Baculoviruses are large viruses with circular double-stranded DNA (dsDNA) genomes. They specifically propagate amongst insect cells, playing a natural role in regulating insect populations and therefore are used as biological control agents in agriculture, a rising alternate to chemical pesticides. Beyond their ecological role, baculoviruses are also valuable biotechnological tools: they are used extensively worldwide, including by EPN campus researchers, as expression systems for producing recombinant proteins in insect cell cultures and as delivery vehicle for gene therapy and vaccine development.
Despite their widespread use, detailed structural studies of baculovirus nucleocapsids, the protein shells that protect and package the viral DNA, were still limited when this project began. To address this, a collaboration was established between Martin Pelosse (EMBL), Eaazhisai Kandiah (ESRF), and Gregory Effantin (IBS) to investigate the most studied baculovirus, Autographa californica Multiple NucleoPolyhedroVirus (AcMNPV).
AcMNPV particles consist of a nucleocapsid surrounded by a lipid envelope decorated with viral glycoproteins. The nucleocapsid itself is composed of hundreds of proteins and exhibits an elongated structure, approximately 50 nm in diameter and 300 nm in length, with two distinct termini: the “apical cap,” through which viral DNA enters and exits, and the “basal” structure. These termini are linked by the capsid body that encloses the viral genome. Despite the wealth of biochemical knowledge on the virus, the project began with the challenge of an unknown nucleocapsid proteome (its protein composition).
Using cryo-electron microscopy (cryo-EM) data collected at the CM01 facility of the ESRF on the baculovirions routinely used at the EMBL Eukaryotic Expression Facility (EEF), the team focused on both the apical and basal regions of the nucleocapsid. Multiple 3D maps, spanning resolutions from medium to high, were determined to assign and position proteins within these structures. The remarkable feature of AcMNPV nucleocapsid is its composition of several distinct protein sub-assemblies, each with a different symmetry. By elucidating the various symmetries within these sub-assemblies, a composite pseudo-atomic model representing the complete AcMNPV nucleocapsid was obtained for the first time [1]. To delineate the proteome of these individual sub-assemblies, cutting-edge modelling tools were employed. At resolutions better than 4 Å, protein identities were determined using Modelangelo, a machine learning program designed to build atomic models of proteins into cryo-EM density without prior knowledge of the amino acid sequence [2]. For lower resolution maps, AlphaFold2 predictions were essential for determining the identity and placement of several proteins [3]. Beyond predicting the structures of the 155 proteins encoded by the AcMNPV genome, Alphafold was also used to identify possible interaction partners, thus facilitating the interpretation of complex assemblies.
This integrative approach led to the elucidation of the peculiar structure of the apical cap and the discovery of eight previously uncharacterized proteins as well as a fragment of the viral genome at the apex. These findings provide direct experimental support for the proposed role of the apical cap as the entry and exit point of viral DNA. The unambiguous presence of two dsDNA strands at the apical cap not only suggests that the baculoviral genome is packaged as covalently closed circular dsDNA (cccdsDNA) but also provides, for the first time, insights into the dsDNA packaging mechanism of baculoviruses and other cccdsDNA viruses.
This study delivers a near-atomic resolution model of the entire nucleocapsid of AcMNPV. Beyond offering fundamental insight into baculovirus architecture and function, it significantly enriches the structural database of this virus and opens the way for more rational design of baculovirus-based biotechnological applications.
G. Effantin (IBS), E. Kandiah (ESRF), M. Pelosse (EMBL)
[1] Effantin G, Kandiah E, Pelosse M (2025) Nat Commun., 16, 4844.
[2] Jamali K, Käll L, Zhang R, Brown A et al. (2024) Nature, 628, 450–457.
[3] Jumper J, Evans R, Pritzel A, Green T et al. (2025) Nature, 596, 583–589.
Figure 1: Superimposition of a composite 3D map of the complete nucleocapsid of AcMNPV over a cryo-EM image of an intact AcMNPV viral particle.
Figure 2: Structures of the AcMNPV nucleocapsid obtained by cryo-EM. Left panel: electron micrograph of an AcMNPV virion with enlarged 3D reconstructions of the apical cap, the capsid sheath and the basal structure. Right panels, upper row: isosurface representations of the 3D composite map of the apical cap: – side v iew (left), top v iew (center) and bottom view (right). Lower row: isosurface representations of the 3D composite map of the basal structure: side v iew (left), top view (center) and bottom view (right).