Western Michigan University ScholarWorks at WMU Master's Theses Graduate College 4-1998 Characterization of Swinepox Virus Secretory Polypeptides Takeshi Shimamura Follow this and additional works at: https://scholarworks.wmich.edu/masters_theses Part of the Biology Commons Recommended Citation Shimamura, Takeshi, "Characterization of Swinepox Virus Secretory Polypeptides" (1998). Master's Theses. 4576. https://scholarworks.wmich.edu/masters_theses/4576 This Masters Thesis-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Master's Theses by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. CHARACTERIZATION OF SWINEPOX VIRUS SECRETORY POLYPEPTIDES by Takeshi Shimamura A Thesis Submitted to the Faculty of The Graduate College in partial fulfillment of the requirements for the Degree of Master of Science Department of Biological Sciences Western Michigan University Kalamazoo, Michigan April 1998 Copyright by Takeshi Shimamura 1998 ACKNOWLEDGEMENTS I wish to express my high regard and appreciation to my advisor and mentor, Dr. Karim Essani for his guidance and help throughout this study. I am thankful to Dr. Robert C. Eisenberg, and Dr. Ronald C. Shebuski for taking the time to be a part of my thesis committee. Cardiovascular studies were carried out in Dr. Ronald Shebuski's laboratory. I also thank Dr. Anthony Manning forhis generosity to allow me to work in his laboratory on the cell adhesion molecules. Thanks also to Mr. William R. Humphrey for his help in the cardiovascular study, Ms. Jennifer L. Northrup, Ms. Cynthia J. Mills, and Ms. Carol A. Simmons for assistance with the morphological and statistical studies on the cardiovascular project. I also wish to thank my parents for their continued financial and moral support throughout my graduate studies. I also appreciate the warm support from my other graduate student colleagues, Bina Garimella, Scott Haller, Mini Paulose, Celene Spangler and Debora Williams throughout my graduate studies. Finally, I would like to acknowledge everyone I have met throughout my visit in the United States of America who have all been gracious hosts during this period. Finally, I dedicate this thesis to my mother who, while courageously fighting breast cancer, has continued her strong support of my education. Takeshi Shimamura 11 CHARACTERIZATION OF SWINEPOX VIRUS SECRETORY POLYPEPTIDES Takeshi Shimamura, M.S. WesternMichigan University, 1998 Swinepox virus (SPV) is the only known member of the genus Suispoxvirus. SPV causes a mild disease in swine characterized by slight fever, and weight loss, associated with lesions restricted to the skin. There have been few studies on SPV. Other poxviruses, such as vaccinia virus, have been well characterized and the biological activities of their secretory polypeptides have been analyzed. This study describes a major SPV secretory polypeptide with an apparent molecular mass of 35kDa. Poxvirus encoded polypeptides secreted during infection are known to exhibit immunomodulatory activities. The 35kDa SPV secretory polypeptide was tested.for its ability to inhibit hIFN-y, hTNF-a, and hIL-1� activities. Results from these experiments were inconclusive for SPV polypeptide immunomodulatory activity on these human cytokines. Unavailability of swine cytokines restricted additional studies. Since myxoma virus secretory polypeptide, which is a serine proteinase inhibitor, is shown to reduce restenosis caused after percutaneous transluminal coronary angioplasty, SPV polypeptide was tested for similar activities. SPV secretory polypeptide did not reduce restenosis and lack of evident immunomodulatory functioncould explain only mild porcine pathogenesis of SPV. TABLE OF CONTENTS ACKNOWLEDGMENTS...................................................................................... n LIST OF TABLES................................................................................................... Vil LIST OF FIGURES ................................................................................................ Vlll INTRODUCTION.................................................................................................. 1 REVIEW OF LITERATURE................................................................................. 3 General Characteristics of Swinepox Virus................................................... 3 Historical Background . .. .. .. .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 3 Epidemiology................................................................................................. 4 Clinical Signs, Gross Lesions, and Pathogenesis . .. ..... ..... ... .. ..... .. ... .. .. .. .. .. .. 5 Microscopic Lesions...................................................................................... 5 Kinetics of SPV Infection.............................................................................. 6 Viral Genome................................................................................................. 7 Virus Entry..................................................................................................... 9 Viral Polypeptides ......................................................................................... 10 Virus Host Interactions.................................................................................. 11 Inhibition of Host Macromolecular Synthesis...................................... 11 Stimulatory Effectson Cell Growth .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 12 Viral Defense Molecules...................................................................... 13 Viral Protein - SERPIN Superfamily ............................................................ 13 111 Table of Contents - Continued IL-1 and INF Receptor Homologues. ... .. ..... ..... .. ..... ..... ..... ....... ... ..... ....... ..... 14 Inhibitors of Interferon .. ....... .. .. ... .. ..... .. ... ..... .. .. ... .. ..... .. ... ... .. .. ..... ... .. .......... ... 15 Complement Regulatory Protein . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 15 Cell Adhesion Molecules............................... .-............................................... 16 Re stenosis and Percutaneous Transluminal Coronary Angioplasty . ............. 18 Clinical Significanceof Viral Polypeptides in Restenosis .. .. ... ..... ..... ... .. ... .. 19 Antiviral Therapy in the Treatment of Restenosis After Angioplasty........... 20 MATERIALS AND METHODS ........................................................................... 22 Virus and Cells .. .... .. ... .. .. ... .. ... .. .. .. .. .. .. ... .. ..... .. .. .. ... .. .. .. ... .. .. ... .. .. .. ..... ... .. ... 22 Titration of SPV. .. .. .. .. .. ..... .. .. .. ... .. .. ... .. .. ... ..... .. ... .. ..... ... .. ... .. .. .. ... .. ... .. .. .. .. .. .. 23 Identification of SPV Secretory Proteins....................................................... 24 Metabolic Labeling Studies of SPV Infected Cells.. .. ... .. .. ... .. ... ... .. ... .. 24 Early and Late Protein Experiment....................................................... 25 Pulse - Chase Experiment . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 26 Characterization of SPV Secretory Polypeptides.......................................... 26 Interferon-y Assay................................................................................. 26 Human Recombinant Tumor Necrosis Factor-a and Human Recombinant Interleukin-Ip Assay ... .. ... .. ..... .. ... ... .... ... ........ .. ... .. .. .. 27 Cell Culture........................................................................................... 27 Treatment of HUVEC With Recombinant Human INF-a and SPV Protein Samples or Human Recombinant IL-1p and SPV Protein Samples..................................................................... 28 IV Table of Contents - Continued Atherosclerosis Study ..... ....... .... ... ..... ....... .. ... .. ... .. ....... ..... ..... ....... ..... ........ .. .. 29 Pigs and Diet......................................................................................... 29 SPV Production and Infection Procedures .. .. .............. ... ..... ....... ... ....... 29 Percutaneous Transluminal Coronary Angioplasty (PTCA) ................ 30 Histopathology and Morphometric Analysis of Porcine LAD Coronary Artery.................................................................................... 31 Indirect Immunofluorescence............................................................... 32 ELISA................................................................................................... 34 Production of Monoclonal Antibody (mAb) ................................................. 35 Production ofHybridomas.................................................................... 35 Indirect Immunofluorescence............................................................... 36 RESULTS............................................................................................................... 37 Viral Polypeptides ......................................................................................... 37 SPV Replication in Tissue Culture....................................................... 37 Synthesis of Swinepox Virus Protein................................................... 38 Post-Translational