Divergent Entry, Convergent Trafficking in Defensin-Mediated Adenovirus Infection

Human α-defensins can paradoxically inhibit or enhance human adenovirus (HAdV) infection. Defensin-resistant HAdVs exploit α-defensins as molecular bridges to bypass canonical receptors, but whether this mode of entry redirects intracellular trafficking or contributes to defensin resistance remains unknown. Here we systematically compared intracellular trafficking after canonical receptor-mediated versus defensin-mediated entry using a defensin-resistant chimeric virus based on HAdV-C5 together with the human α-defensins, HD5 and HNP1. Pharmacological perturbations, capsid mutants, and imaging revealed that both entry routes rapidly converge on common checkpoints. Defensin-mediated infection showed the same sensitivity to endosomal acidification blockade and comparable co-localization with early endosomes as canonical entry. Notably, the membrane-lytic activity of HD5 did not substitute for viral protein VI, indicating that endosomal escape still requires intact protein VI function. Downstream transport also overlapped, as defensin-mediated infection, like canonical entry, depended on dynein-driven microtubule transport and passage through the microtubule-organizing center (MTOC) for nuclear entry. Co-infection experiments further showed that defensin-sensitive and defensin-resistant viruses retain their intrinsic phenotypes within the same cell, indicating that defensin effects are virion-autonomous and determined at or before cell entry rather than by downstream trafficking. These findings support a model in which resistance to α-defensin neutralization is governed by capsid features that control defensin binding and its effects on uncoating, not by access to an alternative intracellular pathway.

Human adenoviruses can be neutralized by α-defensins, antimicrobial peptides that bind the capsid and block uncoating. Paradoxically, some defensin-resistant adenoviruses infect cells more efficiently in the presence of defensins, with the peptides acting as molecular bridges that allow the virus to attach independently of its normal receptors. We asked whether this alternative attachment route changes how adenoviruses traffic through the cell, which could contribute to defensin resistance.Using labeled viruses, inhibitors, capsid mutants, and receptor knockout cells, we compared trafficking after canonical versus defensin-mediated entry. Both routes converged at the earliest steps of infection, with identical endosomal acidification requirements, similar early endosome colocalization, and the same dependence on microtubule transport to the nucleus. Co-infection experiments demonstrated that sensitive and resistant viruses retained distinct phenotypes in the same cell, indicating susceptibility is set at or before entry.These results demonstrate that defensin effects are decided at the plasma membrane, where capsid features, not alternative entry routes, drive resistance. Adenovirus entry is flexible at the cell surface, where the virus can attach through multiple mechanisms, but narrows to a single, constrained trafficking route inside the cell. Our findings inform how defensin-resistant vectors may behave in defensin-rich tissues during gene therapy or virotherapy.