Cryo-tomography in cell biology research
According to the Center for Disease Control (CDC), polio was once one of the most feared diseases in the United States, causing more than 15,000 cases of paralysis per year in the early 1950s. But in 1955, people across the country rejoiced at the announcement of an approved poliovirus vaccine, and by 1979 the CDC said that because of widespread vaccination, polio had been eliminated from the United States. Yet, poliovirus has remained an endemic in countries like Pakistan and Afghanistan, and the United States hasn’t been without transmission over the past 40 years, albeit extremely low. Travel still poses a threat, especially in areas with lower rates of vaccination. Unfortunately, just this summer, the virus was detected in New York City wastewater.
We recently had the opportunity to share a virtual platform with Dr. Selma Dahmane, a Marie Sklodowska-Curie postdoctoral fellow at Umea University, to discuss her lab’s cryo-electron tomography (cryo-ET) cell biology research on enteroviruses—the genus of viruses in which polio falls.
Here, we’ll give a brief synopsis of our discussion on enterovirus membrane-assisted assembly and release detailed using cryo-ET. This work demonstrates the power of cryo tomography in cell biology research, and we encourage you to watch the full on-demand webinar for an in-depth look at her results.
Cryo-electron tomography of enteroviruses
For those who are not as familiar with cryo-ET, it is a cryogenic imaging method in which frozen hydrated samples like virus particles or thin slices of cells or tissues are imaged to get the 2D projections from which 3D structures of biological molecules are obtained computationally.
Cryo-ET is an extremely powerful cryo-electron microscopy (cryo-EM) technique that allows structure determination in situ. It enables researchers to determine the components within their native environments rather than using isolated/purified samples, as is commonly done for single particle cryo-EM.
Dr. Selma Dahmane’s work focuses on enteroviruses: “non-enveloped, positive-sense single strand RNA viruses that are extremely small and very contagious, causing diseases ranging from the common cold to heart inflammation and polio,” she shared while setting the stage for our discussion.
Her most recent study narrows in on poliovirus and more specifically the assembly, replication, and release of poliovirus infected cells.
Cell biology research into virus assembly
For decades, the precise location and mechanism of enterovirus assembly within the host cell and exit from the cell remained unclear.
Poliovirus consists of a single stranded RNA encapsulated in an icosahedral shaped protein coat. Upon attachment to the host, poliovirus injects its RNA into the cytosol and hijacks the host translation machinery to make several key proteins, including coat proteins and RNA-dependent RNA polymerase for making new copies of viral genome. Somehow, the proteins and RNA molecules come in close proximity and are packaged into new viral particles ready to leave the cell and infect other neighboring cells.
Dr. Dahmane et al. observed that virion assembly and RNA-loading occur while tethered to replication membranes and a solid selectivity to release only properly loaded virions via autophagy.
What are the roles of autophagy in virus replication and release?
Dr. Dahmane found a complex interplay between autophagy and viral replication by pharmacologically interfering with the assembly and egress pathway.
“We tried to find factors that support virus assembly,” said Dr. Dahmane. “In this case, we used a drug that inhibits VPS34—a lipid protein kinase that is very important in autophagy activation as it helps the maturation and formation of autophagy membranes. What we saw was very surprising: the virus is still able to activate autophagy” even when the protein was inhibited.
Next, they used an inhibitor of ULK1/ULK2, two protein kinases which are the initiator of canonical autophagy.
“In this case, we see that the virus goes completely crazy,” said Dr. Dahmane. “It forms this large area in the cytoplasm… where more than 600 particles stack on top of each other in this really beautiful crystal-like arrangement. This was true for half of the cells treated with this drug compared to untreated cells.”
According to Dr. Dahmane, these results show that unlike VPS34, which is a required host factor for complete poliovirus assembly, inhibiting ULK shifts the balance toward a cellular environment much more favorable for virus assembly and release.
Why advances in cryo-ET technology matter for virology research
“It is beautiful to see how dedicated scientists like Selma Dahmane and her team leverage the recent hardware, software, and methodologic advances cryo-ET has seen,” said Kristian Wadel, product marketing manager at Thermo Fisher Scientific and co-host from our recent webinar. “They are demystifying longstanding questions in host-pathogen interactions, and the wealth of, at times surprising, information gained from their cryo-ET data could provide the basis for future translational research into tackling these diseases.”
Thanks to cryo-ET, continuous innovation in technology and workflows, and dedicated scientists including and especially Dr. Selma Dahmane and her team, we’re demystifying what have long been mysteries and providing a framework for drug discovery and the management of such longstanding diseases.
UPDATE as of fall 2022: New cryo-EM research out of Umea University demonstrates for the first time how the poliovirus forms and takes over human cells. Learn more >>
This blog was written in collaboration with Thermo Fisher Scientific Product Marketing Manager Kristian Wadel and with source content from our Spring 2022 webinar with Dr. Dahmane.