Diamond Light Source - Annual Review 2022/23

24 25 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 2 / 2 3 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 2 / 2 3 Herpes simplex virusmakes itself at home by rearranging the contents of your cells Technique Development – Life Sciences & Biotech - Pathogens – Health &Wellbeing – Structural biology Herpes simplex virus (HSV)-1 is a highly prevalent human pathogen that causes life-long infections that usually manifest as cold sores or genital herpes. However, HSV-1 infection can cause life-threatening disease (encephalitis), and people with weakened immune systems such as neonates, the elderly and patients on certain immunosuppressive drugs tend to be more at risk. HSV-1 has also been developed as a potential therapeutic; it is already in use as an anti-cancer agent (T-VEC) and is under development as a delivery vector for gene therapy. Given both the biotechnology potential of engineered HSV-1 and the detrimental health effects of infection with the wild virus, it is essential that we understand more about how this virus interacts with infected human cells. Infection of human cells with HSV-1 has been extensively studied using imaging techniques like electron and fluorescence microscopy, but these techniques can generally only image thin sections of the cell or work at low resolution. Additionally, many of these techniques rely upon sample processing that can distort the shape of the cell and its components. Researchers from the University of Cambridge sought to image infected cells that are as close as possible to how they would look in the cellular context. Generally, before imaging cells undergo a series of steps to aid in their imaging and can lead to changes in cell structure that lead to artefacts and generate images that are not representative of their cellular state. Elimination of these states and flash- cooling of cells to liquid nitrogen temperatures preserves the cells structures and provides a way to image cells in a 'near-native' state. This opens up the possibility to understand, in this instance, how the shape and arrangement of cellular compartments change through the course of virus infection. They used the facilities on Diamond’s B24 beamline to flash-freeze infected cells in liquid ethane, cryogenically preserving them in a ‘near-native’ state, and to perform cryo-soft-X-ray tomography (cryo-SXT). The cryo-SXT analysis allowed them to reconstruct 3D images of infected cells at very high resolution. Combining this analysis with a special fluorescent ‘timestamp’ strain of HSV- 1, constructed in Cambridge, allowed them to work out the stage of infection each cell was in when it was frozen. Using B24, they were able to identify individual virus particles within the infected cells and to see how virus infection progressively changes the shape and distribution of mitochondria (the powerhouses of the cell), vesicles (the cell’s internal delivery system) and lipid droplets (both energy stores and signalling stations for the immune system). Importantly, the high throughput imaging afforded by B24 allowed them to define these changes numerically. This study demonstrates the power of cryo-SXT for monitoring virus infection and highlights which organelles to focus on as we study themolecular characteristics of herpesvirus infection. Related publication: 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: e1010629 (2022). DOI: 10.1371/journal. ppat.1010629 Funding acknowledgement: Co-fund DLS - Department of Pathology, University of Cambridge Forschungsgemeinschaft (SCHE 1672/2-1) EPSRC (EP/H018301/1) MRC (MR/K015850/1 and MR/K02292X/1) BBSRC (BB/M021424/1) Sir Henry Dale Fellowship, jointly funded by theWellcome Trust and the Royal Society (098406/Z/12/B) Corresponding authors: Colin M. Crump, University of Cambridge, [email protected] Stephen C. Graham, University of Cambridge, [email protected] Biological Cryo-Imaging Group Beamline B24 Herpes simplex virus infected cell as visualised using cryo-soft-X-ray tomography (left) allows identification of cellular structures (middle) that can be visualised in 3D (right), showing vesicles (rings) and mitochondria (tubes) in the infected cell.