A Single Surface-Exposed Amino Acid Determines Differential Neutralization of AAV1 and AAV6 by Human Alpha-Defensins.

Adeno-associated viruses (AAVs) are being developed as gene therapy vectors due to their low pathogenicity and tissue tropism properties. However, the efficacy of these vectors is impeded by interactions with the host immune system. One potential immune barrier to vector transduction is innate immune host defense peptides, such as alpha-defensins, which are potent antiviral agents against other nonenveloped viruses. To investigate the interaction between AAVs and alpha-defensins, we utilized two closely related AAV serotypes, AAV1 and AAV6. Although their capsids differ by only six residues, these two serotypes exhibit markedly different tissue tropisms and transduction efficiencies. Using two abundant human alpha-defensins, enteric human defensin 5 (HD5) and myeloid human neutrophil peptide 1 (HNP1), we found both serotype-specific and defensin-specific effects on AAV infection. AAV6 infection was uniformly neutralized by both defensins at low micromolar concentrations; however, inhibition of AAV1 infection was profoundly influenced by the timing of defensin exposure to the virus relative to viral attachment to the cell. Remarkably, these differences in the defensin-dependent infection phenotype between the viruses are completely dictated by the identity of a single, surface-exposed amino acid (position 531) that varies between the two serotypes. These findings reveal a determinant for defensin activity against a virus with unprecedented precision. Furthermore, they provide a rationale for the investigation of other AAV serotypes not only to understand the mechanism of neutralization of defensins against AAVs but also to design more efficient vectors.