Diamond Annual Review 2023/24

22 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 3 / 2 4 A new dimension in viral studies: 3D insights into HSV-1 assembly with correlative light X-ray tomography 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). 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 biggest finding from the study was an insight into the mechanism behind howthe capsid becomes enveloped in the cytoplasm. In 2D,TEMstudies 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.With X-ray tomography, researchers 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 they 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. Nahas, K.L. et al. DOI: 10.1101/2024.03.13.584906. Biological Cryo-Imaging Group eBIC Targeting a key COVID protein with antivirals The SARS-CoV-2 virus that was responsible for the COVID-19 pandemic synthesises a large number of its proteins in a single polyprotein chain, and the virus relies heavily on a viral enzyme called the main protease to clip the polyproteins into individual units. The importance of the main protease makes it an appealing target for antivirals, but an in-depth understanding of the protease’s structure is required to determine how drugs may interfere with the enzyme. In a recent paper, Andre Schützer de Godoy at the University of São Paulo in Brazil and his colleagues collaborated with the Electron Bio-Imaging Centre (eBIC) at Diamond Light Source to determine the structure of themain protease bound to its substrate by cryo-electron microscopy (cryoEM). This was one of the smallest protein structures to be solved at eBIC. This dimer has a molecular weight of approximately 68 kiloDaltons, only 25 kiloDaltons larger than the current world-record for the smallest protein to have its structure solved with this technique. It led to important insights into how the active site engages with the polyproteins that it then cleaves into individual viral proteins which are essential for the viral replication. With their cryoEM structure in tow, the researchers explored how different antivirals interfere with the protease at the molecular level, including a drug developed by the COVID Moonshot Initiative in collaboration with Diamond. Their findings reveal that separate antivirals have forced the structure of the protease to change in distinct ways, paving the way to understanding how these drugs interfere with the enzyme at the molecular level. Noske, G.D. et al. DOI: 10.1038/s41467-023-37035-5 Figure: CryoEM at eBIC was used to visualise how two copies of the SARS-Cov-2 main protease pair up to form heart-shaped dimers in their active form (blue). A fragment of a viral polyprotein (red) was detected in the dimer’s active site, allowing the team to study how this protease cleaves proteins. Image Credit: Andre Schützer de Godoy. Figure: Soft X-ray tomography imaging at cryogenic temperatures of HSV-1-infected HFF-hTERT cells identifies virus particles. HFF-hTERT cells were grown on EM grids, infected (MOI 2) with HSV-1 or mock-infected, and plunge cryocooled 16 hpi. All tomograms were reconstructed from X-ray projections collected using 25 nm ( A ) or 40 nm ( C, D, G–I ) zone plate objectives; scale bars = 1 μm. ( A ) The nucleus (Nuc) has a largely uniform X-ray absorbance in uninfected HFF-hTERT cells. Cyto, cytoplasm. ( B ) Schematic of infection workflow. ( C ) In HSV-1 infected cells many dark puncta are evident in the nucleus, consistent with these puncta being newly assembled HSV-1 capsids. ( D ) Dark puncta were also observed within the perinuclear space of the nuclear envelope, consistent with these being HSV-1 capsids undergoing primary envelopment/de-envelopment to leave the nuclear space. ( E ) Segmentation of a perinuclear viral particle (magenta) and the two membranes of the nuclear envelope (cyan). The perinuclear viral particle expands the nuclear envelope.