By Juliana W. Noronha
Conjugation of Genetically Engineered HEV-VLPs to Gold Nano-Clusters (Au102(pMBA)44) for VLP Tracking in Cells
The capsid structure of Hepatitis E Virus (HEV) was modified to produce non-infectious virus-like particles (HEV-VLPs). VLPs retain the antigenicity, structural stability and cell binding capacity of the native virus. An Aspargine residue on the surface of the VLP was mutated to Cysteine (N573C), allowing the particle to covalently bind and expose ligands. This modulatable HEV capsid has potential to become a stable antigen and/or drug delivery platform. To study the capacity and efficiency of this platform, tracking VLPs in cells is critical. While purified virus-sized proteins are visible in cryo-electron microscopy (cryo-EM), distinguishing virus sized particles in cells remains a challenge due to labelling limitations. This study investigates conjugation of gold nano-clusters, Au102(pMBA)44, to the HEVVLP cysteine via ligand exchange. Au102(pMBA)44 particles are ligand protected mono-dispersed nano-gold clusters with high electron density, allowing VLP structure detection and tracking in cryo-EM. For conjugation optimization, incubation time, temperature and reducing agents were compared. Following Au102(pMBA)44 conjugation to HEV-VLP, the focus turns to cryo-EM and 3D reconstruction to assess the degree of Au102(pMBA)44 occupancy on the VLP cysteine sites.