Written by Nóra Lehotai, Lars-Anders Carlson and Selma Dahmane
How a virus hijacks a host cell to make new virus particles is a key question in the prevention and treatment of virus-caused diseases. Dr Selma Dahmane and Associate Professor Lars-Anders Carlson at Umeå University are the first and last authors of a freshly published paper in Nature Communications that sheds new light on this process. They show how new poliovirus particles are formed inside the infected cell thanks to the latest advancements in cryo-electron tomography. Poliovirus belongs to the group of enteroviruses, a big group of RNA viruses that also includes for example the common cold-causing rhinoviruses, or enterovirus D68 that can cause severe respiratory disease and paralysis in children. Polio itself has caused disease and death in humans for millennia, and the poliovirus was not long ago hoped to be eradicated. But the recent detection of poliovirus in London sewage, and a case of poliomyelitis in New York, has highlighted the difficulty in eliminating poliovirus even in developed countries.
Picture: Selma Dahmane and Lars-Anders Carlson analyzing pictures from an electron microscope at UCEM, Umeå University. Credit: Nóra Lehotai.
“This was the first cryo-EM project to be started in my new group, and one of the first cryo-electron tomography projects to start with the newly installed microscopes at Umeå Centre for Electron Microscopy (UCEM) in 2017”, starts Lars-Anders Carlson, an associate professor at Umeå University, and MIMS Investigator. “MIMS and Umeå’s wider infection biology community had made great efforts to establish the new cryo-EM facility, and the microscopes still smelled new when Selma joined my group and we started this project. Working at UCEM is very special: the facility staff take care of all the maintenance so when you go there, everything is ready to be used and you can just focus on your research question. The facility staff also guide the users by sharing their knowledge and allowing time for discussions. This means that frequent users eventually can become independent in using even the most advanced cryo-EM instrumentation.”
“Since fluorescent or chemical labelling disrupts the replication of the virus inside the cell, we needed cryo conditions to be able to observe this process. I learned the whole cryo-EM workflow from Lars and the UCEM staff”, says Selma Dahmane. “We were the first ones to do cryo-tomography with ion beam milling to slice the cells and image their internal structures using the Titan Krios cryo-electron microscope at UCEM. It took me almost two years to perfect the entire workflow including all the instruments and sample preparation. One especially crucial step is the plunge freezing process where I need to vitrify the infected cells at different time points to study the progression of virus replication. It is a step where if something goes wrong, you can lose all your samples in one day. But if it goes well, you are eventually sitting at the microscope and you see structures inside a cell which no one has seen before, this is what I enjoy the most!”
And what did they see? They saw the step-by-step replication, assembly, and release of the poliovirus inside the cell for the very first time.
Lars-Anders continues: “Since we did cryo-electron tomography, we were able to see a lot of new details about what is going on inside poliovirus-infected cells. The mechanism is quite surprising.”
To understand why it is surprising, we need to tell the tale of lipid membranes and viruses: if a virus is surrounded by a lipid membrane, it is called an enveloped virus, while without such membrane, it is called a non-enveloped virus. Enteroviruses were known to be non-enveloped viruses.
“The twist in the story came when we saw that new virus capsids are in fact formed on membranes of the host cells”, Selma and Lars-Anders continue. “We see virus assembly intermediates, half capsids, on these membranes. The virus infection transforms the cell’s membranes into virus factories.”
Picture: Selma Dahmane using one of the electron microscopes at UCEM, Umeå University. Credit: Nóra Lehotai.
They also explain that the membranes where new viruses are made are associated with autophagy. Autophagy is a naturally occurring process to remove components from the cell by degrading them. It can be used as a means to survive starvation, or as a defense mechanism to eliminate harmful components such as viruses.
Lars-Anders continues: “We saw how enteroviruses hijack autophagy to assist their replication. Combining our imaging approach with chemical inhibition of autophagy proteins, we identified one autophagy factor that is necessary for new virions to assemble completely, whereas another autophagy protein actually puts a break on the virion production. It’s thus possible that future generations of autophagy-targeting drugs could be used as antivirals against enteroviruses.”
We learned, so far, that a non-enveloped virus not only uses membranes of the host cell to make new virus particles during infection, but also that these membranes are related to autophagy, and that the poliovirus selectively uses or inactivates autophagy components to optimise its replication. The last piece missing is how the newly formed viruses get released so they can start their infectious journey.
Selma and Lars tell that this is another twist in the storyline.
“The virus uses the autophagy pathway also for this, to release itself. Autophagy can induce vesicle formation which means that the unwanted component will be encapsulated, then digested to get rid of it. Another form of autophagy, called secretory autophagy, instead leads to release of the formed vesicles from the cell. It was known that enteroviruses use secretory autophagy for their release. Interestingly, Lars-Anders says, the cryo-electron tomograms are so detailed that we can distinguish when a virus capsid is filled with a genome or not. While empty capsids are always present in the cell, it came as a total surprise when we saw that the virus manages to select only genome-containing (i.e. infectious) capsids to be sent into vesicles for secretion. This is a new mechanism that the virus uses to increase its infectivity.”
This big project, in which Selma combined skills in virology, structural biology and microscopy, is now concluded in a publication, but it is also just at the beginning.
Lars-Anders concludes: “The new cryo-EM methods that Selma used allowed us to see many unexpected processes in enterovirus-infected cells for the first time. Some of them we could explore in depth in this paper, but for several others we have merely scratched the surface. Much interesting work remains.”
Original scientific article is available here: https://www.nature.com/articles/s41467-022-33483-7
Find the official press release by Umeå University here: https://www.umu.se/en/news/research-shows-how-poliovirus-takes-over-cells-from-within_11671585/
Read a blog post by Thermo Fischer Scientific about the discovery with fantastic videos included here: https://bit.ly/3EMYWdL
Webpage of Lars-Anders Carlson (UmU): https://www.umu.se/en/staff/lars-anders-carlson/
Webpage of Lars-Anders Carlson (MIMS): https://www.mims.umu.se/groups/research-groups-lars-anders-carlson.html
Website of the Carlson lab: http://www.carlsonlab.se/
UCEM webpage: https://www.umu.se/en/research/infrastructure/umea-centre-for-electron-microscopy-ucem/