Unraveling the architecture of the poxvirus core

The recent re-emergence and outbreak of Mpox has re-emerged poxviruses as a public health threat and highlighted important knowledge gaps at their core. Now, a team of researchers from the Austrian Institute of Science and Technology (ISTA) has solved the mystery of the core structure of poxviruses by combining various cryo-electron microscopy techniques and molecular modeling. The survey results are Structural biology and molecular biology of naturecould facilitate future research into therapies targeting the core of poxviruses.

The variola virus is the most notorious poxvirus and one of the deadliest viruses to afflict humans, causing smallpox and wreaking havoc until it was eradicated in 1980. Eradication was successful thanks to a massive vaccination campaign using another poxvirus, aptly named vaccinia virus. The re-emergence and outbreak of the Mpox virus from 2022 to 2023 has once again reminded us that viruses are finding their way back to the forefront as public health threats. Importantly, this highlights fundamental questions about poxviruses that remain unanswered to date.

One such fundamental question is literally at the heart of the matter. ”We know that for poxviruses to be infectious, the viral core must be properly formed. But what is the core of this poxvirus made of, and how do its individual components come together to function?” asks ISTA assistant professor Florian Schur, corresponding author of the study.

Schull and his team have now focused on the missing link: a protein called A10. Interestingly, A10 is common to all clinically relevant poxviruses. Furthermore, the researchers discovered that A10 functions as one of the major components of the poxvirus core. This knowledge could inform future research into therapies targeting the core of poxviruses.

“The most advanced cryo-EM technology currently available”

The viral core is one factor common to all infectious poxvirus types.

Previous experiments in virology, biochemistry, and genetics suggested several possible poxvirus core proteins, but no ly derived structures were available. ”

Julia Datler, ISTA PhD student and one of the study's co-lead authors

So the team started by computationally predicting models of key core protein candidates using the now famous AI-based molecular modeling tool AlphaFold. In parallel, Datler drew on his background in virology and his group's main expertise in cryogenic electron microscopy, or cryo-EM, to establish the biochemical and structural basis for the project. I was there. “We have integrated many of the most advanced cryo-EM techniques currently available with his AlphaFold molecular modeling. This allows us to explore the core of the poxvirus, a 'safe' or 'bioreactor' located inside the virus. For the first time, a detailed picture of the “It breaks down the viral genome and releases it into the infected cell,” Schull said. “It was a bit of a gamble, but in the end we were able to find the right combination of techniques to investigate this complex question,” said postdoctoral fellow Jesse Hansen, co-lead author of the study. speaks. Critical to the project.

Overall 3D view of poxvirus

ISTA researchers examined “live” mature vaccinia virus particles and purified poxvirus cores from literally every angle. “We combined 'classical' single-particle cryo-EM, cryo-electron tomography, subtomogram averaging, and AlphaFold analysis to get a complete picture of the poxvirus nucleus,” Datler said. talk. Using cryo-electron tomography, researchers can reconstruct a 3D volume of a biological sample as large as an entire virus by gradually tilting the sample while acquiring images. “It's like doing a CT scan for a virus,” Hansen said. “Through cryo-electron tomography, which is our laboratory's 'specialty', we were able to obtain nanometer-level resolution of the entire virus, its core, and its interior,” Schurr says. Additionally, the researchers were able to puzzle-fit his AlphaFold model to the observed shapes and identify the molecules that make up the core of the poxvirus. Among these, the core protein candidate A10 stands out as one of the major components. “We found that A10 defines a key structural element in the core of poxviruses,” Dutler said. Schuur added, “These findings are an excellent resource for interpreting the fragments of structural and virological data generated over the past several decades.”

The rocky road to uncovering the core of poxviruses

The path to these discoveries was mostly easy. “We had to find our way from the beginning,” Dutler said. Leveraging expertise in biochemistry, virology, and structural biology, Dutler isolated, propagated, and purified samples of his vaccinia virus and established a protocol to purify the core of the complete virus, while simultaneously constructing We have optimized these samples for research purposes. “Structurally, it was very difficult to study the core of these viruses, but fortunately our perseverance and optimism paid off,” Hansen says.

ISTA researchers believe their findings can provide a knowledge platform for future treatments targeting the core of poxviruses. “For example, we could think of drugs that prevent nuclear assembly, or even drugs that break down and release viral DNA during infection. Ultimately, basic virus research like the one being done here will help We can better prepare for possible future virus outbreaks,” Schur concludes.