Human adenoviruses (HAdV) are nonenveloped dsDNA viruses that infect a variety of cell types. Species C HAdV5 is the most commonly used vector for gene therapy and vaccine applications in the clinic. Recently, it has been shown that association of species A and C HAdVs with several blood coagulation factors (e.g., Factors X, IX) following intravenous delivery allow these viruses to gain access to epithelial cells and liver hepatocytes in vivo, respectively. This clotting factor-mediated pathway results in the unintended retargeting of C-type HAdVs to hepatocytes. Remarkably, this retargeting overrides the selectivity of natural receptor (CAR) for the virus fiber protein. Blocking of the hexon-FX interaction, either by pharmacological intervention or mutation of HAd5 hexon protein, abolishes liver transduction in vivo. Although low-resolution structural information on HAdV-FX association is available from cryoEM studies, detailed knowledge on the molecular interactions between the GLA-domain of FX with the hexon on the HAd capsid is still lacking. We recently determined the crystal structure for a HAdV5 based vector, designated Ad35F, at near atomic resolution by X- ray diffraction. Despite this new structural information, we still lack important knowledge of how the virus capsid influences tissue tropism in vivo. The absence of an accurate model of HAdV-FX interactions hampers the development of antivirals and gene therapy vectors. Preliminary diffraction experiments on Ad35F crystals soaked with chemically synthesized GLA (cs-GLA) peptide have been quite positive. Initial Fo-Fc maps at 6? resolution indicate that footprint of th GLA domain binding site on the hexon subunits is different from the location previously suggested. This proposal seeks to greatly improve our understanding of adenovirus host cell tropism in vivo by 1) determining the structure of HAdV in complex with the FX-GLA domain at near atomic resolution by employing X-ray diffraction methods. These analyses will build on the extensive knowledge and expertise that we gained in solving the crystal structure of Ad35F at 3.5 ? resolution and 2) analyzing the structure of single point mutant (E451Q) in the hexon subunit that drastically reduces FX binding to HAdV suggesting that the region of the hexon containing this mutation is crucial for clotting factor association. We anticipate that these investigations will provide greater understanding of mechanism and underlying molecular interactions involved in adenovirus transduction of liver hepatocytes.

Public Health Relevance

Human adenoviruses (HAdV) are nonenveloped dsDNA viruses that infect a variety of cell types and species. C HAdV5 is the most commonly used vector for gene therapy and vaccine applications in the clinic. Recently, it has been shown that association of species C HAdVs with several blood coagulation factors (e.g., Factors, X, IX) enable these viruses to gain access to liver hepatocytes in vivo. This proposal seeks to greatly improve our understanding of molecular interactions involved in adenovirus infection in vivo, employing X-ray diffraction methods.

Agency
National Institute of Health (NIH)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI103692-02
Application #
8731791
Study Section
Virology - A Study Section (VIRA)
Program Officer
Park, Eun-Chung
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037