Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization

Abstract

The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitinrich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides.ost entomopoxviruses (EV) produce two types of intracellular crystals. Virus-containing spheroids are the main infectious form of EV (1) and are functionally analogous to polyhedra of cypovirus (2) and baculovirus (3, 4). In contrast, the function of the second type of crystals is less clear. These crystals of the viral fusolin protein, called "spindles" because of their characteristic shape, assemble in the endoplasmic reticulum of infected cells and for some species also occur embedded within the crystalline lattice of spheroids (5). Purified spindles are not infectious but strongly enhance the infectivity of EV by a mechanism that involves disruption of the peritrophic matrix, a physical barrier that protects the midgut epithelium of insects against oral pathogens (6, 7). Remarkably, in larval cofeeding experiments, spindles also enhance the insecticidal activity of unrelated oral pathogens such as baculovirus (8) and the Bacillus thuringiensis (Bt) toxin (9) by up to three orders of magnitude. This effect on virulence prompted their use as synergistic additives to common bioinsecticides, for instance by transgenic expression of spindles in plants to improve the effectiveness of baculovirus insecticides (10).Fusolin proteins have a signal sequence that targets them to the endoplasmic reticulum, and the mature protein has a mass of 36-44 kDa. Some fusolins are glycosylated, and the glycosylation site of the fusolin produced by Anomala cuprea EV (ACEV) is required for full virulence (11). Sequence analysis shows that the N-terminal regio...

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus

“…CjLPMO10A toward chitin (and not cellulose) is intriguing, and further studies of this enzyme may eventually allow a deeper insight into the determinants for LPMO substrate specificity. Interestingly, comparison of CjLPMO10A with the recently published AA10 structure of viral origin (68) revealed an intriguing similarity between the catalytic centers and substrate-binding surfaces of these enzymes (Fig. 4, C and D), even though the overall sequence similarity is low (27% sequence identity).…”

“…4D), whose activity has not been demonstrated yet (68). It should be noted that a cavity formed by the non-conserved residues Thr-76, Glu-79, Arg-103, Ala-104, and Asn-105 is present on the putative binding surface (Fig.…”

“…4, C and D), even though the overall sequence similarity is low (27% sequence identity). The viral AA10s are essential for viral penetration of the chitinous insect gut peritrophic matrix (that contains ␣-chitin), which enables infection by virus particles (68). An interesting property of CjLPMO10A is the presence of two CBMs, a common CBM5 chitin-binding module and a previously uncharacterized C-terminal domain (Fig.…”

De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure

“…7c, d). An analogous mechanism was proposed for the dissolution of the ultra-stable viral spindle crystals, which also contain an unusually high frequency of tyrosines (8.6%), some located strategically at crystal contacts 25 .…”

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars

“…78–80 The presence of genes corresponding to LPMO profiles have been reported for Drosophila melanogaster , bivalves, stony corals, and sea anemones. 71 …”