Structural characterization of site-modified nanocapsid with monodispersed gold clusters

Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au(102) pMBA(44) (Au(102)) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au(102) was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au(102) was functionalized with a maleimide linker (Au(102)_C(6)MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au(102)_C(6)MI to bind nanocapsid more efficiently, while Au(102) remained more soluble over time. Nanocapsids conjugated to Au(102)_C(6)MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling.