Structure of AcMNPV nucleocapsid reveals DNA portal organization and packaging apparatus of circular dsDNA baculovirus

Baculoviruses are large DNA viruses found in nature propagating amongst insects and lepidoptera in particular. Their viral cycle has been studied and characterized over decades and they are nowadays considered as invaluable biotechnology tools widely used as biopesticides, recombinant expression systems or delivery vehicle for gene therapy. However, very little is known about the baculovirus nucleocapsid assembly at a molecular level. Here, we solved the whole structure of the nucleocapsid of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by applying cryo-electron microscopy (CryoEM) combined to de novo modelling and Alphafold2 predictions. Our structure completes prior observations from the helical sheath and the basal structure and elucidate the intricate architecture of the apical cap, unravelling for the first time the organization of a baculoviral DNA portal featuring intriguing symmetry mismatches between its core and vertex. The core, closing the capsid at the apex, holds two antiparallel DNA helices of the covalently closed circular (ccc) DNA genome tethered to Ac54 proteins. Different symmetry components at the apical cap and basal structure are constituted of the same building block, made of Ac101/Ac144, proving the versatility of this modular pair. The crown forming the portal vertex displays a C21 symmetry and contains, amongst others, the motor-like protein Ac66. Our findings support the viral portal to be involved in DNA packaging, most probably in conjunction with other parts of a larger DNA packaging apparatus that are not bound to mature virions and await further elucidation.