMBUSVIRUS REPLICATION Jozsef Stork University of Kentucky Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Stork, Jozsef, "ROLE OF P33 IN TOMBUSVIRUS REPLICATION" (2009). University of Kentucky Doctoral Dissertations. 684. https://uknowledge.uky.edu/gradschool_diss/684 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Jozsef Stork The Graduate School University of Kentucky 2009 ROLE OF P33 IN TOMBUSVIRUS REPLICATION _________________________________________________ ABSTRACT OF DISSERTATION _________________________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Jozsef Stork Lexington, Kentucky Director: Dr. Peter D. Nagy, Associate Professor of Plant Pathology Lexington, Kentucky 2009 Copyright 2009 ELSEVIER ABSTRACT OF DISSERTATION ROLE OF P33 IN TOMBUSVIRUS REPLICATION Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In my thesis I describe (i) the effect of phosphorylation of p33, (ii) the RNA chaperone-like activity of p33, and (iii) the role of HSP70s a host proteins in the viral replication. To test the effect of phosphorylation on p33 function, I used in vitro phosphorylated p33. I found that phosphorylation inhibited the ability of p33 to bind to the viral RNA. Phosphorylation-mimicking mutations rendered p33 nonfunctional in plant protoplasts and in yeast. Based on these results, I propose that the primary function of phosphorylation of p33 is to regulate its RNA binding capacity, which could affect the assembly of new viral replicase complexes, recruitment of the viral RNA template into replication and/or release of viral RNA from replication. Thus, phosphorylation of p33 might help in switching the role of the viral RNA from replication to other processes, such as viral RNA encapsidation and cell-to-cell movement. Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, small RNA viruses might use RNA chaperones for replication as shown here for the p33 replication protein. In vitro experiments demonstrated that the purified recombinant p33 facilitated RNA synthesis on plus- stranded and double-stranded (ds)RNA templates up to 5-fold. In addition, p33 rendered dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNA. Altogether, the RNA chaperone activity of p33 might perform similar biological functions to the helicases. SSa a yeast HSP70 found in the viral replication complex and shown to facilitate viral replication (Serva and Nagy, 2006)To dissect the mode of action of SSA in the viral replication I used temperature sensitive and deletion mutants. Both showed miss localization of p33 compared to the wild type. Purified SSA rendered non functional bacterial expressed p92 functional in an in vitro replication assay. SSa might play a role in the transportation and assembly of viral replication proteins. KEYWORDS: Tombusvirus, phosphorylation, RNA, chaperone, host factor. Jozsef Stork 02/13/2009 ROLE OF P33 IN TOMBUSVIRUS REPLICATION By Jozsef Stork ____________________________ Dr Peter Nagy 02/13/2009 ____________________________ Dr Lisa Vaillancourt 02/13/2009 ____________________________ RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open to inspection, but are to be used only with the due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries may be published only with the permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for the use by its patrons is expected to secure the signature of each user. Name Date __________________________________________________________________ _________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ DISSERTATION Jozsef Stork The Graduate School University of Kentucky 2009 ROLE OF P33 IN TOMBUSVIRUS REPLICATION _________________________________________________ DISSERTATION _________________________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Jozsef Stork Lexington, Kentucky Director: Dr. Peter D. Nagy, Associate Professor of Plant Pathology Lexington, Kentucky 2009 Copyright 2009 ELSEVIER Table of Contents List of figures ................................................................................................................ v CHAPTER I .................................................................................................................. 1 General introduction ..................................................................................................... 1 Goals and hypothesizes ............................................................................................. 6 CHAPTER II ............................................................................................................... 10 Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication ................................ 10 Introduction ............................................................................................................. 10 Results ..................................................................................................................... 13 Discussion ............................................................................................................... 21 Materials and methods ............................................................................................ 24 CHAPTER III ............................................................................................................. 40 Inhibition of in vitro RNA binding and replicase activity by phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus ....................................... 40 Introduction ............................................................................................................. 40 Results ..................................................................................................................... 41 Discussion ............................................................................................................... 48 Materials and methods ............................................................................................ 53 CHAPTER IV ............................................................................................................. 67 RNA chaperone activity of the tombusviral p33 replication protein facilitates initiation of RNA synthesis by the viral RdRp ........................................................... 67 Introduction ............................................................................................................. 67 Results ..................................................................................................................... 69 Discussion ............................................................................................................... 75 Materials and Methods ............................................................................................ 78 CHAPTER V ............................................................................................................. 96 Role of a host HSP70 protein in viral replication ....................................................... 96 Introduction ............................................................................................................. 96 Results ..................................................................................................................... 98 Discussion ............................................................................................................. 102 Materials and methods .......................................................................................... 104 CHAPTER VI ........................................................................................................... 117 Summary ................................................................................................................... 117 iii Future
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