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Stiv F93 Protein Is a Transcription Factor That Regulates C92

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Stiv F93 Protein Is a Transcription Factor That Regulates C92 STIV F93 PROTEIN IS A TRANSCRIPTION FACTOR THAT REGULATES C92 EXPRESSION PLAYING ROLE IN VIRAL GENOME REPLICATION A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Prudencio A. Merino Carreon 2019 SIGNATURE PAGE THESIS: STIV F93 PROTEIN IS A TRANSCRIPTION FACTOR THAT REGULATES C92 EXPRESSION PLAYING ROLE IN VIRAL GENOME REPLICATION AUTHOR: Prudencio A. Merino Carreon DATE SUBMITTED: Spring 2019 Department of Biological Sciences Dr. Jamie C. Snyder Thesis Committee Chair Biological Sciences Dr. Nancy E. Buckley Biological Sciences Dr. Christos Stathopoulos Biological Sciences ii ACKNOWLEDGEMENTS It is a sincere pleasure to express my deep sense of thanks and gratitude to my mentor and professor Dr. Jamie C. Snyder for giving me the opportunity to be part of her amazing research team. Dr. Snyder’s dedication and keen interest above her awesome attitude to help her students had been responsible for trying to excel and completing my work despite my rough start at the beginning of this journey. Her timely and scholarly advice, and scientific approach have helped me immensely to accomplish this challenging task. A very special thanks to my lab colleagues Michael S. Overton and Christian E. Pirijanian who without a doubt helped me get through many questions and obstacles along the span of my research. Michael S. Overton a.k.a. Mike, was like a second mentor always willing to help out in answering questions that anyone would have in the lab. Having had the opportunity to work alongside both these gentlemen is yet another reason why working in this lab was such an incredible experience. I’m eternally grateful to my small army of researchers: Benjamin Clock, Gabriel Martinez and the newest recruit Peter Youssef, for their contribution and dedication in helping me with my research. This amazing group of scientists, always offered their support, if it wasn’t to come into the lab during the week it was during the weekend to get work done, work that they didn’t have to do. I respect and admire their dedication to the lab; I know without a doubt they will be very successful in their scientific endeavors. I would like to give a special thanks Dr. Stathopoulos and Dr. Nancy E. Buckley for accepting to be part of my thesis committee and providing great insight into my project. These two individuals are not only great professors but also very kind human iii beings. I would also like extend my gratitude to Dr. Buckley’s lab members and to the rest of my lab colleagues from the Snyder squad for their collaboration in helping me revise sections of my thesis and being great friends over all. Last but not least, I would like to thank my family, because without their support this journey would have been much more difficult to accomplish. I am forever thankful for all the lessons my parents have thought me, remaining humble and true to my roots. I could not finish acknowledgements without thanking my siblings: Erika, Claudia, Luciano, Alfonso and Juan who have been extremely supportive of my educational achievements. iv ABSTRACT It is a biological constant that viruses are found associated with all known life and that they play an essential role in the ecology and evolution of all life-forms. Compared to viruses that infect Eukarya and Bacteria, little is known about the viruses that infect Archaea. One archaeal virus-host system, STIV-Sulfolobus, has developed into a model system for studying archaeal cell biology and virology. However, despite that, we still don’t fully understand the replication cycle of this virus, or any other archaeal virus. Sulfolobus turreted icosahedral virus (STIV) was first isolated from an acidic hot spring in Yellowstone National Park. STIV contains a double-stranded DNA genome in which 38 open reading frames (ORFs) have been identified. In crystallographic studies of the STIV proteome, one protein (F93) was found to be a 93-residue winged-helix-turn-helix (wHTH) protein. Because of its structural similarity to other proteins, it has been suggested that F93 is most likely a transcriptional regulator of several viral genes. Studies have revealed potential binding sites for F93 within the STIV genome consisting of two boxes of nearly palindromic DNA sequence with the consensus sequence including a region upstream of C92 and F93. Using site directed mutagenesis, a series of mutations were made to the F93 binding site upstream of C92. The findings suggest that by altering the conserved regions within the F93 binding site upstream of C92, the virus is still able to carry out a complete replication. However, these mutations do play a role in the level of viruses being produced. It is most likely that C92 is being regulated by F93 based on the changes made on the binding site. In addition, a series of mutations in the binding site upstream of F93 and within F93 protein were also created. These mutations will continue v to help us better understand the role of F93 in the viral genome replication cycle and provide further insights on gene regulation within Archaea. vi TABLE OF CONTENTS SIGNATURE PAGE ......................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................ iii ABSTRACT ....................................................................................................................... v LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES ......................................................................................................... ix CHAPTER 1 ...................................................................................................................... 1 INTRODUCTION......................................................................................................... 1 CHAPTER 2 .................................................................................................................... 18 MATERIALS AND METHODS ............................................................................... 18 CHAPTER 3 .................................................................................................................... 39 RESULTS .................................................................................................................... 39 CHAPTER 4 .................................................................................................................... 46 DISCUSSION .............................................................................................................. 46 REFERENCES ................................................................................................................ 53 vii LIST OF TABLES Table 1. Classification and morphotypes of archaeal viruses. Morphotypes in green indicate viruses isolated from crenarchaea and euryarchaea; Morphotypes in blue represent viruses isolated from only crenarchaea; Morphotypes in purple represent viruses isolated from only crenarchaea; Morphotypes in purple represent viruses isolated from euroarchaea and bacteria; Morphotypes in red represent viruses isolated from euryarchaea (Snyder etal.,2015)…………………………………………………………………………………6 Table 2. Primers designed for the study of F93 binding site upstream C92……….. ……18 Table 3. F93 binding site sequence upstream C92 represented by Box1 and Box2 and base pair changes on conserved and non-conserved regions………………………..…..19 Table 4. Primers designed with changes to the binding site upstream of F93…………..27 Table 5. Binding site sequence upstream F93 represented by Box1 and Box2 and base pair changes in conserved and non-conserved regions………………….……………….28 Table 6. Primers designed with changes to the STIV F93 protein……………………....33 viii LIST OF FIGURES Figure 1. A. Microscopy image of Sulfolobus Sulfataricus from the Phylum Crenarchaeota (D. Janckovik and W. Zillg). B. Methanogen (Methanosarcina)/Halophile (Halobacterium) from phylum Euryarchaeota (Whitehead Institute Center of Genome Research). C. Enriched culture with Korarchaeotal cells (Barns et al. 1996). D. Two Nanoarchaeum equitans cells and its larger host Ignicoccus from phylum Nanoarchaeota (Huber et al. 2002). .................................................................................... 3 Figure 2. A) Cryoelectron microscopy image of the STIV particle with a cutaway view of the T=31 icosahedral capsid with turret-like projections that extend from each of the 5- fold vertices. Portions of the protein shell (blue) and inner lipid layer (yellow) have been removed to reveal the interior (Lawrence et al., 2009). B) Scanning electron microscopy image of closed pyramid-like lysis structures on the surface of a Sulfolobus cell indicated by the red arrow (C-y. Fu et. al., 2010). C) Immunolocalization of overexpressed c92 in Sulfolobus PH1-16 showing localization to pyramid-like lysis structures (red arrows indicate gold particles on pyramid structure, and black arrows indicate internal membrane structures near the base of the pyramids). Bar scale 100 nm (Snyder, Brumfield, et al., 2011). D) Scanning electron microscopy image of opened pyramid lysis structures on the surface of a Sulfolobus cell indicated by the red arrow (C-y. Fu et. al., 2010). ............... 10 Figure 3. A. Structure of the F93 homodimer. Chain A (red), chain B (blue).
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