Abstract
Human α-defensins are potent anti-microbial peptides with the ability to neutralize bacterial and viral targets. Single alanine mutagenesis has been used to identify determinants of anti-bacterial activity and binding to bacterial proteins such as anthrax lethal factor. Similar analyses of α-defensin interactions with non-enveloped viruses are limited. We used a comprehensive set of human α-defensin 5 (HD5) and human neutrophil peptide 1 (HNP1) alanine scan mutants in a combination of binding and neutralization assays with human adenovirus (AdV) and human papillomavirus (HPV). We have identified a core of critical hydrophobic residues that are common determinants for all of the virus-defensin interactions that were analyzed, while specificity in viral recognition is conferred by specific surface-exposed charged residues. The hydrophobic residues serve multiple roles in maintaining the tertiary and quaternary structure of the defensins as well as forming an interface for virus binding. Many of the important solvent-exposed residues of HD5 group together to form a critical surface. However, a single discrete binding face was not identified for HNP1. In lieu of whole AdV, we used a recombinant capsid subunit comprised of penton base and fiber in quantitative binding studies and determined that the anti-viral potency of HD5 was a function of stoichiometry rather than affinity. Our studies support a mechanism in which α-defensins depend on hydrophobic and charge-charge interactions to bind at high copy number to these non-enveloped viruses to neutralize infection and provide insight into properties that guide α-defensin anti-viral activity.
Highlights
Human a- and b-defensins are small (3–5 kDa), cationic peptides of the innate immune system with broad anti-microbial activity [1]
We have shown that specific arginine residues and the hydrophobicity of residue 29 are important for human a-defensin 5 (HD5) to function as an anti-viral molecule against human AdV and human papillomavirus (HPV) [16]
Human AdVs cause a broad spectrum of diseases, many serotypes are transmitted by the fecal-oral route, where they may encounter intestinal HD5 [9,17,18]
Summary
Human a- and b-defensins are small (3–5 kDa), cationic peptides of the innate immune system with broad anti-microbial activity [1]. The six human a-defensins can be further divided by expression pattern and gene structure into myeloid [human neutrophil peptides (HNPs) 1–4] or enteric [human a-defensins (HDs) 5 and 6] classes [2,3] Despite their variable sequences, adefensins share common structural features including a triplestranded b-sheet fold, three intramolecular disulfide bonds, and a salt bridge [4]. The activity of a-defensins against both gramnegative and gram-positive bacterial pathogens has been well characterized, while their anti-viral properties are less well understood [4,5] Their capacity to neutralize enveloped viruses can be explained in part through properties identified in antibacterial studies, including lipid perturbation and their ability to function as lectins, other mechanisms have been proposed [5,6]. Recent studies have identified post-entry blocks of HPV and JC polyomavirus infection by HD5 [11,12], suggesting that common mechanisms may govern a-defensin neutralization of non-enveloped viruses
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