The world is full of treasure troves of hoarded scientific samples – everything from pressed plants in herbaria and pinned insects in museum drawers to rocks from the Moon and asteroids in air-tight vaults. Generations of scientists have contributed to collecting, preserving and studying such specimens, amassing an immense amount of knowledge about our natural world and how it changes over time. As we develop ever more powerful technologies to explore them, these historical samples can lead us to new discoveries, as an international team of researchers recently found. Their work, published in Nature Communications, began with a 70-year-old viral sample tracked down in a low temperature freezer. This viral sample was of a Nudivirus that are economically important in agriculture, where they are used as biological agents to control some insect pests. However, they can be pests themselves, damaging large-scale crustacean and insect farming efforts. Using X-ray crystallography, the research team solved the lattice structure of a polyhedrin (occlusion body) from Nudiviridae, which adds to our knowledge of how viruses use protein self-assembly to protect themselves from the environment.
Two families of insect viruses – the double-stranded DNA Baculoviridae and double-stranded RNA cypoviruses – are known for using protein crystals, called polyhedra, to encase virus particles. The notable feature of the polyhedra is their remarkable resilience, protecting the viruses inside from environmental factors such as temperature, light, and water. When insects consume contaminated foliage, the viral polyhedra dissolve in their digestive tracts to produce a new viral infection. New crystals of viral polyhedra grow inside the infected insects, which when they die contaminate more foliage and spread the virus. The Baculoviridae and cypoviruses evolved polyhedra separately, essentially using different proteins to arrive at the same biological solution.
Although Nudiviridae are related to Baculoviridae, they were originally given the prefix nudi- because they were thought to lack the polyhedrin protein. However, recent research has found that at least one genus of nudiviruses form polyhedra that were not previously recognised.
Dr Jeremy Keown at the University of Oxford said:
We were interested in investigating the structure of these nudivirus polyhedra and so we got in touch with Dr Annie Bézier at the Institut de Recherche sur la Biologie de l’Insecte in Tours, who had done some initial characterisation of these samples. She had frozen samples originally purified in the 1960s. We used the I24 beamline to carry out initial X-ray diffraction experiments, and those results showed us that the crystal lattice structure was different from that found in other viruses.
We were able to make a mutant version of the protein that included methionines and that was crucial to our research. It enabled us to grow the cells in a medium containing selenomethionines – a sort of derivatised version of the protein – and that eventually allowed us to solve the structure.
In October 2019, the team returned to Diamond, this time to the new VMXm beamline.
Dr Keown said:
At this point, we were working with large numbers of very small crystals. We had a lot of help from beamline scientists Dr Adam Crawshaw and Dr Jose Trincao, preparing slurries of microcrystals in cryo-EM grids, which we then vitrified. Crystals this small are rapidly destroyed in the X-ray beam, so we collected small wedges of data from lots and lots of crystals. When the Covid-19 pandemic hit, it meant we couldn’t collect data in person, so I dropped samples off at Diamond, and the beamline staff ran the experiments.
Read more on Diamond website
Image : a) Scanning electron micrograph of native ToNV occlusion bodies prepared for diffraction experiments. Images have been cropped but are otherwise unedited. b) A single polyhedrin molecule coloured from the N-terminus (blue) to the C-terminus (red) and annotated with secondary structure features. A calcium ion is shown (green). The dashed line shows the missing loop 171–174. c) The dimeric unit (red and blue chains) of the OB lattice are shown in two orientations. The two-fold symmetric axis is indicated by the black ellipse.