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Biological Chemistry

Directed evolution suggests way viruses could have evolved

Researchers evolve bacterial enzyme to package and protect its own RNA, as viruses do

by Celia Henry Arnaud
June 10, 2021 | A version of this story appeared in Volume 99, Issue 22

The original lumazine synthase and four evolved artificial nucleocapsids, NC-1, -2, and -4. Through directed evolution, the structure changes so that the capsid contains more subunits and has smaller pores.
Credit: Stephan Tetter
Directed evolution transformed Aquifex aeolicus lumazine synthase (AaLS) into a structure that could package and protect its own RNA, functioning as viral capsids do. Each iteration (NC-1, NC-2, and NC-4) had more subunits and smaller pores. Different conformations of the structures’ subunits are shown in different colors.

Among the hypotheses for how viruses came to be is that they started as self-replicating nucleic acids that co-opted host proteins for packaging and protection, leading eventually to the viral capsid—the protein shell that holds a virus’s genetic material—in today’s viruses.

Donald Hilvert of the Swiss Federal Institute of Technology (ETH) Zurich and his colleagues show that the this hypothesis is plausible by making it happen in the lab (Science 2021, DOI: 10.1126/science.abg2822). “You can take a bacterial protein that has no starting affinity for nucleic acids and teach it how to bind its own encoding mRNA, package it, and protect it against nucleases,” Hilvert says.

The researchers started with a bacterial enzyme called lumazine synthase, which naturally forms 60-subunit nanoscale cages. The researchers redesigned the protein by adding a peptide that tightly binds an RNA sequence called BoxB; this peptide was designed to trap the RNA the team hoped to evolve the protein to package. Meanwhile, they added BoxB to both ends of the RNA sequence encoding the protein. They then subjected the protein to multiple rounds of directed evolution in which they randomly mutated the gene encoding the protein and selected for variants that could package the target RNA and protect it from nucleic acid–cleaving enzymes.

The resulting container combined 240 protein subunits and efficiently packaged the full-length RNA. Intermediate versions had fewer subunits with large pores that let nucleic acid–cleaving enzymes in. During evolution, the protein underwent structural changes in which monomers interlaced with one another by swapping domains and forming trimeric building blocks. Those building blocks further assembled into pentamers of trimers. The final capsid consisted of 12 pentamers of trimers plus an additional 20 trimers in a spherical shape with small pores. The interlacing reduces the size of the pores compared to the original, which helps block nucleases.

The mutations in the RNA sequence for the evolved protein also caused the RNA to fold differently, which the researchers say enhanced packaging efficiency and specificity. The RNA folded in such a way that it formed packaging signals—multiple loops with affinity for the protein, beyond the BoxB sequence . “In the final version we get better yields. We get more efficient assembly. The whole structure is a lot more stable,” Hilvert says. “We think it’s really because this cassette of packaging signals, which copies what natural RNA viruses do, is critical.”

The work is a “multidimensional tour de force that experimentally recapitulates processes by which viruses might have emerged from free-living organisms,” says Charles W. Carter Jr., a structural biologist at the University of North Carolina at Chapel Hill.



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