Adenoviruses (Ads) are common human viruses that cause mainly respiratory infections and have greatpotential for vector-based gene delivery and vaccination strategies. Ads provide numerous advantagesfor vector design, including the fact that they have been well studied as a model system in cell andmolecular biology and we have a good understanding of their life cycle. In the previous funding period,we made exciting new discoveries related to the role of fiber flexibility in receptor usage and cell entry;determined a 6 resolution cryoEM structure of an Ad vector; localized the membrane-lytic protein VIand protein IX, which has been used as a retargeting platform, within the Ad capsid; identified likelybinding sites on hexon for the human blood coagulation Factor X, which plays an important role intargeting Ad to the liver; investigated the nature of the cell entry defect of the temperature sensitivemutant Ad2ts1; and shown that v integrins undergo significant conformational change after binding toAd. We have also developed a script assisted microscopy package, which facilitates semi-automatedcryoEM data acquisition, and collaborated on the development of cryoEM guided de novo protein foldelucidation methods.
The specific aims of this proposal are to determine high resolution cryoEMstructures of: 1) Ad complexed with human defensin 5, a component of the innate immune system withboth antimicrobial and antiviral activity; 2) Ad complexed with Factor X, to determine the key residues ofhexon that are involved in the interaction and might be mutated to ablate Factor X binding and livertargeting; and 3) the multiprotein complex that binds the Ad DNA packaging sequence and is critical forAd assembly. Cryoelectron tomography, combined with antibody labeling, will be used to locate the DNApackaging complex within the Ad virion. In addition, we will perform non-icosahedral averaging on theprotein VI density in the Ad capsid to facilitate atomic modeling of this membrane-lytic factor; and applycryoEM guided de novo methods to enhance structure determination of the Ad capsid and the Ad-integrin receptor complex. These studies will contribute to a greater understanding of the Ad life cycleincluding key interactions with cell receptors, antimicrobial peptides, and tissue targeting factors in theblood. This knowledge may in turn lead to novel strategies for antiviral drug design, Ad vectorretargeting, and shielding of Ad vectors from the host immune system. The long term goal of thesestudies is to provide structural information that will help guide the redirection of Ad for numerous potentialbiomedical applications.
of this project to human health is that Adenovirus is a common human pathogen that typically causes respiratory infections and has great potential for gene delivery and vector- based vaccination strategies. Understanding Adenovirus structure, cell entry, and assembly, will ultimately help guide its redirection for numerous potential biomedical applications.
Showing the most recent 10 out of 15 publications
