379. Identification of Structural Aspects of Adenovirus Interactions with Host Factors In Vivo Using Tandem Mass Spectrometry

Abstract

Intensive studies of adenovirus infectivity and bio-distribution in animal models demonstrated that liver tissue is responsible for clearance of the bulk of intravenously applied vector. We recently demonstrated that adenovirus binding to blood factors, specifically coagulation factor IX and C4 binding protein, leads to high level hepatocyte transduction and virus trapping by Kupffer cells. Apparently, virus interaction with the coxsackie- and adenovirus receptor, CAR, was not required for virus trapping by hepatic cells, although the fiber knob domain was identified as the primary moiety, mediating virus interaction with blood factors in vivo. Because adenovirus hepatocyte transduction and trapping by Kupffer cells is associated with the severe innate immune and inflammatory host responses observed following systemic adenovirus administration, we hypothesized that preventing virus interaction with blood factors should dramatically improve both the circulatory persistence and toxicity profile of adenovirus vectors. To better understand the structural basis of Ad fiber knob domain interaction with FIX, we first analyzed the efficiency of FIX-Ad knob association at different ionic strengths using dynamic light scattering. These analyses revealed that formation of the Ad fiber knob-FIX complex is highly sensitive to ionic strength and most efficient at low salt concentrations. Salt concentrations exceeding 0.5M, on the other hand, completely prevent complex formation. These data suggest that interaction between hydrophilic charged amino acids in FIX and Ad fiber knob domain, rather than hydrophobic amino acids, plays the major role in complex formation. To further identify the precise areas of protein-protein interaction, we cross-linked FIX with purified recombinant Ad5 or Ad9 fiber knob domains. Using PAGE protein gels we confirmed that upon incubation with different cross linkers, FIX and Ad fiber knobs efficiently form complexes that migrate within the expected mass range. Proteolytic digestion of cross-linked Ad fiber knob-FIX complex and subsequent analysis of obtained peptides using ESI-MS and MS/MS revealed several candidate peptides participating in the cross-linking reaction. These peptides define the interface of Ad fiber knob-FIX interaction. To confirm mass spectrometry analysis data, we are currently generating a panel of Ad fiber knobs possessing amino acid mutations within the putative protein interface area. The approach of protein cross-linking followed by mass spectrometry analyses may be widely applied to study interactions of virus proteins with host factors in vitro and in vivo. The development of Ad vectors ablated for binding to blood factors should ultimately allow for the construction of safe and efficient adenovirus vectors for therapeutic application in humans.