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Title page for ETD etd-07202009-180045

Type of Document Dissertation
Author Silvestry Ramos, Mariena
URN etd-07202009-180045
Title Insight into adenovirus programmed disassembly from cryoem: the structures of Ad2ts1 and the Ad35f+defensin HD5 complex
Degree PhD
Department Molecular Physiology and Biophysics
Advisory Committee
Advisor Name Title
Charles E. Cobb Committee Chair
Albert H. Beth Committee Member
Hassane S. Mchaourab Committee Member
Jens Meiler Committee Member
Phoebe L. Stewart Committee Member
Terence S. Dermody Committee Member
  • cryoEM
  • electron microscopy
  • structural biology
  • structure
  • adenovirus
Date of Defense 2008-07-09
Availability unrestricted
Adenoviruses (Ads) are a promising tool for gene and vaccine delivery. Though known for over 50 years, details about Ad structure and intermediates of its lifecycle remain elusive. CryoEM, a technique suitable for studying this large non-enveloped virus (>900Å in diameter), has increased our understanding of Ad structure, in particular of the components for which we do not have atomic resolutions structures. The cryoEM structure of a temperature-sensitive mutant, Ad2ts1, was determined to structurally characterize this intermediate in the Ad maturation process. Ad2ts1 fails to incorporate the viral protease and contains the precursor forms of multiple structural proteins when produced at non-permissive temperatures. It also has a cell entry defect and is defective in endosomal escape. The 10.5Å resolution cryoEM structure of Ad2ts1 shows that the core of the virus remains connected to the capsid via preproteins IIIa and VI, which are normally cleaved by the protease. In a second study, a cryoEM structure of an Ad vector, Ad35f, complexed with human ¦Á-defensin HD5, was determined to visualize the binding sites for defensin on the Ad capsid and to characterize the mechanism of neutralization. HD5 binds at negatively charged regions of the hexon, penton base, and fiber. Based on sequence analysis and information on which Ad types are neutralized by HD5, a critical binding site for neutralization is proposed, comprising 4 polar and negatively charged residues in the fiber N-terminal region and additional residues in the RGD-loop of the penton base. The mechanism of defensin neutralization may involve bridging the fiber and penton base and blocking programmed disassembly. These cryoEM studies highlight the importance of the exquisitely timed disassembly of the virion and release of viral proteins during cell entry.
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