FUNCTIONAL GENOMIC AND PROTEOMIC PROFILES OF MACROPHAGE RESERVOIRS FOR HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 By JOSEPH NICHOLS BROWN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1 © 2007 Joseph Nichols Brown 2 To my fiancée, Roslyn Frank, my mother, Darnell Brown, and my father, George Brown. 3 ACKNOWLEDGMENTS I have had an outstanding graduate experience at the University of Florida College of Medicine, and for this I must thank my excellent mentor, Dr. Maureen M. Goodenow. Through her guidance, I learned much, especially to enjoy learning and science. Great thanks should also be given to all of my committee members Dr. Henry Baker, Dr. Ayalew Mergia, Dr. William Farmerie, Dr. David Ostrov, Dr. Gregory Schultz, and Dr. Peggy Wallace, who have always been available and supportive. Dr. James Kohler, a former post-doc in the lab, has been instrumental in much of the work presented here, and has been an outstanding mentor, providing training, encouragement, and friendship during my graduate career. Dr. Li Yin, an assistant professor in the lab, has provided invaluable support and guidance. I would also like to acknowledge Cecilia Lopez who helped me with the microarrays and functional genomics. I greatly appreciate Bhavna Bhardwaj and Neal Benson at the UF Flow Cytometry Core for all their help and patience. Dr. Jeremiah Tipton at Scripps Florida deserves many thanks for his work performing the iTRAQ experiments and the many hours he provided teaching about mass spectrometry. I would also like to thank Brian Krastins and Dr. David Sarracino at Harvard University Proteomics Core for their helpfulness and work performing the mass spectrometry on the 1D gel slices. Lauren McIntyre deserves great thanks for all her work performing the statistical analyses on the gp120 microarrays. Finally, I would like to thank UF Shand’s Cancer Center for providing a generous amount of funding that made much of this research possible. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................9 LIST OF FIGURES .......................................................................................................................11 CHAPTER 1 INTRODUCTION ..................................................................................................................15 1.1 Rationale ......................................................................................................................15 1.2 Specific Aims...............................................................................................................16 1.2.1 Analyze the Global Genetic and Proteomic Networks Perturbed by HIV-1 Infection in Macrophages ............................................................................................16 1.2.2 Determine Functional Impact of Viral Envelope-mediated Coreceptor Usage on Macrophages................................................................................................17 1.3 Background..................................................................................................................17 1.3.1 Physical Characteristics of the Human Immunodeficiency Virus Type-1 (HIV-1) Virion.............................................................................................................17 1.3.2 Differences in the HIV-1 Lifecycle between T-lymphocytes and Macrophages................................................................................................................19 1.3.2.1 HIV-1 entry ...............................................................................................19 1.3.2.2 Reverse transcription and nuclear translocation........................................20 1.3.2.3 Proviral integration and expression...........................................................22 1.3.2.4 Viral assembly and release........................................................................22 1.3.3 Phenotypic Characterization of HIV-1 Isolates .................................................23 1.3.4 Cellular Signal Transduction Cascades Activated by HIV................................24 1.3.5 Influence of Cellular Factors on HIV infection .................................................26 1.3.6 Acquired Immunodeficiency Syndrome and Related Malignancies .................27 1.3.7 Functional Genomics and HIV ..........................................................................28 1.3.8 Proteomics and HIV...........................................................................................30 2 IMPACT ON GENETIC NETWORKS IN HUMAN MACROPHAGES BY HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 ............................................................................37 2.1 Introduction..................................................................................................................37 2.2 Experimental Design....................................................................................................38 2.3 Results..........................................................................................................................38 2.3.1 Viral Replication and Spread in Monocyte-derived Macrophage (MDM) Cultures........................................................................................................................38 2.3.2 Temporal Expression of Genes in HIV-treated Macrophage Cultures..............39 2.3.3 Genes Expressed Exclusively in Virus-treated or Mock-treated Macrophage Cultures...................................................................................................40 2.3.4 Functional Categories of Genes Modulated by HIV..........................................40 5 2.3.5 HIV Impacts Similar Genes in Lymphocytes and Macrophages.......................41 2.3.6 HIV-1 Impact on Genes Involved in Regulation of the Cell Cycle in Macrophage Cultures...................................................................................................42 2.3.7 Qualitative Identification of Genes Involved in Cell Cycle Regulation Checkpoints..................................................................................................................42 2.3.8 Altered Protein Expression in Macrophages Treated with Virus. .....................43 2.4 Discussion....................................................................................................................44 3 NETWORK ANALYSIS REVEALS HIV-1 ACTIVATES MACROPHAGES INDEPENDENT OF TOLL-LIKE RECEPTORS .................................................................59 3.1 Introduction..................................................................................................................59 3.2 Experimental Design....................................................................................................60 3.3 Results..........................................................................................................................61 3.3.1 Temporal Expression of Genes in HIV-treated Macrophages...........................61 3.3.2 HIV Activates Genes Involved in Calcium Signaling. ......................................62 3.3.3 Temporal Activation of Apoptotic Signaling Pathways by HIV in Macrophages................................................................................................................63 3.3.4 HIV Activates the Mitogen-activated Protein Kinase (MAPK) Pathway. ........63 3.3.5 HIV Induces Expression and Secretion of Type-2 Cytokines/Chemokines in Macrophages............................................................................................................64 3.3.6 Activation of Macrophages is Independent of Cell Proliferation. .....................65 3.3.7 HIV Activates Macrophages Independent of Toll-like Receptors (TLR). ........65 3.4 Discussion....................................................................................................................67 4 COMPARATIVE METHODS OF PROTEOMIC PROFILING of PRIMARY MACROPHAGES: IDENTIFICATION OF THE MACROPHAGE ANTIVIRAL FACTOR.................................................................................................................................83 4.1 Introduction..................................................................................................................83 4.2 Experimental Design....................................................................................................84 4.3 Results..........................................................................................................................85 4.3.1 Lipopolysaccharide Stimulation of Macrophages Inhibits HIV Replication.....85 4.3.2 Lipopolysaccharide-primed Macrophages Secrete Products Important in Controlling Virus. ........................................................................................................85 4.3.3 Tandem Mass Spectrometry Identification of a Unique High Molecular Weight Band in Lipopolysaccharide-primed Macrophage Supernatants. ...................87 4.3.4 Quantitative Intracellular Proteome Analysis of Macrophages via Isotope- coded Labeling of Peptides..........................................................................................88 4.3.5 Quantitative Intracellular Proteome Analysis