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Open Purdy Dissertation.Pdf The Pennsylvania State University The Graduate School College of Medicine TO ASSEMBLE, OR NOT TO ASSEMBLE: THE INITIATION OF RETROVIRAL CAPSID ASSEMBLY A Dissertation in Microbiology and Immunology by John Gerard Purdy Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2009 The dissertation of John Gerard Purdy was reviewed and approved* by the following: Rebecca C. Craven Associate Professor of Microbiology and Immunology Dissertation Adviser Chair of Committee Sarah K. Bronson Associate Professor of Cellular and Molecular Physiology Ira J. Ropson Associate Professor of Biochemistry and Molecular Biology David J. Spector Professor and Distinguished Educator of Microbiology and Immunology John W. Wills Distinguished Professor of Microbiology and Immunology Richard J. Courtney Professor, Chair and Distinguished Educator of Microbiology and Immunology *Signatures are on file in the Graduate School ii ABSTRACT During the complex multistep process of retroviral maturation the immature Gag lattice is cleaved apart prior to the formation of the mature capsid shell. Proper maturation and capsid assembly are required for infectivity. The capsid protein (CA) contains two domains [N-terminal domain (NTD) and C-terminal domain (CTD)] and self-assembles to enclose the genomic RNA and other viral proteins. Retroviral capsids are highly polymorphic, exhibiting a wide-range of structures, and were previously modeled as a lattice consisting of 12 CA pentamers unequally distributed among 150 - 300 hexameric rings. Three CA-CA interfaces form between assembled subunits in the mature capsid: NTD-NTD, NTD-CTD, and CTD-CTD. Very little is known about the mechanism and pathway of assembly that form the mature capsid despite being attractive targets for antiviral therapy. A full understanding of events occurring during maturation, including capsid assembly will require further advances such as: 1. establishing how the cleavage products of Gag reorganize to stimulate capsid assembly during maturation, 2. defining how the CA subunits self- assemble, 3. characterizing the CA pentamer and its interaction with the previously characterized hexamer, and 4. visualizing the capsid lattice at an atomic resolution to guide inhibitor development. Understanding capsid assembly and structure will help to elucidate how the capsid interacts with other viral components to provide necessary functions during the early stages of viral replication. Ancillary information suggests that the assembly of the capsid is nucleated at a specific location in the maturing virion following Gag cleavage. The presumptive nucleation event is explored in greater detail in this thesis using protein from Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) and a novel in vitro system that, at least for RSV, recapitulates many features of capsid assembly occurring in the virus. Results clearly demonstrate that RSV CA assembles via a nucleation-driven mechanism in vitro. Assembly was demonstrated to be electrostatically controlled and multivalent anionic salts were iii demonstrated to induce CA to form structures resembling authentic mature capsids. A complex as small as a CA dimer acted as a seed for assembly was demonstrated by isolating intermediates of the capsid assembly pathway. Biochemical evidence combined with genetic analyses supports the proposal that a similar nucleation step happens in the maturing virion. The conserved major homology region (MHR) in the CTD was shown to be required for proper initiation of capsid assembly. Lethal MHR mutations were suppressed by secondary substitutions located in the NTD and CTD that increased the propensity of CA to assemble in vitro. The results are consistent with CA undergoing structural transformations during maturation. Proteolytic cleavage of Gag results in the accumulation of a transient CA- SP (spacer peptide, C-terminal of CA) intermediate. CA-SP enhanced the kinetics of in vitro assembly. The data suggest that CA-SP participates in nucleation of capsid assembly and the MHR participates in an early step of assembly. Identifying icosahedral particles formed by multimerization of RSV CA protein permitted the development of a pseudo-atomic model for the retroviral CA pentamer. The data are the first description of all three mature CA-CA interactions (NTD-NTD, NTD-CTD, CTD-CTD) forming in pentamers and hexamers. Although further research is needed to understand the polymorphic nature of retroviral capsids, the results provide a major step forward in understanding the architecture of mature capsids at the atomic level. The data further support a model whereby initiation of capsid assembly involves a tripartite interaction between two NTDs and one CTD. The model invokes a critical participatory role not previously described for the interdomain NTD-CTD interaction in forming a functional retroviral capsid. iv TABLE OF CONTENTS LIST OF FIGURES .................................................................................................. ix LIST OF TABLES .................................................................................................. xii LIST OF ABBREVIATIONS .................................................................................xiii ACKNOWLEDGMENTS ...................................................................................... xvi CHAPTER I: LITERATURE REVIEW..……….…………………………………………………...1 A. Introduction to and Aims of the Thesis .................................................................. 2 B. Defining a Viral Capsid and its Functions ............................................................. 5 Definitions and Nomenclature used in this Thesis .................................................. 5 General Functions of Viral Capsids ........................................................................ 6 Genome Packaging and Protection .................................................................... 6 Moving the Genome: Capsid mediated Receptor-Binding and Intracellular Movement .......................................................................................................... 9 Release of the Packaged Genome: Metastability and Disassembly ................... 10 Capsid Participation in Genome Replication ................................................... 11 C. Principles of Viral Capsid Morphology ............................................................... 12 Icosahedral Capsids ............................................................................................. 12 Helical Capsids .................................................................................................... 17 Irregular / Complex Capsids ................................................................................ 18 Notes on classifying capsids into a morphological group ..................................... 19 D. More than just an assembly line: Understanding Capsid Assembly...................... 20 Capsid Protein Self Assembly .............................................................................. 20 Assembly Intermediates ....................................................................................... 21 Assisted Assembly: Scaffolding Proteins ............................................................. 22 A Case Study: Bromoviral Capsid Function, Structure, and Assembly ................. 23 E. Summary of the Three Types of Capsid Morphologies: Functions and Assembly ............................................................................................................. 26 F. Introduction to Retroviruses and their Replication ............................................... 27 Classification and Genetic Organization of Retroviruses ...................................... 27 Early events of Replication: Viral Entry to DNA Integration................................ 29 Late events of Replication: Virion Production ...................................................... 37 G. Functional and Structural roles of CA during Retroviral Replication ................... 39 Genetic Analysis of CA reveals a role of the Capsid during Early Events of Replication .......................................................................................................... 42 Role of CA in Disassembly and in Recognition by Host Restriction Factors ........ 43 Structural Role of CA in Immature Particles ........................................................ 45 CA-CA interactions in Immature Particles ....................................................... 46 NTD Models for Immature Hexameric Interface ........................................... 46 MHR (major homology region) Domain-Swap for Gag Dimerization ........... 49 Spacer Peptide Oligomerization in Immature Particles ................................ 50 Fullerene Model for the Morphology of Mature Capsids .................................. 56 CA-CA interactions in the Mature Retroviral Capsids ...................................... 60 NTD-NTD Hexamers of Capsids .................................................................. 60 CTD-CTD Dimeric Interface ........................................................................ 63 NTD-CTD Interdomain Interface ................................................................. 63 v Pentamers (Has anyone seen a pentamer?) ...................................................... 67 Maturation Switches and the “Hallmarks” of Mature Capsids ......................... 67 H. Modeling in situ Retroviral Capsid Assembly ....................................................
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