Angiogenin Inhibits Human Immunodeficiency Virus type 1 (HIV-1) Replication in Peripheral Blood Mononuclear Cells by a Complex Mechanism that Includes an Intracellular Component Authors Lin, Ping-Hsin Publication Date 2012 Abstract Angiogenin (ANG, also known as ribonuclease 5) is a 14.1kDa polypeptide secreted by T lymphocytes and epithelial cells. Other than its well-characterized function of promoting development of vasculature, ANG also possesses anti- Human Immunodeficienc... Keywords angiogenin; p24; PBMC; HIV-1 Download date 02/10/2021 11:30:10 Link to Item http://hdl.handle.net/10713/2163 Ping-Hsin Lin, M.S. [email protected] Education Master of Science in Medical Research July 2012 University of Maryland, Baltimore MD. Bachelor of Science in Medical Technology June 2004 Chang Gung University, Taoyuan, Taiwan Experience Institute of Human Virology, University of Maryland 09/2010-02/2012 Graduate Research Assistant • Studied the mechanism of human immunodeficiency virus type 1 (HIV-1) inhibition by angiogenin. Department of Medical & Research Technology, University of Maryland 01/2011-05/2011 Graduate Teaching Assistant • Assisted the teaching faculty by preparing lessons and lab experiments by photocopying class materials, and setting up equipment. • Graded students’ tests and homework. • Prepared and delivered lectures on various topics to undergraduate students. Department of Biochemistry and Molecular Biology, University of 03/2010-09/2010 Maryland Laboratory Assistant • Made aseptic buffer solutions and growth media, and autoclave glassware and instruments . Chang Gung University, Taoyuan, Taiwan 09/2006-06/2007 Research Assistant • Supported clinical research by using quantitative and qualitative techniques at the Liver Research Unit, Chang Gung Memorial Hospital, Taoyuan, Taiwan Licensure and Qualification • License for Medical Technician, the Ministry of Health Taiwan. Presentation • Poster, “: Angiogenin Inhibits Human Immunodeficiency Virus type 1 (HIV-1) Replication in Peripheral Blood Mononuclear Cells by a Complex Mechanism that Includes an Intracellular Component.” , Graduate Research Conference 2012 University of Maryland Baltimore Activity • 13th Annual International Meeting of the Institute of Human Virology Abstract Title of Thesis: Angiogenin Inhibits Human Immunodeficiency Virus type 1 (HIV-1) Replication in Peripheral Blood Mononuclear Cells by a Complex Mechanism that Includes an Intracellular Component Ping-Hsin Lin, Master of Science, 2012 Thesis Directed By: Alfredo Garzino-Demo, Ph.D. Associate Professor, Department of Microbiology and Immunology, Institute of Human Virology Angiogenin (ANG, also known as ribonuclease 5) is a 14.1kDa polypeptide secreted by T lymphocytes and epithelial cells. Other than its well-characterized function of promoting development of vasculature, ANG also possesses anti- Human Immunodeficiency Virus- 1 (HIV-1) properties. Previous findings show that ANG inhibits X4 tropic HIV-1 replication in peripheral blood mononuclear cells (PBMCs). Our studies show that ANG also inhibits R5 tropic virus, the most commonly transmitted HIV-1 strain. Inhibition was detected treating cells prior to or after infection by p24 ELISA, and is not mediated by CD4, CXCR4, or CCR5 as determined by flow cytometry assays. Furthermore, we demonstrated that ANG binds to tRNA-Lys, which serves as primer for HIV-1 reverse transcription. These results indicate that ANG inhibits HIV-1 replication by a post-entry mechanism, which might be exploited to develop new antiretroviral strategies. Angiogenin Inhibits Human Immunodeficiency Virus type 1 (HIV-1) Replication in Peripheral Blood Mononuclear Cells by a Complex Mechanism that Includes an Intracellular Component By Ping-Hsin Lin Thesis submitted to the faculty of the Graduate School of the University of Maryland Baltimore in partial fulfillment of the requirements for the degree of Master of Science 2012 © Copyright 2012 by Ping-Hsin Lin All Rights Reserved Acknowledgements It would not have been possible to write this thesis without the help and support of the kind people around me. Since it would be too many to list them all, I would only address some here. First and foremost, I would like to show my deepest gratitude to my mentor, Dr. Alfredo Garzino-Demo, for his constant support, guidance, encouragement, and patience. Professor Garzino-Demo undertook the responsibility as my supervisor in spite of his many academic and professional commitments. His wisdom, knowledge and passion for science has inspired and motivated me. I would also like to thank the members of my dissertation committee: Dr. Jianyuan Luo, Dr. Anthony DeVico, and Dr. Olga Latinovic. Their insightful advice and wonderful support have been invaluable to me both professionally and personally. I also appreciate Dr. Robert Gallo for providing the opportunity to pursue my research at the Institute of Human Virology. I am fortunate not only to meet and but also to work with many wonderful people throughout my time in graduate school. I am truly indebted and thankful all of the members of our lab, Dr. Lingling Sun, Dr. Mark Lafferty, Dr. Mohammad Khan, Dr. Jennifer Bharucha, Aaron Christensen-Quick, and Khrystal McGrant, for their helpful discussions on my project. I would especially like to thank Dr. Mark Lafferty who has generously shared his academic and experimental experiences, and Dr. LingLing Sun who has served as a tremendous source of technical expertise. Appreciation to my dear friends, Rebecca Hu, Yunting Fu, Vogtle Nanh, and Jane Huang for providing good company to make my memory at University of Maryland at Baltimore enjoyable. iii Table of Contents Acknowledgements ........................................................................................................... iii List of Figures .................................................................................................................. vii List of Abbreviations ........................................................................................................ ix Chapter1: Background and hypothesis ........................................................................... 1 Introduction ............................................................................................................. 1 Human Immunodeficiency Virus (HIV) ................................................................. 3 HIV Replication Cycle ............................................................................................ 9 HIV Pathogenesis .................................................................................................. 12 HIV Reverse Transcription ................................................................................... 16 Reverse Transcription ............................................................................... 16 tRNA-Lys3 ................................................................................................ 17 Angiogenin ............................................................................................................ 22 Angiogenin Molecular Structure and Domains ..................................................... 22 Angiogenin Receptor and Binding Protein ........................................................... 25 Angiogenesis ......................................................................................................... 26 Angiogenin RNase activity ................................................................................... 28 Angiogenin Anti-HIV Activity ............................................................................. 30 Specific Aims ........................................................................................................ 31 Chapter 2: Materials and Methods................................................................................ 32 Isolation and culture of primary cells and cell lines.............................................. 32 Virus production .................................................................................................... 32 Angiogenin antiviral assay .................................................................................... 32 Infectivity assays – p24 ......................................................................................... 33 Detecting CD4, CXCR4, and CCR5 by flow cytometry assay ............................. 34 iv Cell metabolism assay ........................................................................................... 34 AMLV pseudo type virus production.................................................................... 35 Virus concentration ............................................................................................... 36 Sucrose Gradient assay.............................................................................. 36 PEG 6000 assay ......................................................................................... 36 Chapter 3: To determine if ANG inhibits X4 and R5 tropic HIV-1 replication by an intracellular mechanism ................................................................................................. 37 3.1 Angiogenin inhibits both X4 and R5 tropic virus in PBMCs ........................ 37 Introduction ........................................................................................................... 37 Experimental Design ............................................................................................. 39 Results ..................................................................................................................