Humanity has always been plagued by herpes simplex virus type 1 (HSV-1), which causes cold sores and genital herpes and is implicated in long-term neuropathologies. To separate ourselves from this pathogen, we need a better understanding of how it hijacks our cells to make copies of itself. However, getting a good view of cells in their natural state is challenging. Traditional imaging techniques such as transmission electron microscopy and fluorescence microscopy often need samples that have been processed – by fixing, staining, and sectioning – and that can introduce artefacts. In work recently published in PLOS Pathogens, researchers from the University of Cambridge have collaborated with Diamond's B24 beamline to exploit a new 3D imaging technique using Cryo Soft X-ray Tomography (Cryo-SXT). Using soft X-rays to produce 3D images of the virus as it infects cells, they've discovered interesting changes to cell morphology. Their results demonstrate the utility of this technique, which will help us in our fight against our ancient foe.
Estimates suggest that around two-thirds of the global population is infected with herpes simplex virus type 1 (HSV-1), the virus that causes cold sores and genital herpes. After the initial infection, the symptoms normally abate. However, the virus hides in the body in a latent form and can reactivate at a later date.
The modus operandi of any virus is to penetrate the cells of another organism and take them over - turning them into factories that churn out more viral particles. HSV-1 and humans have evolved together for millions of years, so this particular virus is very good at invading and modifying our cells. A better understanding of how the virus works would enable us to develop better treatments and bring us closer to a vaccine that can prevent infection and transmission. We might also become better at re-purposing the virus as a cancer treatment or for gene therapy. An HSV-1 based therapy (T-VEC) was the first licensed anti-cancer virus, against melanoma, and it's also being developed as a gene delivery vector.
Segmentation of mitochondria reveals the effect of HSV-1 infection on mitochondrial morphology
CryoSXT data was collected from uninfected U2OS cells and U2OS cells infected for 9 hours with the timestamp HSV-1 virus at an MOI of 1. Mitochondria were segmented and colour-coded using Contour  and appear elongated and branched in cells at late stages of infection. Fields of view are 9.46×9.46 μm.
Video reused from DOI: 10.1371/journal.ppat.1010629 under the CC BY 2.0 license.
HSV-1 is a large, enveloped DNA virus. It interacts with the nucleus and organelles - the machinery inside our cells - to produce virus particles in a complex multi-step process. Researchers commonly use transmission electron microscopy and fluorescence microscopy to study viruses like HSV-1, but these produce images that are only in 2D or have limited resolution. High-resolution 3D images would greatly improve our understanding of the changes the virus makes to the cell's components. Transmission electron microscopy and fluorescence microscopy imaging techniques can also require time-consuming sample processing - such as sectioning, staining and fixing - that can themselves cause changes to the cell.
Researchers from the University of Cambridge have been working closely with Diamond's B24 beamline to develop a new technique for 3D imaging of virus-infected cells. The first author on this paper, Kamal Nahas, is a joint PhD student with Diamond and the University of Cambridge.
The Cambridge team works with a modified version of HSV-1, engineered to fluoresce at different stages of the infection process, referred to as a "timestamp" reporter virus1. That means they can capture a snapshot of what the cell looks like at different times in the infection and replication process. The cells are then flash frozen in liquid ethane, which preserves them in a near-native state.
B24 is a dedicated biology beamline with state-of-the-art equipment for Cryo Soft X-ray Tomography (Cryo-SXT), a technique ideally suited for high-resolution 3D imaging of cell structure. As soft X-rays pass through the sample cell, they are preferentially absorbed by carbon-rich biological structures. Gradually changing the angle at which the X-rays pass through the cell creates a series of images with structural information captured at different angles. These images are then reconstructed into tomograms - like a medical CT scan, but on a much smaller scale - to produce a 3D image volume.
For Dr Colin Crump, from the University of Cambridge, these experiments were his first experience of working at a synchrotron. He said
It's a very different way of doing science to university-based research. Diamond is such a large and well-organised facility - it really is Big Science! I found working with the beamline staff a really valuable experience, seeing the technology in action, and finding out how it works."
These experiments were proof of concept, demonstrating that high-resolution 3D images of cellular compartments within infected cells can be captured in a near-native state using Cryo-SXT.
Mitochondria are often referred to as the "powerhouses" of the cell, and the results showed marked changes during infection, with the mitochondria forming a network across the cell. A more short-lived effect is seen in the distribution of lipid droplets in the cell, which temporarily change their size during infection.
As Dr Stephen Graham from the University of Cambridge explains, one of the benefits of this technique is that it can quickly capture large amounts of data. He said:
With Cryo-SXT, sample preparation and data acquisition are both very quick. We can collect a lot of tomograms, which allows robust numerical analysis of the data. Using this technique, we can not only see the individual virus particles, but we can also see how infection changes the morphology of the cells it infects. These early studies show us which organelles to focus on as we look deeper into how this virus works.
To find out more about the B24 beamline or discuss potential applications, please contact Principal Beamline Scientist Maria Harkiolaki: email@example.com.
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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