The long-term objective of this project is to understand the events involved in adenovirus (Ad) cell entry at the molecular level. The specific goals for the next funding period are to undertake high resolution structural studies of Ad and Ad/integrin complexes, to investigate the geometry of the interaction between Ad and its host cell receptors, and to define the conformational changes induced in alpha-v beta-5 integrin by binding to monovalent and multivalent ligands. The proposed research will definitively test the paradigm that Ad has evolved efficient pathways for infecting specific cell types and for inducing integrin cell signaling events. The results will bridge the knowledge gap between our understanding of Ad molecular biology and the rapidly expanding field of Ad vector based gene therapy. In the previous funding period, we have made exciting new discoveries that have provided a better characterization of Ad structure and its interaction with host cell receptors. An emerging concept is that the precise three-dimensional orientation of the virus with its associated receptors is a contributing factor to viral tropism. The proposed higher resolution studies will enable us to characterize the tertiary protein fold of the Ad penton base protein, which interacts with alpha-v integrins during viral cell entry, as well as the conformation of alpha-v integrin when bound and clustered by the multivalent Ad penton base protein.
The specific aims are designed to address two fundamental questions: 1) What structural features of Ad are critical for efficient binding to host cell receptors? 2) What conformational changes does Ad induce in alpha-v beta-5 integrin to initiate signaling pathways? Advances in cryo-electron microscopy (cryo-EM) have made determining a high resolution structure of an icosahedral virus and cryoelectron tomography of Ad/receptor vesicle complexes feasible. These advances include the development of automated data acquisition software, computer-controlled tomography software, parallelized image processing software, and microscopes with liquid-helium-cooled specimen stages. Cryo-EM methods have also recently been extended to detergent solubilized membrane proteins and we will apply this approach to determine structures of alpha-v beta-5 integrin and an Ad/alpha-v beta-5 integrin complex. Increased knowledge of the Ad cell entry process may provide an opportunity to develop antivirals that block viral cell entry and will facilitate the rational design of targeted Ad vectors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI042929-09
Application #
7004577
Study Section
Virology Study Section (VR)
Program Officer
Park, Eun-Chung
Project Start
1997-08-01
Project End
2008-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
9
Fiscal Year
2006
Total Cost
$368,629
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Flatt, Justin W; Kim, Robert; Smith, Jason G et al. (2013) An intrinsically disordered region of the adenovirus capsid is implicated in neutralization by human alpha defensin 5. PLoS One 8:e61571
Nemerow, Glen R; Stewart, Phoebe L; Reddy, Vijay S (2012) Structure of human adenovirus. Curr Opin Virol 2:115-21
Woetzel, Nils; Lindert, Steffen; Stewart, Phoebe L et al. (2011) BCL::EM-Fit: rigid body fitting of atomic structures into density maps using geometric hashing and real space refinement. J Struct Biol 175:264-76
Reddy, Vijay S; Natchiar, S Kundhavai; Stewart, Phoebe L et al. (2010) Crystal structure of human adenovirus at 3.5 A resolution. Science 329:1071-5
Smith, Jason G; Silvestry, Mariena; Lindert, Steffen et al. (2010) Insight into the mechanisms of adenovirus capsid disassembly from studies of defensin neutralization. PLoS Pathog 6:e1000959
Lindert, Steffen; Silvestry, Mariena; Mullen, Tina-Marie et al. (2009) Cryo-electron microscopy structure of an adenovirus-integrin complex indicates conformational changes in both penton base and integrin. J Virol 83:11491-501
Lindert, Steffen; Stewart, Phoebe L; Meiler, Jens (2009) Hybrid approaches: applying computational methods in cryo-electron microscopy. Curr Opin Struct Biol 19:218-25
Lindert, Steffen; Staritzbichler, René; Wötzel, Nils et al. (2009) EM-fold: De novo folding of alpha-helical proteins guided by intermediate-resolution electron microscopy density maps. Structure 17:990-1003
Silvestry, Mariena; Lindert, Steffen; Smith, Jason G et al. (2009) Cryo-electron microscopy structure of adenovirus type 2 temperature-sensitive mutant 1 reveals insight into the cell entry defect. J Virol 83:7375-83
Nemerow, G R; Pache, L; Reddy, V et al. (2009) Insights into adenovirus host cell interactions from structural studies. Virology 384:380-8

Showing the most recent 10 out of 15 publications