Tracking Viral Pathogens With Cryo-Electron Tomography
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The COVID-19 pandemic has shown the entire world just how critical virus research can be. Years of work investigating SARS and other coronaviruses proved to be the invaluable foundation that allowed researchers to determine the structure of the SARS-CoV-2 spike protein and quickly develop vaccines that have saved countless lives.
In many ways, the field of virology is experiencing a renaissance, with numerous fundamental techniques having rapidly evolved over the past ten years. A significant contributor to this, other than the 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 hazardous virus species in biosafety laboratories.
Early virus analysis
Historically, cell and virus samples have been preserved through chemical fixation or crystallization. Where whole virus analysis was impractical or impossible, the sample was broken down into key proteins for in-depth structural analysis. While these techniques have provided valuable insights into the structure, behavior and function of viruses, they are divorced from the native context in which the virus acts. And while this was acceptable for isolated, mature virus particles, it left us with gaps in our understanding of virus-cell interactions, particularly once the virus has been internalized into the cell.
The reprogramming of cellular machinery for virus replication is an intricate and highly transient process. Even if intracellular imaging is possible with chemical fixation, it is challenging to capture these fleeting events, particularly with sufficient detail to deconvolute what is happening.
In cryogenic sample preparation, aqueous specimens are rapidly frozen in order to preserve them in a suspension of amorphous (vitreous) ice. Unlike crystalline ice, which expands as it forms, vitreous ice molecules generally remain in their liquid-phase positions, preserving the surrounding molecular structure of the sample. This essentially takes a “snapshot” of the specimen in a near-native state.
For virus analysis, cryo-preservation has been instrumental in the development of a range of electron microscopy techniques, broadly described as cryo-EM. The most common of these is single particle analysis, whose developers were awarded the 2017 Nobel Prize in Chemistry. Single particle analysis uses averaging to produce 3D molecular structures from hundreds of 2D transmission electron microscopy (TEM) images of different copies of the sample, oriented randomly within the ice. It is broadly considered complimentary to X-ray crystallography, as it can readily generate structures for protein and complexes that are challenging to crystallize.
While single particle analysis has filled a vital niche in the world of structural analysis, it is still performed on fragmented systems isolated from their physiological context. Luckily, this need for in situ observation can be accommodated by another complementary cryo-EM technique, cryo-electron tomography (cryo-ET).
In electron tomography, a thin sample is progressively 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 specimen. Whereas single particle analysis relies on many copies of the same target molecule to capture its various orientations, tomography must utilize a precision stage that reliably tilts a single sample at discreet increments instead.
Notably, this means that cryo-ET can be performed on a segment of a whole cryogenically preserved cell. While early cryo-ET experiments were limited to inherently thin samples (or thin regions of thicker samples), the advent of various cryo-thinning techniques allowed “windows” to be cut directly into frozen cells to peer into their intracellular machinery.
For virology, this presents a unique opportunity to capture elusive viruses red-handed after they enter the cellular cytoplasm. In particular, cryo-ET has been used to study the behavior of coronavirus replication within cells. In a collaboration led by the Leiden University Medical Center, researchers were able to visualize the virus-induced formation of double-membrane vesicles that are used to shelter viral RNA replication. This included pore structures on the surface of the vesicles that are likely responsible for RNA transport, marking them as prospective drug targets.
Cryo-electron tomography revealing novel features of poliovirus replication.
Tomogram 3D visualization of a poliovirus-infected HeLa cell. Empty capsids: pink, RNA-filled capsids: red, protein complexes tethering 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, is naturally of great interest to researchers due to the clear and present danger that viral pathogens pose to global human health.
However, biosafety laboratories have defined discrete levels of hazard that various virus samples present. Work on pathogens that pose a risk for disease requires a higher biosafety containment facility unless 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 set levels of decontamination). As a result, high-end cryo-EM instruments are not yet commonly found in these higher biosafety level facilities.
Nevertheless, some labs have shown promising results in their exploration of pathogenic species. For example, the Carlson research group at Umeå University has used cryo-ET to study the poliovirus as a model enterovirus. In their work, they were able to capture much of the replication cycle of the virus within its host cell. They observed the ways in which 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 instance, the 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-ET, opens exciting avenues, not just in therapeutic development, but in our fundamental understanding of host-pathogen interactions.
“Imagine following a virus from its contact with a host, down to cell binding, all the way through replication and release. Only a decade ago this may have been considered a fanciful dream, but we are steadily approaching a more complete understanding of viral behavior.” – Selma Dahmane, Marie Skłodowska-Curie Postdoctoral Fellow, Umeå University
Cryo-ET opens the door to new insights into both "known" viruses whose structures might have already been determined, as well as the plethora of viruses that have yet to be studied. Many questions about their life cycles and interactions with host cells remain unanswered. Next to the determination of virus structure itself, cryo-EM, including cryo-ET, enables a close look at the virus life cycle within a host cell, e.g., entry, replication, assembly and release. There is also much to learn about how cells respond 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 poised to enter a new era of discovery with near-native-state observations enabled by cryo-preservation and analysis. Cryo-electron tomography, in particular, can provide insights that bridge our current knowledge of molecular structure and cellular interaction. Installing cryo-electron microscopes in higher level biosafety facilities would enable research into a broader range of virus species. With a full, multi-scale understanding of virus action we can design better, more targeted and efficient treatments. Accessing this information will not be without its challenges, but the joint effort of experimentalists and instrument developers will surely drive us onward to this exciting future.
About the authors
Alex Ilitchev is lead scientific editor, Materials and Structural Analysis, at Thermo Fisher Scientific.
Kristian Wadel is product marketing manager, Materials and Structural Analysis, at Thermo Fisher Scientific.