The Effect of Androstenediol on the Influenza Virus APR8/34 in A549 Cells THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Scott Franklin Wray, B.A. Graduate Program in Pathology The Ohio State University 2012 Master's Examination Committee: W. James Waldman, Advisor John F. Sheridan Copyright by Scott Franklin Wray 2012 Abstract A major problem in fighting influenza virus infections with the current trivalent vaccines and antivirals is the inherent genetic instability of the RNA-encoded proteins of the virus. Current vaccines induce neutralizing antibodies specific for two major surface antigens expressed on the virion. These two proteins are encoded by the hemagglutinin and neuraminidase genes respectively, and in these genes, there is a high rate of point mutation resulting in antigenic drift of the expressed proteins. A similar problem arises with antivirals where resistant strains develop or are selected for when antivirals are used in a widespread fashion. In other words, the virions that are sensitive to the antiviral are killed, whereas those that are resistant to the antiviral are free to infect susceptible individuals. Therefore, it would be beneficial to develop anti-influenza strategies that do not rely on treatments directed against the variable genes encoded by the virus. Based on previous work from our laboratory, we hypothesize that androstenediol inhibits influenza replication by modifying the host cell’s interaction with the virus. Our proposal is that treatment with androstenediol (AED), may lead to an approach that is not directed at the variable genes encoded by the viruses. Our results suggest that AED treatment may inhibit viral replication, as indicated by the influenza viral matrix protein, M1, gene expression. Additionally, our results exclude certain cellular mechanisms and suggest other possible mechanisms that will need to be examined in more detail. ii Dedication This document is dedicated to my family and friends. iii Acknowledgments I would like to acknowledge all my advisors, fellow laboratory members, and other faculty members for their guidance, encouragement, and support during the course of my training. iv Vita May 1999 .......................................................Liberty Union-Thurston High 2003................................................................B.A. Biology, Capital University 2003 to 2004 ..................................................Graduate Research Associate, Department of Integrated Biomedical Science, The Ohio State University 2004 to 2009 ..................................................Graduate Research Fellow, Department of Oral Biology, The Ohio State University 2009 to 2011 ..................................................Graduate Research Associate, Department of Integrated Biomedical Science, The Ohio State University 2011 to present ..............................................Master’s Student, Department of Pathology, The Ohio State University Publication(s) 1. Integrin engagement increases histone H3 acetylation and reduces histone H1 association with DNA in murine lung endothelial cells. Molecular Pharmacology. 68(2): 439-46, 2005. v Fields of Study Major Field: Pathology vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v List of Tables .................................................................................................................... iix List of Figures ..................................................................................................................... x List of Abbreviations ........................................................................................................ xii Chapter 1: Introduction ...................................................................................................... 1 Chapter 2: Androstenediol Suppresses Influenza Virus-Encoded M1 Gene Expression in Human Respiratory Epithelial Cells ................................................................................. 19 Introduction ................................................................................................................... 19 Methods ......................................................................................................................... 23 Results ........................................................................................................................... 28 Discussion ..................................................................................................................... 31 Chapter 3: Androstenediol’s Effect on Cellular Signaling and a Coalescing of Pattern Recognition Receptors ...................................................................................................... 42 vii Introduction ................................................................................................................... 42 Methods ......................................................................................................................... 44 Results ........................................................................................................................... 48 Discussion ..................................................................................................................... 51 Chapter 4: Determining Whether Androstenediol Modifies Influenza Virus-Encoded M1 Gene Expression Through the Androgen Receptor .......................................................... 57 Introduction ................................................................................................................... 57 Methods ......................................................................................................................... 59 Results ........................................................................................................................... 66 Discussion ..................................................................................................................... 69 Chapter 5: General Discussion......................................................................................... 80 References ......................................................................................................................... 91 viii List of Tables Table 2.1. Primer and TaqMan Probe Sequences of cytokines M1 and 18S for Real Time PCR ................................................................................................................................... 35 Table 3.1. PCR array analysis of AED activated genes in A549 cell culture with exposure to influenza virus Primer and TaqMan Probe Sequences of cytokines M1 and 18S for Real Time PCR .................................................................................................... 56 ix List of Figures Figure 2.1 Dose Curve of AED and its Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells .............................................................................................. 36 Figure 2.2 Time Course of AED and its Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells .......................................................................................... 37 Figure 2.3 Data of Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells ........................................................................................................................................... 38 Figure 2.4 Multiple Steroid Agonists and Antagonists Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells .............................................................. 39 Figure 2.5 The above images were used to determine whether AED had an effect on influenza viral entry .......................................................................................................... 40 Figure 2.6 The above images were used to determine whether AED had an effect on influenza viral entry .......................................................................................................... 41 Figure 3.1 Data of Interferon Beta Gene Expression in A549 Lung Epithelial Cells ..... 53 Figure 3.2 Focused microarray analysis of AED activated genes in A549 cell culture without exposure to influenza virus .................................................................................. 54 Figure 3.3 Data of Interferon Beta Gene Expression in A549 Lung Epithelial Cells With Exposure to Influenza Virus ............................................................................................. 55 Figure 4.1 Data of Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells Treated With a Dose Curve of Methyltrienolone (R1881) ............................................... 74 x Figure 4.2 Time Course of AED and its Effect on Influenza Virus M1 Gene Expression in A549 Lung Epithelial Cells .......................................................................................... 75 Figure