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THE ROLE of the HERPES SIMPLEX VIRUS TYPE 1 UL28 PROTEIN in TERMINASE COMPLEX ASSEMBLY and FUNCTION by Jason Don Heming Bachelor

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THE ROLE of the HERPES SIMPLEX VIRUS TYPE 1 UL28 PROTEIN in TERMINASE COMPLEX ASSEMBLY and FUNCTION by Jason Don Heming Bachelor THE ROLE OF THE HERPES SIMPLEX VIRUS TYPE 1 UL28 PROTEIN IN TERMINASE COMPLEX ASSEMBLY AND FUNCTION by Jason Don Heming Bachelor of Science, Clarion University of Pennsylvania, 2004 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 2013 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Jason Don Heming It was defended on April 18, 2013 and approved by Michael Cascio, Associate Professor, Bayer School of Natural and Environmental Sciences James Conway, Associate Professor, Department of Structural Biology Neal DeLuca, Professor, Department of Microbiology and Molecular Genetics Saleem Khan, Professor, Department of Microbiology and Molecular Genetics Dissertation Advisor: Fred Homa, Associate Professor, Department of Microbiology and Molecular Genetics ii Copyright © by Jason Don Heming 2013 iii THE ROLE OF THE HERPES SIMPLEX VIRUS TYPE 1 UL28 PROTEIN IN TERMINASE COMPLEX ASSEMBLY AND FUNCTION Jason Don Heming, PhD University of Pittsburgh, 2013 Herpes simplex virus type I (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. During productive lytic infection, over 80 viral proteins are expressed in a highly regulated manner, resulting in the replication of viral genomes and assembly of progeny virions. Cleavage and packaging of replicated, concatemeric viral DNA into newly assembled capsids is critical to virus proliferation and requires seven viral genes: UL6, UL15, UL17, UL25, UL28, UL32, and UL33. Analogy with the well-characterized cleavage and packaging systems of double-stranded DNA bacteriophage suggests that HSV-1 encodes for a viral terminase complex to perform these essential functions, and several studies have indicated that this complex consists of the viral UL15, UL28, and UL33 proteins. However, the inability to purify the terminase proteins has hampered biochemical analysis of these proteins. The goal of the following studies was to isolate a functional terminase complex from HSV-1-infected cells by affinity chromatography using a virus expressing a UL28-TAP fusion protein. The tandem affinity purification (TAP) procedure resulted in the isolation of soluble UL28 complexes containing the UL15 and UL33 proteins. Biochemical studies were performed to determine the protein composition and stoichiometry of the purified complex, and the associated nuclease activity was examined. Mass spectrometry was utilized to identify viral and cellular proteins that associate with the complex during infection. Finally, mutations or deletions within the nuclease domain of UL15 or the iv metal-binding domain of UL28 were introduced into the genome of the NTAP-UL28 fusion virus. Characterization of these viruses followed by the isolation of terminase complexes revealed that the domain mutations did not preclude complex formation but each virus was deficient in viral DNA cleavage, further demonstrating the importance of these domains during DNA encapsidation. The ability to purify the endogenous terminase complex is novel to the field and we view these studies as a critical step in understanding how the terminase complex functions in the context of productive HSV-1 infection. v TABLE OF CONTENTS PREFACE .................................................................................................................................... XI 1.0 INTRODUCTION ........................................................................................................ 1 1.1 HSV-1 LINEAGE AND PATHOGENESIS ...................................................... 1 1.2 THE HSV-1 VIRION .......................................................................................... 5 1.3 MOLECULAR BIOLOGY OF HSV-1 LYTIC INFECTION ........................ 9 1.4 HSV-1 CAPSID ASSEMBLY AND DNA ENCAPSIDATION ..................... 17 1.5 THE HSV-1 TERMINASE COMPLEX .......................................................... 30 1.6 SPECIFIC AIMS AND RATIONALE ............................................................ 40 2.0 AFFINITY PURIFICATION OF THE HERPES SIMPLEX VIRUS TYPE I TERMINASE COMPLEX ......................................................................................................... 42 2.1 ABSTRACT........................................................................................................ 42 2.2 INTRODUCTION ............................................................................................. 43 2.3 MATERIALS AND METHODS ...................................................................... 46 2.4 RESULTS ........................................................................................................... 54 2.5 DISCUSSION ..................................................................................................... 72 3.0 MUTATIONAL ANALYSIS OF ESSENTIAL RESIDUES WITHIN THE HERPES SIMPLEX VIRUS TYPE I UL15 AND UL28 TERMINASE SUBUNITS ........... 80 3.1 ABSTRACT........................................................................................................ 80 vi 3.2 INTRODUCTION ............................................................................................. 81 3.3 MATERIALS AND METHODS ...................................................................... 85 3.4 RESULTS ........................................................................................................... 92 3.5 DISCUSSION ................................................................................................... 102 4.0 SUMMARY AND CONCLUSIONS ...................................................................... 108 APPENDIX A ............................................................................................................................ 123 BIBLIOGRAPHY ..................................................................................................................... 130 vii LIST OF TABLES Table 1. The human Herpesviridae ................................................................................................ 2 Table 2. Essential viral replication proteins .................................................................................. 13 Table 3. Protein components of the four capsid types. ................................................................. 19 Table 4. Role of the four capsid types during infection. ............................................................... 20 Table 5. PCR primers for generation of recombinant viruses ...................................................... 47 Table 6. Viral stock titers: NTAP-UL28 fusion mutants .............................................................. 56 Table 7. Mass spectrometry confirmation and analysis of specific interacting proteins .............. 65 Table 8. HSV-1 interacting proteins ............................................................................................. 66 Table 9. Interacting cellular proteins ............................................................................................ 68 Table 10. PCR primers for generation of recombinant domain mutant viruses ........................... 87 Table 11. Viral stock titers: UL15 and UL28 domain mutants..................................................... 94 Table 12. Interacting HSV-1 protein (complete) ........................................................................ 123 Table 13. Interacting cellular proteins (complete) ...................................................................... 124 viii LIST OF FIGURES Figure 1. Model of recurrent HSV-1 infection. .............................................................................. 3 Figure 2. Structure of the HSV-1 virion. ........................................................................................ 5 Figure 3. The capsid structural proteins. ......................................................................................... 7 Figure 4. Structure and sequence arrangement of the HSV-1 genome. .......................................... 8 Figure 5. Isolation and morphology of the four capsid types. ...................................................... 19 Figure 6. Proteolytic processing of the scaffolding proteins by the maturational protease. ......... 21 Figure 7. Capsid assembly and DNA packaging. ......................................................................... 24 Figure 8. Structure and essential cis-acting elements within the viral a sequences. .................... 26 Figure 9. Conserved amino acid domains within the UL15 protein. ............................................ 36 Figure 10. Conserved amino acid domains within the UL28 protein. .......................................... 37 Figure 11. UL33 protein domain mutations affecting terminase activity. .................................... 38 Figure 12. Recombinant UL28 virus constructs. .......................................................................... 55 Figure 13. Recombinant NTAP-UL28 virus single-step growth curves....................................... 57 Figure 14. Recombinant NTAP-UL28 fusion protein expression. ............................................... 58 Figure 15. Recombinant NTAP-UL28 fusion protein expression time course. ............................ 59 Figure 16. TAP of NTAP-UL28 fusion proteins. ......................................................................... 61 Figure 17. Sedimentation velocity analysis of TAP-purified UL28
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