Unraveling the architecture of poxvirus cores

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The recent resurgence and outbreak of Mpox has brought poxviruses back as a public health threat, underscoring an important knowledge gap in their origins. Now, a team of researchers from the Institute of Science and Technology Austria (ISTA) has unraveled the mystery of poxviral core architecture by combining various cryo-electron microscopy techniques with molecular modeling. Results, published Nature Structural and Molecular BiologyTargeting the poxvirus core may facilitate future research on therapeutics.

Variola virus, the most notorious poxvirus and one of the deadliest viruses to infect humans, wreaked havoc with smallpox until it was eradicated in 1980. Eradication was successful thanks to a mass vaccination campaign using another poxvirus, the vaccinia virus. The 2022-2023 resurgence and outbreak of the Mpox virus reminded us once again that viruses find ways to return to the forefront as a public health threat. Importantly, it highlighted fundamental questions about boxviruses that remain unanswered to date.

Such a fundamental question is, quite literally, at the heart of the matter: “We know that for boxviruses to be infectious, their viral core must be properly formed. But what is this boxviral core made of and how do its individual components work together?” asks Assistant Professor Florian Schur, corresponding author of the ISTA study.

Schur and his team now have their finger on the missing link: a protein called A10. Importantly, A10 is common to all clinically relevant boxviruses. Additionally, the researchers found that A10 serves as one of the main building blocks of the boxviral core. This knowledge may be helpful for future research on therapeutics targeting the poxviral core.

“The most advanced cryo-EM technique available today”

The viral core is one of the common causes of all infectious forms of poxviruses.

Previous experiments in virology, biochemistry, and genetics suggested key protein candidates for poxviruses, but no experimentally derived structures were available.”


Julia Datler, an ISTA PhD student, is one of the study’s co-first authors

Thus, the team began by computationally predicting models of key protein candidates using the now-famous AI-based molecular modeling tool AlphaFold. In parallel, Dattler was laying the biochemical and structural foundations of the project, drawing on his background in virology and one of Schur’s group’s core expertise: cryogenic electron microscopy, or cryo-EM for short. “We integrated many of the advanced cryo-EM techniques available today with alphafold molecular modeling. This gave us, for the first time, a detailed holistic view of the boxviral core – the ‘safe’ or ‘bioreactor’ viral genome that resides inside the virus and releases it into infected cells,” Shur said. “It was a gamble, but we were finally able to find the right mix of techniques to test this complex question,” said postdoc Jesse Hansen, co-first author of the study whose expertise in various structural biology techniques and image processing methods was critical to the project.

Poxvirus is a global 3D view

ISTA researchers examined “live” vaccinia virus mature virions and purified poxviral cores under every possible angle—quite literally. “We combined ‘classic’ single-particle cryo-EM, cryo-electron tomography, subtomogram averaging and alphafold analysis to achieve a holistic view of the poxviral core,” said Dattler. With cryo-electron tomography, researchers can reconstruct a 3D volume of a biological sample as large as an entire virus by acquiring images while slowly tilting the sample. “It’s like doing a CT scan of the virus,” Hansen says. “Cryo-electron tomography, our lab’s ‘specialty,’ allows us to achieve nanometer-level resolution of the entire virus, its core and interior,” Schur said. In addition, researchers can fit the alphafold models to the observed shapes like a puzzle and identify the molecules that make up the boxviral core. Among these, the key protein candidate A10 stands out as one of the main components. “We found that A10 defines key structural elements of boxviruses,” said Dattler “These results are a great resource for interpreting bits of structural and virological data generated over the past few decades,” adds Schur.

A difficult path to uncovering the poxviral core

The path to this search was simple. “We had to find our own way from the beginning,” Dattler said. Utilizing his expertise in biochemistry, virology, and structural biology, Dattler isolated, propagated, and purified samples of vaccinia virus and established protocols for purifying entire viral cores, all while optimizing these samples for structural studies. “Structurally, these virus cores were extremely difficult to study. But fortunately, our persistence and optimism paid off,” says Hansen.

The ISTA researchers are confident that their findings can provide a knowledge platform for future therapeutics that seek to target poxviral cores. “For example, one could think of drugs that prevent the core from assembling — even dissociating and releasing viral DNA during infection. Ultimately, basic virus research, like the one done here, allows us to be better prepared against possible future viral outbreaks. ,” concludes this.

Source:

Journal Reference:

Dutler, J., etc. (2024). Multi-model cryo-EM reveals trimers of protein A10 forming the palisade layer in the poxvirus core. Nature Structural and Molecular Biology. doi.org/10.1038/s41594-023-01201-6.



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