For decades, when scientists have wanted to study cell architecture in granular detail and to study how viruses assemble inside the cell, they've turned to transmission electron microscopy (TEM), a high-resolution imaging technique. However, TEM studies are limited to 2D sections and offer no information regarding the 3D geometry of different features. They also look at thin slivers rather than a whole cell, and slow throughput means TEM studies are conducted on a small number of samples. On Diamond’s B24 beamline, researchers can perform correlative fluorescence microscopy and X-ray tomography, imaging proteins using structured illumination microscopy under cryogenic conditions (cryoSIM) and capturing cellular ultrastructure from the same cells using cryo-soft-X-ray tomography (cryoSXT). In a preprint recently published on bioRxiv, an international team of researchers used this correlative light X-ray tomography (CLXT) approach to study the roles of nine genes in virus assembly in herpes simplex virus-1 (HSV-1). This multi-modal imaging strategy allowed a 3D study of viral assembly, highlighting the contributions that key HSV-1 proteins make to virus assembly and underscoring the power of correlative fluorescence and X-ray tomography cryo-imaging for studies of this type.
The WHO estimates that, globally, 3.7 billion people under the age of 50 are infected with HSV-1, which can cause oral and genital herpes. Most people have mild symptoms, or none at all. However, HSV-1 infection occasionally leads to more severe complications such as encephalitis (brain infection) or keratitis (eye infection), and although treatments are available, it is not curable. A more thorough understanding of the virus could allow us to develop better treatments, or a vaccine that could prevent infection.
Previous studies have shown that a number of viral genes are involved in assembly of HSV-1, but haven’t fully characterised their relative importance and functions. In this study, researchers conducted a comparative analysis of nine HSV-1 mutants lacking specific structural proteins using CLXT, revealing the relative roles of each viral protein in virus assembly.
Lead author Dr Kamal Nahas, a beamline scientist on B24, says:
There are many ways in which TEM is a little bit better than X-ray tomography. TEM gives you a better resolution and you can see more detail. On the other hand, using cryoSXT allows you to capture more examples of different things because you're imaging the entire depth of the cell, and to see the 3D morphology of features. Those extra details really helped us clarify certain aspects of the virus's life cycle in the cell, such as how it undergoes envelopment.
The key to this correlative technique is that both imaging techniques use the same grid of prepared samples. The first stage is use cryoSIM to identify fluorescent markers, essentially giving researchers a map that shows the location of features they want to explore further. In this case, it allowed them to discriminate between unenveloped and enveloped particles in the cell.
The sample grid can then be moved over to the X-ray beamline for cryoSXT on those exact same positions. The two complementary data sets then correlate together.
Dr Nahas says:
Using cryoSIM allowed me to identify the virus capsid, the virus envelope protein, and then using those markers, I could track and trace where the virus was in the X-ray data. I used this experimental protocol to explore various different virus mutants to try to assess the relative role and importance of different viral genes in the assembly pathway. I could see some huge defects when certain genes were removed, and much more minor differences for others. That’s great for weighing up the relative importance of each of the genes, which is something that TEM has struggled with a little bit, because the lower throughput means you spend less time looking at multiple different conditions. Here we were able to look at nine different mutants in one go.
The biggest finding from the study was an insight into the mechanism behind how the capsid becomes enveloped in the cytoplasm. In 2D, TEM studies show the capsid being wrapped by what looks like a C-shaped membrane. That could mean that a tubular membrane wraps around the capsid and fuses. However, this 2D image is actually compatible with more than one 3D interpretation. For example, it could also be the cross section of a capsid budding into a spherical membrane.
Dr Nahas concludes:
With X-ray tomography, we were able to discern which of those two models was correct by imaging it in 3D. We were able to see that the capsid budding into a spherical membrane is actually the true model. And that was true for every example that we found, so it wasn't a case of it being a mix between the two. These are important findings from the perspective of herpes virus research. More generally, scientists have relied on EM for decades to do ultra structural imaging. That's given them one view on architectural detail in the cell, but there are certain blind spots with that technique. Our work here has demonstrated the potential of CLXT to give a 3D perspective of virus cells and provide crucial new information.
To find out more about the B24 beamline or discuss potential applications, please contact Deputy Director of Life Sciences Martin Walsh, [email protected]
Nahas KL et al. Applying 3D correlative structured illumination microscopy and X-ray tomography to characterise herpes simplex virus-1 morphogenesis. bioRxiv (2024): 2024-03. DOI: 10.1101/2024.03.13.584906.
Nahas KL et al. Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection. PLoS pathogens 18.7 (2022). DOI: 10.1371/journal.ppat.1010629.
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