CHARACTERIZATION OF THE HUMAN T-CELL LEUKEMIA VIRUS TYPE-2 P28 ACCESSORY PROTEIN DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Rami Doueiri MSc. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Dissertation Committee: Dr. Patrick L. Green, Advisor Dr. Mamuka Kvaratskhelia Dr. Michael Oglesbee Dr. David Bisaro Copyright by Rami Doueiri 2012 ABSTRACT Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) was the first identified human retrovirus that causes malignancy, and further research identified it as the etiological agent of several neurodegenerative and inflammatory diseases. On the other hand, HTLV-2, which shares high sequence and structural homology with HTLV-1, is less pathogenic with few reported cases of neurodegenerative diseases. HTLVs encode structural and enzymatic proteins, Gag, Pro and Env, regulatory proteins, Tax and Rex and several accessory genes required for in vivo viral persistence. Extensive comparative studies of the regulatory genes elucidated the role of these proteins in the viral life cycle and in part explained the different pathology of both viruses. However, increasing amount of evidence describes an essential role for the accessory genes in the viral pathogenic process. p30 and p28 are two accessory proteins encoded by HTLV-1 and HTLV-2, respectively. p30 and p28 are encoded from the second open reading frame (ORF-II) and share some amino acid homology. They are post-transcriptional negative regulators of viral replication, and act by retaining tax/rex mRNA in the nucleus. However, unlike p30, p28 is devoid of transcriptional activity. Several binding proteins have been identified for p30 that helped to elucidate its role in the viral life cycle; however, no binding partners for p28 have been identified to date. In addition, functional domains and post-translational modifications of p30 have been described, but this data is ii lacking for the related p28. Identification and characterization of p28 structure/function would facilitate comparative studies between p28 and p30, allow us to better understand the role of accessory genes in the viral life cycle, and ultimately to better understand the different pathobiology of HTLV-1 and HTLV-2. In Chapter 2 of this dissertation, we identified binding partners of p30 and p28 using mass spectrometry, and then verified our data using molecular methods. We identified NIP30 as a unique binding partner for p30, while hnRNP H1 solely interacted with p28, and PRMT5 interacted with both proteins. We then knocked down PRMT5 in vivo to assess the role of PRMT5 in the viral life cycle. Our data suggest that PRMT5 seems to be involved in post-transcriptional control of HTLV-2 replication, while its effect on HTLV-1 might be at the level of DNA damage and cell cycle control. In Chapter 3, we identified and characterized the effect of phosphorylation on p28 in vivo. We conducted phosphoryl mapping of mammalian-expressed p28 protein using a combination of affinity purification, tandem mass spectrometry, and site-directed substitution mutational analysis. We identified seven phosphorylation events on p28 at Ser-33, Ser-160, Ser-170, Ser-172, Thr-199, Ser-200 and Ser-203. We evaluated the functional significance of these phosphorylation sites and found that phosphorylation at Ser-160 and Thr-199 reduced the ability of p28 to dimerize, while phosphorylation did not affect the post-transcriptional activity of p28 or its ability to interact with hnRNP H1 or PRMT5. iii In Chapter 4, we report the identification of functional domains of p28. We created six p28 deletion mutations p28ΔN (deletion of amino acid (Δ)1-50), p28ΔM1(Δ51-100), p28ΔM2 (Δ101-150), p28ΔC (Δ151-216), p28ΔM2-C (Δ101-216) and p28ΔM1-M2-C(Δ51-216), and evaluated the functional significance of these domains. We identified a tripartite nuclear localization sequence (NLS) in regions N-M2- C (amino acids 1-50 and 100-216), while p28 interaction with hnRNP H1 requires domains N (amino acids 1-50) and C (amino acids 151-216), and its dimerization requires domains M1 (amino acids 51-100) and C (amino acids 151-216). Finally, we identified that p28 C terminus (150-216) exerts an inhibitory effect on p28 post-transcriptional function. Taken together this work is the first to identify p28 binding partners, phosphorylation sites and functional domains, providing important insight into p28 mechanism of action to better understand its role in HTLV-2 replication and pathogenesis. iv Dedicated to my parents, my brothers, my aunt, and Masi v ACKNOWLEDGEMENTS I would like to thank my advisor, Dr Patrick Green for his valuable help, insight and guidance throughout my PhD. He taught me how to think like a scientist, behave like one and most importantly how to clearly present and explain my data. I have learned how to be relaxed yet effective in my pursuit of my goals. Thank you for this great positive learning opportunity and for helping reach higher. I would like to thank my committee members, Dr Mamuka Kvaratskhelia, Dr Michael Oglesbee and Dr David Bisaro for their valuable advice, encouragement and guidance throughout the years. I would like to thank all the Green lab members, past and present, Dr Matt Kesic, Dr Min Li, Dr Brenda Yamamoto, Dr Priya Kannian, Dr Amanda Robinson, Nathan Dissigner, Jacob Al-Saleem, Krissy Landes and Sneha Gupta for the great times and the great fun we had that helped endure the stress over the years! A great thank you to Dr Raj Anupam for the great times and without whom there wouldn’t be a comparative paper. I would like to thank Dr Kvaratskhelia and his lab, Dr Michael Lairmore, Dr Boris-Lawrie and her lab and Dr Li Wu and his lab for their great insight and resources that helped me to progress in my research. Special thanks for Dr Nikolozi Shkriabai, Dr vi Amit Sharma, Dr Arnaz Ranji, Dr Corine St-Gelais (the best editor) and Wei Jing for all their help and for making the basement a brighter place to be. Finally, my dad Abdul Hafiz, my mom Wafica, my brother Ziad and his family and my brother Dany and his family for being next to me, believing in me and encouraging me for the past 30 years of my life, you are the perfect family to have. My girlfriend Masi, thank you for the great time we spent together, the joy you brought to my life and the years we will spend together, without you the past four years would have been unbearable. My friends Ali Assi and his family, Mohammad Hotait and his family, Mohammad Kabbout and Zakaria Chehab, thank you enormously, you made Columbus a warm place to be and I wish you the best wherever you are. vii VITA 2001-2003………………………………. Bachelor of Arts and Science The American University of Beirut Beirut, Lebanon. 2003-2005...……………………………...Masters of Science The American University of Beirut Beirut, Lebanon 2003-present...…………………………...Graduate Research Assistant Department of Molecular, Cellular and Developmental Biology The Ohio State University PUBLICATIONS Doueiri R, Anupam R, Kvaratskhelia M, Green-Church K, Lairmore MD, Green PL. Comparative host protein interactions with HTLV-1 p30 and HTLV-2 p28: insights into difference in pathobiology of human retroviruses. Retrovirology 2012, in press Doueiri R, Green, PL. Human T-Cell Leukemia Virus Type 2 (HTLV-2) Biology and Pathogenesis. 2012 Cancer-Associated Virus, Robertson, Erle (Ed.) 1st Edition. XII, 1026 p. Hardcover, ISBN 978-1-4419-9999-3 Kannian P, Yin H, Doueiri R, Lairmore MD, Fernandez S, Green PL: Distinct transformation tropism exhibited by Human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) is the result of post-infection T cell clonal expansion. J Virol 2012 86(7):3757-66. PMCID:PMC3302488 Kannian P, Yin H, Doueiri R, Green PL: Cellular tropism exhibited by human T lymphotropic virus type 1 and type 2 (HTLV-2). Retrovirology 2011, 8 (Suppl 1):A176. PMCID: PMC3112649 Kesic M, Doueiri R, Ward MD, Semmes OJ, Green PL: Phosphorylation regulates human T-cell leukemia virus type 1 Rex function. Retrovirology 6:105, 2009 doi:10.1186/1742-4690-6-105. PMCID:PMC2780990 viii FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology Area of emphasis: Molecular Virology ix TABLE OF CONTENTS Abstract ...........................................................................................................................ii Dedication .......................................................................................................................v Acknowledgements ........................................................................................................vi Vita ..................................................................................................................................viii List of Tables ..................................................................................................................xii List of Figures .................................................................................................................xiii Chapter 1: HTLV2 Background...................................................................................1 1.1. Discovery of HTLV-1 ...................................................................................1 1.2. Discovery of HTLV-2 ...................................................................................2 1.3. HTLV-3 and HTLV 3-4 ................................................................................3 1.4. HTLV-2 virion