Viral occlusion bodies (OBs), also known as polyhedra, are stable protein crystals crucial to the lifecycle of certain viruses. These structures, found in insect viruses from families like Baculoviridae, Reoviridae, and Poxviridae, encapsulate newly assembled virions, protecting them from environmental hazards like dehydration, temperature fluctuations, and enzymatic degradation. Initially described in the early 20th century for their reflective properties and polyhedron geometry, electron microscopy later confirmed their role in virion containment. Further research, using proteomics and genome sequencing, established that these OBs primarily consist of a single protein, polyhedrin. Baculoviridae, a large family of circular double-stranded DNA (dsDNA) viruses, forms OBs in the nucleus, while cypoviruses (segmented linear dsRNA viruses from the Reoviridae family) form cytoplasmic OBs. Although initially thought unique to these families, OB formation has been reported in nudiviruses. Nudiviruses, circular dsDNA viruses infecting various insects and aquatic arthropods, were initially considered a baculovirus subgroup but were later reclassified due to differences in host range, genome structure, and cytopathology. The discovery of OBs in some nudivirus species, such as *Penaeus monodon* nudivirus (PmNV) and *Tipula oleracea* nudivirus (ToNV), challenged the initial classification criteria. This study focuses on ToNV polyhedrin to understand the structural features and lattice constraints conserved in nudivirus OBs, exploring the convergent evolution of polyhedra design principles.

Previous structural characterization of native OBs purified from moth larvae infected with cypoviruses (Reoviridae) and baculoviruses, alongside intracellular polyhedrin microcrystals from recombinant expression in Sf9 cells, revealed that while polyhedrin amino acid sequences show little conservation, baculovirus and cypovirus OBs exhibit remarkably similar crystal lattices (cubic space group symmetry I23, unit cell dimensions between 101 and 106 Å). Despite the lack of sequence homology between the two, both structures share a predominantly β-strand fold with α-helical extensions, although their secondary structure topologies differ. Reports of OB formation in nudivirus-infected cells prompted further investigation into the structural characteristics of these OBs, especially given the economic importance of nudiviruses as biocontrol agents (e.g., *Oryctes rhinoceros* nudivirus (OrNV) for coconut palm beetle control). However, early studies on nudivirus polyhedrins indicated little homology to baculovirus or cypovirus polyhedrins. This study aims to bridge this gap and understand whether the previously observed structural features and lattice constraints are conserved in nudivirus OBs.

The researchers utilized both a 70-year-old archival sample of ToNV polyhedra and recombinantly expressed and assembled protein for structural analysis via X-ray crystallography. The archival sample, initially purified from *Tipula oleracea* larvae, yielded high-quality diffraction patterns. For the recombinant protein, the wild-type ToNV polyhedrin gene was cloned into an insect cell expression vector. Three hydrophobic residues (F104/L105/L137) were mutated to methionine to facilitate phasing using single wavelength anomalous diffraction (SAD). Selenomethionine-derivatized crystals, alongside native polyhedra, were prepared for diffraction experiments at the VMXm beamline (Diamond Light Source). Crystals were mounted on cryo-EM grids and data collected from numerous small crystals, then merged into final datasets. The final model used native data, which offered greater completeness and resolution. Molecular replacement attempts using previously determined polyhedra structures, in silico models (trRosetta and AlphaFold), or secondary structure fragments were unsuccessful. The structure was ultimately solved using SAD data from the selenomethionine crystals. The stability of the recombinant crystals was tested under various conditions (urea, SDS, acids, bases, organic solvents, temperature, reducing/oxidizing agents) by assessing diffraction after treatment. SDS-PAGE analysis assessed stability under reducing and non-reducing conditions at different pH levels. The researchers also analyzed crystal structures of both native and recombinant crystals to understand the lattice arrangement and the role of various interactions (disulfide bonds, domain swapping, electrostatic and hydrophobic interactions).

The ToNV polyhedrin is predominantly helical (ten helices and a short two-stranded antiparallel β-sheet), with a unique 3D fold lacking homology to known structures. The polypeptide (residues 4-237) forms a dimeric repeating unit within a trigonal P3221 space group with a = b = 53.7 Å, c = 105.6 Å. This dimeric interface is stabilized by extensive hydrophobic and electrostatic interactions, a salt bridge, hydrogen bonds, and a hydrophobic interface. Each polyhedrin molecule interacts with eight neighbors, forming disulfide bonds, hydrogen bonds, and salt bridges. The lattice's 3D organization involves domain swapping of the N-terminal region, interlocking the sheets. The ToNV polyhedra lattice is extremely dense, with minimal solvent content and the absence of channels. Recombinant ToNV polyhedra exhibited high stability under various environmental stresses (urea, ethanol, reducing/oxidizing agents, high temperatures) but dissolved in high pH conditions (20 mM NaH2CO3, pH 10.5) and 10% SDS, similar to cypovirus and baculovirus polyhedra. SDS-PAGE analysis confirmed this pH sensitivity. Tyrosine residues, many located at inter-subunit interfaces, are suggested to play a role in pH-mediated dissolution. Analysis of a secondary crystal form (10% of the crystal population), with increased unit cell dimensions and disordered regions, suggests possible intermediate states in the crystal assembly/disassembly process. Comparison with baculovirus and cypovirus polyhedra revealed differences in protein structure (α-helical vs. predominantly β-strand), space group (P3221 vs. I23), and unit cell dimensions. Although differing in structure, all three types of polyhedra utilize common assembly principles including hydrophobic and electrostatic interactions, domain swapping, and a high number of tyrosine residues at interfaces. In silico modeling methods (AlphaFold and trRosetta) proved insufficient to accurately predict the ToNV polyhedrin structure, illustrating the complexity of this unique structure.

This study provides the first high-resolution structure of a nudivirus occlusion body, revealing a unique protein structure and lattice organization distinct from previously characterized baculovirus and cypovirus polyhedra. This highlights the convergent evolution of structurally diverse proteins to perform a common biological function. The high density and structural integrity of the ToNV polyhedra, maintained through extensive interactions (disulfide bonds, domain swapping, hydrophobic and electrostatic interactions), effectively protect virions from environmental stressors. The observed pH sensitivity, potentially mediated by tyrosine residues, is consistent with the alkaline conditions of the insect midgut, enabling virion release and infection. The two key requirements for effective OB formation—robust protein assembly and accommodation of asymmetric virions—are met through the dense, channel-free lattice and the highly symmetric P3221 space group. While the reason for the structural divergence between nudivirus and baculovirus polyhedra, both encapsulating rod-shaped virions, requires further investigation, it may involve other viral proteins. Future research should examine the packaging mechanisms and signals to better understand the packaging of nudiviruses within their polyhedral structures.

This research provides a detailed atomic structure of the ToNV polyhedrin, revealing a novel protein fold and lattice organization in viral occlusion bodies. While differing significantly from baculovirus and cypovirus polyhedra, it demonstrates a remarkable instance of convergent evolution, where different protein structures achieve the same protective function. The study highlights the importance of investigating diverse viral systems to broaden our understanding of virus structure and function. Future research should explore the role of other viral proteins and the mechanism of virion packaging within these structures. Further examination of various nudivirus polyhedra will help elucidate the diversity of protein solutions for occlusion body formation.

The study focuses primarily on recombinant ToNV polyhedra, with comparisons to native material limited. Although the recombinant polyhedra showed high stability, there's a potential for minor differences in stability or behavior compared to naturally formed OBs. The observed secondary crystal form with disordered regions could be either an assembly intermediate, a disassembly product or an artifact of purification. The function of the disordered C-terminus and the precise nature of disulfide bond formation between the C-termini and other cysteine residues remain to be fully elucidated. In silico predictions failed to provide accurate structural models, implying that current methods need further refinement for predicting such unique protein structures.