Cryo-ET to understand how enteroviruses replicate

Enteroviruses cause a range of diverse diseases in humans, like polio or viral myocarditis. While there is a vaccine available for polio, the replication mechanisms for enteroviruses are not yet (fully) understood. Studying how enteroviruses, such as the polio virus, are formed inside human cells and how the assembled virus particles ‘escape’ and are transmitted to surrounding cells are important steps in the infection process, and support the development of vaccines against enteroviruses. That is why Marie Sorin and Bina Kumari Singh of the Carlson Lab at University of Umeå in Sweden are zooming into the processes by means of high-resolution EM techniques. They brought their infectious disease project to the EMBL Imaging Centre, part of Euro-BioImaging’s EMBL Node, with funding support from ISIDORe.

“In October 2022, scientists from the Carlson Lab published a paper in Nature Communications entitled 'Membrane-assisted assembly and selective secretory autophagy of enteroviruses,'” explains Marie Sorin, a postdoctoral researcher in the Carlson Lab. “This paper provides an excellent overview of viral assembly, but it also raises many questions. For example, in this study, our colleagues noticed a class of autophagosomes containing bundles of protein filaments, but they were unable to  identify the protein. I want to use new techniques in cryo-ET to determine the structure and composition of these filaments to understand if they are linked to the virus’s escape mechanism. We have developed a sample preparation method that takes advantage of the specific timepoints identified by our colleagues. We will create a series of pictures with Electron Microscopy, or tomograms, to understand how the proteins interact within the polio-infected cells. This approach is relevant for cells infected with other enteroviruses and also other viruses, like tick-borne encephalitis virus, which we study in our lab.”

To carry out this approach, Marie Sorrin and her colleague Bina Kumari Singh, a fellow postdoctoral researcher in the Carlson Lab, applied to use the EMBL Imaging Centre. “We became interested in the EMBL Imaging Centre because we were looking for a facility that accepted external users, had a BSL-2 working environment, state-of-the art cryo-electron microscopy systems, and a new generation FIB-milling machine with automation. When we heard about the ISIDORe funding opportunity, it really sealed the deal,” explained Bina.

ISIDORe is a large-scale EU-funded project providing free transnational access to a portfolio of cutting-edge research services for studies on epidemic-prone disease. As part of the ISIDORe project via Euro-BioImaging, the EMBL Imaging Centre offers open access to a synergistic portfolio of imaging services including cryo-EM, super-resolution and intravital microscopy.

“We wanted to use cryo-electron tomography in our project because this method is capable of visualising the macromolecular architecture of the interior of cells at (sub)nanometre resolution. That is really unique,” explains Marie. “We would perform subtomogram averaging and hope to see the autophagy pathway. The idea behind this study is to look at the filaments and try to identify what they are and understand their role in infection. Having more, high-quality Cryo-ET data should make this possible,” she continues.

In their PhD projects, Marie and Bina mostly use x-ray crystallography. But they want to know more about cryo-ET and were attracted to the full support they could get at EMBL. “That’s why it’s really interesting to be here. It’s an opportunity to get to know other facilities in the cryo-ET world and learn from the experts,” says Bina.

But conducting an infectious-disease related experiment far from home is not trivial. “Our cells are considered to be contagious,” explains Marie. “So we must work in BSL-2 conditions. We have these conditions in Sweden, and also at the EMBL IC.”

The sample preparation process they developed is very long, so Marie got started at her home institute. “First, we infect the cells,” says Marie. “Then we wait about 7 hours. At that time, we can see the assembly and the start of the virus release. That’s when we freeze our cells on the grid.” 

“I prepared lots of samples,” says Marie. “As I had never shipped them before, I was afraid we would lose some. But we worked with a shipping company that is specialized in frozen samples. Our samples arrived one day later. Everything was perfectly intact, they were still frozen.” 

As soon as Marie and Bina arrived at EMBL, they started the FIB-milling, a crucial phase in the sample preparation process. They were curious to try out the EMBL Imaging Centre’s state-of-art FIB-milling system, Aquilos2, because of its ability to automate the preparation of the thin, electron-transparent lamellae they needed for high-resolution cryo-electron tomography.

“The idea is to make the cells thin enough that EM can look at what’s inside,” explains Marie. “The lamellae are only 150-200 nanometers thick.” 

And the new machine lived up to their expectations. “This system allowed us to prepare the lamellae so much faster than we would be able to do at home,” Bina declared enthusiastically. 

“The automation we are seeing here is amazing,” says Marie. “This is something we would like to adopt in the future in our facility.”

Once the cryo-lamellae were prepared, data was acquired by the Krios transmission electron microscope (TEM), which has the latest technology for high-end single-particle and tomography data acquisition. It is configured for high-throughput automated single particle and tomography data acquisition, so they can record more and higher quality cryo-ET data than in the previous study.

“The imaging experience was very similar to what we are used to in Sweden, where we have a partnership with the Umeå Centre for Electron Microscopy (also part of Euro-BioImaging). The whole imaging pipeline is the same as what we have in Umeå, including the software for data analysis,” explains Marie. “So we felt very much at home.” The last step was to transfer the data generated to the file server in Sweden for analysis. “The whole process was very fast and super practical,” says Marie. Altogether, the project was completed in four days.

The next phase of the project will be more time-consuming, as Marie and the team work on the improved subtomogram averaging to identify the structure. Meticulous work, “But the images are very promising,” she says.

Enterovirus replication is one of the key research topics in the Carlson Lab, and understanding this protein filament bundle has become one of the more pressing questions they are working on. 

“We really want to validate this approach and identify the protein,” concludes Marie. “Previous research indicates that all enteroviruses, since they are part of the same family, will act similarly during replication. So these protein filaments may be an interesting candidate for broad-spectrum antivirals.”

If they are successful, the plan is to carry out the same tests on other viruses. “The idea is to apply the method we are developing now to viruses like tick-borne encephalitis virus, the virus I’m studying,” says Bina. With the new skills they acquired at EMBL IC thanks to ISIDORe project funding, these researchers may shed light on the unknown phase of viral assembly and replication.