Tracking viral pathogens with cryo-electron tomography

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The COVID-19 pandemic has shown the world how critical virus research can be. Years of work on SARS and other coronaviruses proved to be the invaluable foundation that allowed researchers to determine the structure of SARS-CoV-2’s spike protein and rapidly develop vaccines that saved countless lives.

In many ways, the field of virology is experiencing a renaissance, with many fundamental techniques rapidly evolving over the past decade. A significant contributor to this, other than growth catalyzed by a global pandemic, is the standardization and commercialization of cryogenic sample preservation and analysis. In this article, we will discuss the integration of cryo-electron microscopy (cryo-EM) into virology research and the unique challenges posed by working with dangerous viral species in biosafety laboratories.

Early virus analysis

Historically, cell and virus samples have been preserved by chemical fixation or crystallization. Where analysis of the whole virus was impractical or impossible, the sample was broken down into key proteins for in-depth structural analysis. Although these techniques have provided valuable insight into the structure, behavior and function of viruses, they are divorced from the native context in which the virus operates. And while this is acceptable for isolated, mature virus particles, it has left us with gaps in our understanding of virus-cell interactions, especially once the virus has been internalized into the cell.

Reprogramming the cellular machinery for viral replication is a complex and highly transient process. Even though intracellular imaging is possible with chemical fixation, it is difficult to capture these fleeting events, especially in enough detail to deconvolute what is happening.

Electron cryo-microscopy

In cryogenic sample preparation, aqueous samples are rapidly frozen in order to preserve them in a suspension of amorphous (glassy) ice. Unlike crystalline ice, which expands as it forms, vitreous ice molecules generally remain in their positions in the liquid phase, preserving the surrounding molecular structure of the sample. This essentially takes a “snapshot” of the specimen in a near-native state.

For the analysis of viruses, cryopreservation has been instrumental in the development of a range of electron microscopy techniques, generally described as cryo-EM. The most common of these is single particle analysis, the developers of which were awarded the 2017 Nobel Prize in Chemistry. Single particle analysis uses averaging to produce 3D molecular structures from hundreds of images of 2D transmission electron microscopy (TEM) of different copies of the sample, randomly oriented in ice. It is widely considered complementary to X-ray crystallography, as it can easily generate structures for hard-to-crystallize proteins and complexes.

While single particle analysis has occupied a vital niche in the world of structural analysis, it is still performed on fragmented systems isolated from their physiological context. Fortunately, this need for on the spot observation can be supported by another complementary cryo-EM technique, cryo-electron tomography (cryo-ET).

Electron cryo-tomography

In electron tomography, a thin sample is gradually tilted and imaged inside a TEM. These images represent different cross-sections of the sample, which can be combined into a single high-resolution 3D representation of the sample. Whereas single particle analysis relies on many copies of the same target molecule to capture its different orientations, tomography must use a precision stage that reliably tilts a single sample at discrete increments instead.

Notably, this means that cryo-ET can be performed on a segment of a cryogenically preserved whole cell. While early cryo-ET experiments were limited to inherently thin specimens (or thin regions of thicker specimens), the advent of various cryo-thinning techniques made it possible to cut “windows” directly into frozen cells to scrutinize their intracellular machinery.

For virology, this presents a unique opportunity to capture elusive viruses red-handed after they have entered the cell cytoplasm. In particular, cryo-ET has been used to study the behavior of coronavirus replication in cells. In a collaboration led by Leiden University Medical Center, researchers were able to visualize the virus-induced formation of double-membrane vesicles that are used to harbor viral RNA replication. This included pore structures on the surface of the vesicles that are likely responsible for transporting RNA, marking them as potential drug targets.

Cryo-electron tomography revealing new features of poliovirus replication.

3D visualization by tomography of a HeLa cell infected with poliovirus. Empty capsids: pink, RNA-filled capsids: red, protein complexes attaching capsids to membrane: yellow, intraluminal densities: orange, and dense granules co-packaged with virions: blue. Image courtesy of Selma Dahmane, Umeå University.

Biosafety Laboratories – working with the enemy

Cryo-ET of pathogenic whole viruses and virus-cell interactions are naturally of great interest to researchers because of the clear and present danger that viral pathogens pose to human health worldwide.

However, biosafety labs have defined discrete levels of danger that various virus samples present. Work on pathogens that pose a disease risk requires a higher biosafety containment facility, unless the work is limited to isolated proteins or inactivated samples. To ensure the safety of everyone working in these facilities, researchers must take specific precautions and adhere to special protocols, including distinct instrument requirements (such as defined decontamination levels). As a result, high-end cryo-EM instruments are not yet commonly found in these higher biosafety level facilities.

Nevertheless, some laboratories have shown promising results in their exploration of pathogenic species. For example, the Carlson research group at Umeå University used cryo-ET to study poliovirus as a model enterovirus. In their work, they were able to capture much of the replication cycle of the virus in its host cell. They observed how the virus transformed the intracellular environment to facilitate viral replication. By visualizing these discrete mechanisms, they were also able to see the interplay between cellular defense mechanisms and viral assembly. For example, suppression of natural autophagy factors allowed the virus to continue replicating despite cellular triggers to destroy them. This work, which was only possible thanks to cryo-TE, opens exciting avenues, not only in therapeutic development, but in our fundamental understanding of host-pathogen interactions.

“Imagine following a virus from contact with a host, through cell binding, through replication and release. Only a decade ago this might have been considered a fanciful dream, but we are gradually approaching a fuller understanding of viral behavior. – Selma Dahmane, Marie Skłodowska-Curie Postdoctoral Fellow, University of Umea

Cryo-ET opens the door to new insights into “known” viruses whose structures may have already been determined, as well as the plethora of viruses that have yet to be studied. Many questions about their life cycles and their interactions with host cells remain unanswered. Besides determining the viral structure itself, cryo-EM, including cryo-ET, allows close examination of the life cycle of the virus in a host cell, e.g. entry, replication, l assembly and release. There is also a lot to learn about how cells react and defend themselves against viral infection. Likewise, studying the effects of drugs or drug candidates on virus life cycles and cellular defenses could better prepare us against the next major virus.


Virology is about to enter a new era of discovery with near-native observations made possible by cryopreservation and analysis. Cryo-electron tomography, in particular, can provide information that links our current knowledge of molecular structure and cellular interaction. The installation of cryo-electron microscopes in higher level biosafety facilities would allow research on a wider range of viral species. With a comprehensive, multi-scale understanding of virus action, we can design better, more targeted, and more effective treatments. Accessing this information will not be without its challenges, but the joint effort of experimenters and instrument developers will surely lead us into this exciting future.

About the authors

Alex Ilitchev is scientific editor-in-chief, Materials and Structural Analysis, at Thermo Fisher Scientific.

Kristian Wadel is Product Marketing Manager, Materials and Structural Analysis, at Thermo Fisher Scientific.

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