Recently, the re-emergence and outbreak of Mpox have once again brought poxviruses to the forefront as a public health threat, highlighting a critical gap in knowledge. Now, researchers from the Institute of Science and Technology Austria (ISTA) have unveiled the secrets of the core architecture of poxviruses by combining various cryo-electron microscopy techniques with molecular modeling. The results, published in Nature Structural & Molecular Biology, could facilitate future research on therapeutics targeting the core of the poxvirus.
The Variola virus, the most notorious poxvirus and one of the deadliest viruses to ever affect humans, caused devastating damage through the emergence of smallpox until it was eradicated in 1980. Smallpox’s eradication was achieved through an extensive vaccination campaign using another poxvirus, the aptly named Vaccinia virus. The re-emergence and outbreak of the Mpox virus in 2022–2023 served as a reminder that viruses find ways to resurface as a public health threat. Importantly, this brought to light the fundamental questions about poxviruses that have remained unanswered until today.
One such fundamental question was at the core of the matter: „We know that the viral core of poxviruses must be properly formed for them to be infectious. But what is the composition of this poxviral core, and how do its individual components come together and function?“ asked ISTA Assistant Professor Florian Schur, the corresponding author of the study.
Schur and his team have now pinpointed the missing link: a protein called A10. Interestingly, A10 is common to all clinically relevant poxviruses. Furthermore, the researchers found that A10 serves as one of the main building blocks of the poxvirus core. This knowledge could be crucial for future research targeting the core of the poxvirus with therapeutics.
„The most advanced cryo-EM techniques available today“
The viral core is one of the factors common to all infectious forms of poxvirus.
Previous experiments in virology, biochemistry, and genetics all pointed to several core protein candidates for poxviruses, but there were no experimentally derived structures available.“
Julia Datler, ISTA PhD student, one of the co-first authors of the study
Therefore, the team began by computationally predicting models of the main core protein candidates using the now-famous AI-based molecular modeling tool, AlphaFold. In parallel, Datler laid the biochemical and structural groundwork of the project, drawing on her background in virology and the Schur group’s main expertise: cryogenic electron microscopy, or cryo-EM. „We integrated many of the most advanced cryo-EM techniques available today into AlphaFold’s molecular modeling. This finally gave us a detailed overview of the poxvirus core – the ’safe‘ or ‚bioreactor‘ inside the virus that encloses its viral genome and releases it in infected cells,“ says Schur. „It was a bit of a gamble, but we eventually found the right mix of techniques to investigate this complex question,“ says postdoc Jesse Hansen, co-first author of the study, knowledgeable in various structural biology techniques and image processing methods crucial to the project.
A global 3D view of the poxvirus
The ISTA researchers examined „live“ mature virions of the Vaccinia virus and purified poxvirus cores from every possible angle. „We combined ‚classic‘ single-particle cryo-EM, cryo-electron tomography, subtomogram averaging, and AlphaFold analysis to gain a comprehensive overview of the poxvirus core,“ says Datler. Cryo-electron tomography allows researchers to reconstruct 3D volumes of a biological sample the size of an entire virus by capturing images while tilting the sample stepwise. „It’s like taking a CT scan of the virus,“ says Hansen. „Our lab’s ’specialty‘ cryo-electron tomography allowed us to achieve nanometer-scale resolutions of the entire virus, its core, and its interior,“ says Schur. Furthermore, the researchers were able to fit the AlphaFold models like a puzzle into the observed shapes and identify molecules that make up the poxvirus core. Among these, the core protein candidate A10 emerged as one of the major components. „We found that A10 defines important structural elements of poxvirus cores,“ says Datler. Schur adds, „These results are a great source for interpreting structural and virological data collected over the past few decades.“
A challenging path to discovering poxvirus cores
The path to these findings was anything but straightforward. „We had to find our own way from the beginning,“ says Datler. Leveraging her expertise in biochemistry, virology, and structural biology, Datler isolated, amplified, and purified samples of the Vaccinia virus while optimizing protocols for purifying whole virus cores, preparing these samples for structural studies. „Structurally, it was extremely challenging to examine these virus cores. But thankfully, our persistence and optimism paid off,“ says Hansen.
The ISTA researchers are confident that their findings could provide a knowledge platform for future therapeutics targeting poxvirus cores. „One could think, for example, of drugs that prevent the assembly of the core, or even dismantle and release the viral DNA during an infection. Ultimately, the fundamental virus research conducted here allows us to be better prepared for potential future virus outbreaks,“ Schur concludes.
Institute of Science and Technology Austria
Datler, J., et al. (2024). Multimodal cryo-EM reveals trimers of protein A10 forming the palisade layer in poxvirus cores. Nature Structural & Molecular Biology. doi.org/10.1038/s41594-023-01201-6.