An integrated structural cell biology to unravel the vesicle and membrane coat formation mechanism at the inner nuclear envelope

Our group uses electron cryo microscopy (cryoEM) / tomography (cryoET) and combines these with complementary techniques to understand biological processes mechanistically. The power of such an integrated structural biology approach to cell biology at the macromolecular level will be exemplified along the group's analyses of crucial steps in the molecular interactions between viruses and their host cells . The main emphasis will be on understanding mechanisms of membrane modulation in vesicle formation at the inner nuclear envelope in cargo egress from the nucleus [1,2]. Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Vesicular nucleo-cytoplasmic transport might also be a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins (pUL31 and pUL34), forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane- distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM. We further solved the crystal structure of the pseudorabies virus NEC. Fitting of the NEC crystal structure into the cryoEM-derived hexagonal lattice found in situ, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling. Altogether, this multimodal imaging study exemplified the power of an integrated structural cell biology approach to reveal novel mechanistic insights by combining in-situ data with information on the isolated macromolecules mediating the respective biological process of interest.