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The Role of the Herpes Simplex Virus Type 1 Ul25 Protein in Dna Packaging and Virion Assembly

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The Role of the Herpes Simplex Virus Type 1 Ul25 Protein in Dna Packaging and Virion Assembly THE ROLE OF THE HERPES SIMPLEX VIRUS TYPE 1 UL25 PROTEIN IN DNA PACKAGING AND VIRION ASSEMBLY by Shelley Kristen Cockrell Bachelor of Science, Duke University, 2003 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2010 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Shelley Kristen Cockrell It was defended on August 23, 2010 and approved by James Conway, Associate Professor, Department of Structural Biology Roger Hendrix, Professor, Department of Biological Sciences Neal DeLuca, Professor, Department of Microbiology and Molecular Genetics Thomas Smithgall, Professor and Chair, Department of Microbiology and Molecular Genetics Dissertation Advisor: Fred Homa, Associate Professor, Department of Microbiology and Molecular Genetics ii Copyright © by Shelley Kristen Cockrell 2010 iii THE ROLE OF THE HERPES SIMPLEX VIRUS TYPE 1 UL25 PROTEIN IN DNA PACKAGING AND VIRION ASSEMBLY Shelley Kristen Cockrell, PhD University of Pittsburgh, 2010 Herpes simplex virus type 1 replicates its DNA and builds progeny nucleocapsids in the nucleus of the infected cell. Replicated viral DNA is a concatemer that is cleaved to unit-length genomes and packaged into capsid precursors, and successful DNA packaging is required for the mature capsid to exit the nucleus and become incorporated into virions. The DNA cleavage and packaging machinery is highly conserved across herpesvirus subfamilies; they represent novel targets for anti-herpesviral treatments. Seven genes have been identified that are essential for the packaging reaction. Of these, the UL25 gene product is unique because it is required for the completion rather than the initiation of DNA packaging. In the absence of functional UL25, the nuclei of infected cells accumulate empty capsids and free, close to genome-length viral DNA. This phenotype differs from null mutations of the other packaging genes, which block cleavage of the concatemer and expulsion of the capsid scaffold protein. While the functions of several members of the packaging machinery have been deduced by analogy with the dsDNA bacteriophages, the role of UL25 protein (pUL25) in this process is unclear. pUL25 is stably associated with the capsid vertices, where it forms a heterodimer with the packaging protein UL17. The goal of this project is to elucidate the pUL25 capsid-binding mechanism and its significance for DNA packaging and virion assembly. Mutational analysis of UL25 mapped the capsid-binding domain to the pUL25 N-terminus. pUL25 interactions with the capsid surface proteins were visualized with cryo-electron microscopy and 3D image reconstructions of capsids iv containing pUL25 fusion proteins. Finally, we demonstrated that UL25 mutants aberrantly cleave viral DNA before the second packaging signal. These data support a model in which pUL25 provides structural support to capsid vertices without interacting directly with DNA during the packaging reaction. v TABLE OF CONTENTS TABLE OF CONTENTS ...........................................................................................................VI LIST OF TABLES ...................................................................................................................VIII LIST OF FIGURES ....................................................................................................................IX PREFACE....................................................................................................................................XI 1.0 INTRODUCTION ............................................................................................................... 1 1.1 HERPESVIRUSES AND DISEASE.......................................................................... 1 1.2 HSV-1 PATHOGENESIS........................................................................................... 4 1.3 THE HSV-1 PARTICLE ............................................................................................ 6 1.4 OVERVIEW OF HSV-1 LYTIC INFECTION........................................................ 9 1.5 FORMATION AND MATURATION OF THE CAPSID..................................... 13 1.6 THE HSV-1 DNA PACKAGING REACTION...................................................... 17 1.7 COMPLETION OF PACKAGING AND BEYOND: ADDITION OF THE CAPSID-STABILIZING PROTEIN PUL25.................................................................... 25 1.8 SPECIFIC AIMS AND RATIONALE .................................................................... 33 2.0 UL25 PROTEIN ATTACHES TO CAPSIDS THROUGH A SMALL N-TERMINAL DOMAIN ..................................................................................................................................... 36 2.1 ABSTRACT ............................................................................................................... 36 2.2 INTRODUCTION ..................................................................................................... 37 vi 2.3 MATERIALS AND METHODS.............................................................................. 40 2.4 RESULTS................................................................................................................... 49 2.5 DISCUSSION............................................................................................................. 73 3.0 STRUCTURAL ANALYSES OF CAPSIDS CONTAINING PUL25 FUSION PROTEINS DEMONSTRATE THAT THE PUL25 N-TERMINUS CONTACTS HEXONS AND TRIPLEXES ADJACENT TO THE PENTON ............................................................. 79 3.1 ABSTRACT ............................................................................................................... 79 3.2 INTRODUCTION ..................................................................................................... 80 3.3 MATERIALS AND METHODS.............................................................................. 84 3.4 RESULTS................................................................................................................... 88 3.5 DISCUSSION........................................................................................................... 101 4.0 CAPSID-ASSOCIATED UL25 PROTEIN IS REQUIRED FOR SECONDARY CLEAVAGE OF THE VIRAL GENOME............................................................................. 105 4.1 ABSTRACT ............................................................................................................. 105 4.2 INTRODUCTION ................................................................................................... 106 4.3 MATERIALS AND METHODS............................................................................ 110 4.4 RESULTS................................................................................................................. 114 4.5 DISCUSSION........................................................................................................... 124 5.0 SUMMARY AND CONCLUSIONS .............................................................................. 129 APPENDIX A: PRIMERS USED FOR CLONING PLASMIDS AND BACS................... 138 APPENDIX B: COPYRIGHTS AND PERMISSIONS......................................................... 141 BIBLIOGRAPHY..................................................................................................................... 142 vii LIST OF TABLES TABLE 1. The order Herpesvirales................................................................................................ 2 TABLE 2. Human Herpesviruses ................................................................................................... 3 TABLE 3. UL25 Homologues...................................................................................................... 26 TABLE 4. Mutational analysis of pUL25 structural features....................................................... 31 TABLE 5. Summary of predicted pUL25 interactions with viral and cellular proteins............... 33 TABLE 6. Virus stock titers: Transposon insertion and N-terminal truncation mutants ............. 58 TABLE 7. Virus stock titers: Small N-terminal deletion and point mutants................................ 66 TABLE 8. Virus stock titers: UL25-fusion mutants..................................................................... 91 TABLE 9. Primers used for plasmid cloning.............................................................................. 138 TABLE 10. Primers used for generation of recombinant HSV-1-BACs with UL25 insertions and point mutations............................................................................................................................ 139 TABLE 11. Primers used for generation of recombinant HSV-1-BACs with UL25 deletions.. 140 viii LIST OF FIGURES Figure 1. The HSV-1 virion............................................................................................................ 7 Figure 2. The HSV-1 genome and a sequence ............................................................................... 8 Figure 3. Precursor capsids mature into three different capsid forms .......................................... 16 Figure 4. DNA packaging in the double-strand DNA bacteriophages ......................................... 18 Figure 5. The a sequence mediates HSV-1 DNA cleavage and packaging.................................. 22 Figure 6. Features of the terminase subunit proteins UL15, UL28, and UL33 ...........................
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