New Results

Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of cytoplasmic vesicles and mitochondria during HSV-1 infection

Kamal L. Nahas, Viv Connor, Maria Harkiolaki, Colin M. Crump, Stephen C. Graham
doi: https://doi.org/10.1101/2021.10.11.463900

Abstract

Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used a 3D imaging technique (soft X-ray tomography) to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments, we used a non-disruptive sample preparation technique involving rapid cryopreservation. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Furthermore, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that altered dynamics of mitochondrial fission/fusion accompany HSV-1 infection. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.

Importance Ultrastructural changes to the morphology and organization of cellular compartments during herpes simplex virus-1 (HSV-1) infection have not been studied under near-physiological conditions. In this study, near-native state X-ray imaging was used to image the ultrastructure of HSV-1 and cellular compartments during infection, identifying striking changes to the abundance and organization of cytoplasmic vesicles and mitochondria. The concentration of vesicles in the juxtanuclear region increased with time post infection, which could represent an increasing supply of vesicles to support capsid envelopment. Mitochondria are dynamic cellular compartments that undergo fusion to share resources and fission followed by mitophagy to recycle damaged components. Here we show that mitochondria tend to elongate and form highly-branched networks as infection progresses, suggesting fusion predominates over fission during HSV-1 infection. These findings offer insight into stages of virion morphogenesis and the cellular response to infection.

Introduction

Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus in the Alphaherpesvirinae subfamily of Herpesviridae that establishes a persistent life-long latent infection in sensory and sympathetic neurons, occasionally reactivating to cause lytic replication in oral or genital mucosal epithelial cells that culminates in cold sores and genital herpes, respectively1. The production of viral particles during lytic replication is a complex process involving multiple cellular compartments2–6.

In the first step of virion morphogenesis, capsid assembly and genome packaging occur in the nucleus7. Fully formed nucleocapsids must cross the nuclear envelope to migrate into the cytoplasm and undergo latter stages of virus assembly – a process which involves close interaction between nucleocapsids and the membranes of the nuclear envelope. Unlike individual proteins, the nucleocapsids are too large to pass through nuclear pores and must therefore first bud into the perinuclear space through the inner-nuclear membrane, forming a perinuclear viral particle (primary envelopment). The envelope of this particle subsequently fuses with the outer-nuclear membrane to release the nucleocapsid into the cytoplasm (de-envelopment)8–11. Numerous copies of multiple (≥ 23) viral proteins deposit on the surface of nucleocapsids in the nucleus and cytoplasm to form an amorphous proteinaceous layer known as the tegument12. Tegument proteins have multiple important roles during infection, including the promotion of virion maturation2,3,6. Several cytoplasmic compartments are essential to virion morphogenesis: viral proteins are synthesized and modified in the endoplasmic reticulum and Golgi complex and, in a process known as secondary envelopment, nucleocapsids acquire their membrane envelope from cytoplasmic vesicles that are thought to be derived from the trans-Golgi network and the endosomal system2,3,13. In addition to compartments directly involved in virion assembly, the cytoskeleton and other cellular organelles, such as mitochondria and lysosomes, can become remodelled in response to infection14–16. Understanding how the morphology and organization of cellular compartments change during infection could illuminate their involvement in virion morphogenesis and in the cellular response to HSV-1 infection.

Previous studies to characterize remodelling of cellular compartments have identified numerous changes that accompany HSV-1 infection, including the fragmentation of the Golgi complex and the condensation of the endoplasmic reticulum around the nuclear rim17,18. A more comprehensive study has recently been carried out using a recombinant form of HSV-1, known as the “timestamp” reporter virus, expressing fluorescent chimeras of the early protein ICP0 and the late protein gC to distinguish between early and late stages of infection16. Eight cellular compartments were compared between uninfected and timestamp virus-infected human TERT-immortalized human foreskin fibroblast (HFF-hTERT) cells, with high-resolution spatial data collected using structured illumination microscopy (SIM) and expansion microscopy. Numerous changes in the morphology of cellular compartments were observed as infection progressed, such as fragmentation of the Golgi complex at late stages of infection, concentration of endosomes and lysosomes at a juxtanuclear compartment, and elongation of mitochondria16. However, the extent to which sample preparation strategies may damage the morphology of cellular structures remains poorly understood and it is possible that disruptive techniques such as immunostaining or sample expansion could introduce artefacts in cellular ultrastructure19–21. Moreover, it is not clear if these changes to cellular compartments are consistent across different cell types used to study HSV-1 infection.

In this study, we investigated changes to cellular compartments during infection with the HSV-1 timestamp reporter virus using soft X-ray tomography of cryopreserved samples (cryoSXT). Soft X-rays used for this analysis have a lower energy (~500 eV)22 and longer wavelength than the “hard” X rays typically used for medical imaging (~15-30 keV)23 or X-ray crystallography (~6–20 keV)24. The wavelengths of soft X-rays used for cryoSXT are in the “water window” where structured carbon-rich structures in the cell such as membranes produce considerable contrast whereas oxygen-rich structures such as the “watery” cytosol remain transparent, thereby enabling cellular compartments to be observed22. We used this label-free technique to image the ultrastructure of infected (and control) cells, monitoring the 3D geometry and organization of cellular compartments25. The ultrastructure of the samples was protected in a near-native state using a non-disruptive cryopreservation protocol26. Our study focused on the ultrastructural changes that accompany HSV-1 infection of human osteosarcoma U2OS cells, allowing comparison with previous reports from infection of HFF-hTERT cells16. Although a few differences were observed between the extent of Golgi fragmentation and the subcellular distribution of ICP0, we determined that remodelling of cytoplasmic vesicles and mitochondria was largely similar between these cultured cells. Furthermore, the high resolution afforded by cryoSXT revealed that mitochondria become highly branched during HSV-1 infection.

Results

HSV-1 viral particles and assembly intermediates are detectable by cryoSXT

While cryoSXT has been used to image virus particles in infected cells25,27, it was unclear whether ‘naked’ HSV-1 capsids, which are approximately 125 nm in diameter28,29, would be large enough and offer sufficient contrast to be observed with this imaging method. To establish a baseline, we grew uninfected HFF-hTERT cells on holey-carbon electron microscopy (EM) grids and plunge cryocooled them for imaging by cryoSXT. Unlike a glass lens that focuses light by refraction, a zone plate was used to focus the X rays by diffraction: a zone plate is a diffraction grating composed of a series of concentric rings in which alternating rings are transparent to X rays and the resolution is determined by the diameter of the outermost ring30. 25 nm zone plates, affording up to 30 nm resolution, were used for our experiments here. To produce a tomogram, a series of X-ray projection images were collected from a single 9.46×9.46 μm field of view in the cell, with each projection collected following rotation of the specimen around an axis normal to the incident X-ray beam (tilt series). For each tomogram the projections spanned up to 120° of rotation with increments of 0.2° or 0.5° per image. To correct for vibrations in the microscope during imaging, the projections in the series were aligned together in the program IMOD31 using gold fiducials or lipid droplets as landmarks for registration. We collected 19 tilt series that were processed into 3D tomograms and we found that the content of nuclei in uninfected cells appear relatively featureless (Fig 1A).

Fig. 1.
Fig. 1. 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 and plunge frozen 16 hpi with 2 plaque-forming units of HSV-1 per cell or mock infection. All tomograms were reconstructed from X-ray projections collected using zone plate objective optics affording 25 nm (A) or 40 nm (C, D, G–I) resolution; 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 distends the nuclear envelope. (F) The width of perinuclear viral particles plus associated membranes is 190.5 ± 6.01 nm SEM (N=11; 20.8 nm SD), which is greater than the width of the nuclear membrane elsewhere (99.8 ± 3.57 nm SEM; N=11; 11.9 nm SD). (G) HSV-1 capsids (arrows) were also observed in the cytoplasm alongside vesicles (arrowheads). (H) Multiple particles are observed along the surface of infected cells, consistent with these being assembled HSV-1 virions that have exited the infected cell. (I) HSV-1 virions are also observed at the junctions between cells. (J) The width of the nuclear capsids is 125.8 ± 1.70 nm SEM (n=80 from 4 tomograms), consistent with these being HSV-1 capsids (~125 nm28,29). The width of the extracellular virions is 198.6 ± 3.48 nm SEM (n=80 from 4 tomograms), consistent with these being fully-enveloped HSV-1 virions (~200 nm36). Due to unequal variance, a Wilcox test was performed to determine a significant difference in the width of nuclear capsids and extracellular virions (W=126, p-value<2.2×10−16). Error bars show mean ± SEM.

Given that the nucleus is the site of capsid assembly, we sought to determine whether an abundance of capsids could be detected in infected cells. To this end, HFF-hTERT cells were cultured on holey-carbon EM grids, infected with 2 plaque-forming units (PFU) per cell of HSV-1 and plunge frozen at 16 hours post-infection (hpi). Infected cells were imaged via cryoSXT using a 40 nm zone plate objective (Fig. 1B), illuminating a 15.14×15.14 μm field of view, using the image acquisition and analysis workflow detailed above. These samples were prepared and cryopreserved on three different occasions and 98 tomograms were collected in total. Numerous dark puncta were observed in the nucleus of infected cells (Fig. 1C). We interpreted these puncta to be HSV-1 capsids because capsids are rich in carbon and phosphorous, being proteinaceous shells surrounding tightly packed DNA genomes, and these elements exhibit strong absorption at the 500 eV X-ray energy used here for imaging22. During virus assembly, capsids enter the perinuclear space by budding at the inner nuclear membrane (primary envelopment), forming a membrane-wrapped perinuclear viral particle that rapidly fuses with the outer nuclear membrane en route to the cytoplasm9. These enveloped virions in the perinuclear space are infrequently observed by EM because they are short-lived and the thin sectioning required for imaging using electron light decreases the probability that such structures will be present within the cellular volume being examined. The high penetrating power of soft X-rays in unstained cryopreserved samples (> 10 μm) removes the requirement for sectioning, allowing the entire depth of the cell to be imaged simultaneously for any given field of view. This increases the likelihood of observing short-lived structures such as primary enveloped viral particles. Dark puncta within the nuclear envelope that are likely to be perinuclear viral particles were found 11 times in 98 tomograms (Fig. 1D). The perinuclear viral particles appear to expand the perinuclear space and distend the nuclear envelope, as shown in a segmented image (Fig. 1E). The width of the nuclear envelope at putative sites of primary envelopment (190.6 ± 6.01 nm SEM; N=11) is significantly greater than the width of the nuclear envelope in other places on the same tomograms (99.8 ± 3.57 nm SEM; N=11; paired t-test p-value=1.93×10−9) (Fig. 1F). This demonstrates the substantial deformation of the nuclear envelope that must occur to accommodate perinuclear viral particles. Viral capsids were also observed in the cytoplasm in close proximity to vesicles that are likely sites of secondary envelopment (Fig. 1G). After secretion, HSV-1 particles commonly remain bound to the cell surface, a property that may be exacerbated by the antiviral restriction factor tetherin32,33. In addition, we expected to see HSV-1 particles between cells because virions are targeted to cell junctions to promote cell-cell spread34. Linear arrays of dark puncta were observed on the cell surface and between cells (Fig. 1H & I) and likely represent released virus particles (extracellular virions). Virus particles accumulate most of their tegument in the cytoplasm and become enveloped before they are released from the cell. We measured the width of nuclear capsids and extracellular virions from 8 tomograms to determine if they could be distinguished based on their size (Fig. 1J). Nuclear capsids had a width of 125.8 ± 1.70 nm SEM (n=80 from 4 tomograms; range 96–160 nm; SD 15.22 nm), which is consistent with high-resolution structural analysis of purified capsids29 (~125 nm) and of capsids inside infected-cell nuclei35. Extracellular virions were larger with a width of 198.6 ± 3.48 nm SEM (n=80 from 4 tomograms; range 128–272 nm; SD 31.15 nm), consistent with previous reports (~175-200 nm28,36). These differences were found to be significant with a Wilcox test for unequal variance (W=126, p-value<2.2×10−16).

Early protein ICP0 and late protein gC have different patterns of spatiotemporal expression in HFF-hTERT and U2OS cells

Recent microscopy and single-cell transcriptomics studies have revealed that, even in a monolayer of cultured cells synchronously infected with HSV-1, individual cells progress through the infection cycle at different rates and the remodelling of cellular compartments varies depending on the stage of infection16,37. To control for this, a recombinant strain of HSV-1 termed the timestamp virus has been developed to allow identification of the stage of infection based on the abundance and subcellular localization of the fluorescently tagged early and late viral proteins, ICP0 and gC respectively16. Fluorescence microscopy of HFF-hTERT cells infected with this timestamp virus allowed characterization of the changes to cellular compartments that accompany progressing HSV-1 infection and categorization of cells into 4 stages of infection. Having confirmed that virus particles could be observed in infected HFF-hTERT cells using a 40 nm zone plate objective, we sought to obtain higher-resolution information on the morphological changes exhibited by timestamp HSV-1-infected cells over the course of infection by cryoSXT imaging using a 25 nm zone plate objective. However, preliminary experiments performed using infected HFF-hTERT cells were unsuccessful as the infected cells proved sensitive to higher flux density of the resultant X-ray beam, leading to localized sample heating and low-quality tomograms. We therefore turned to U2OS osteosarcoma cells, which have been shown to produce consistently high-quality tomograms when imaged by cryoSXT25,26 and have been used previously for HSV-1 ultrastructural analysis38,39.

To compare the temporal profiles of progression of timestamp HSV-1 infection in HFF-hTERT and U2OS cells, we first compared the expression patterns of these proteins between the two cell types. Cells were infected at a multiplicity of infection (MOI) of 1 and 3 and samples were fixed at multiple time points following infection before imaging on a widefield fluorescence microscope (Fig. 2). The immediate early HSV-1 protein ICP0 was used to characterize early stages of infection because it is one of the first viral proteins to be expressed40. In both cell lines, eYFP-ICP0 was expressed in all four stages and its localization was restricted to the nucleus in stage 1. However, the spatial localization of eYFP-ICP0 differed between HFF-hTERT and U2OS cells in stages 2-4: in these stages of HFF-hTERT cell infection, eYFP-ICP0 signal was observed to diminish in the nucleus and continue to increase in the cytoplasm across these three stages, whereas greater retention of eYFP-ICP0 in the nucleus was observed in U2OS cells throughout the entire course of infection (Fig. 2A). This may reflect differences in cellular interactions for ICP0 in U2OS cells, which is consistent with previous observations demonstrating that replication deficits demonstrated by ICP0-null strains of HSV-1 in human fibroblasts are effectively complemented in U2OS cells41. Unlike eYFP-ICP0, the spatial expression of gC-mCherry was similar between HFF-hTERT and U2OS cells. gC is a viral glycoprotein expressed at late stages of virus replication42 that is incorporated into nascent virus particles at sites of virus envelopment43. In both cell types gC-mCherry was enriched at a juxtanuclear site in stage 3 but became dispersed throughout the cytoplasm and at the plasma membrane by stage 4 (Fig. 2A).

Fig. 2.
Fig. 2. Temporal analysis of HSV-1 infection using the dual-fluorescent timestamp virus.

(A) Room temperature widefield fluorescence imaging of timestamp HSV-1 infected HFF-hTERT and U2OS cells was used to delineate between four stages of infection based on the expression and localization of the early protein eYFP-ICP0 and the late protein gC-mCherry16. The spatiotemporal expression of these fusion proteins was similar in HFF-hTERT and U2OS cells, except for partial retention of eYFP-ICP0 in the nucleus of U2OS cells during stages 2–4. Scale bars = 50 μm. (B) The proportion of infected cells in each stage was determined using widefield imaging and different multiplicities of infection (MOIs) in HFF-hTERT and U2OS cells at 9, 12, 16, and 24 hpi.

Next, we probed whether progression through the replication cycle follows the same timecourse in HFF-hTERT and U2OS cells by infecting with different MOIs and imaging at different times post-infection to compare the proportion of infected cells at each stage of infection. Although the spatial expression of gC-mCherry was similar, fewer U2OS cells transitioned between stages 3 and 4 compared to HFF-hTERT cells at MOI 1 and 3, even by the 24-hour timepoint (Fig. 2B). Stage 4 is defined by the dispersal of gC-mCherry throughout the cytoplasm and, given the colocalization of gC with Golgi markers 58K protein and beta-1,4-galactosyltransferase 1, this dispersal is thought to arise via fragmentation of the Golgi complex16. The reduced number of U2OS cells in stage 4 may suggest a delay in, or resistance to, Golgi complex fragmentation. The partial cytoplasmic translocation of eYFP-ICP0 and the reduced gC-mCherry dispersal complicated the distinction between stages 1 and 2, and between stages 3 and 4, respectively. As a result, we combined the stages into two broader early (1+2) and late (3+4) stages in our study.

Vesicles proliferate and mitochondria become elongated and branched during HSV-1 infection

To characterize the changes in morphology of cellular compartments that accompany different stages of virus infection, U2OS cells were grown on holey-carbon EM grids before being infected (or mock infected) with timestamp HSV-1 and cryogenically preserved by plunge freezing in liquid nitrogen-cooled liquid ethane (Fig. 3A). Vitrified samples were analyzed by cryo-wide-field microscopy to classify the stage of infection and then imaged using cryoSXT to correlate the stage of virus infection in a specific cell with observed morphological changes (Fig. 3B). As the proportion of U2OS cells in the early stages of infection (stages 1+2) was consistently lower than the proportion in late stages of infection (stages 3+4; Fig. 2B), grids were infected at MOI 1 and plunge frozen at 9 hpi to achieve a more balanced proportion of cells in early and late stages of infection.

Fig. 3.
Fig. 3. Workflow for multi-modal imaging of HSV-1 infected cells.

(A) Preparation of infected cells samples for multimodal imaging. U2OS cells are cultured on holey-carbon EM grids and infected with recombinant ‘timestamp’ HSV-1, expressing fluorescently tagged proteins eYFP-ICP0 and gC-mCherry that allow identification of the stage of infection for each cell under investigation. At 9 hpi, gold fiducials are overlayed onto the sample to facilitate image registration and grids are cryopreserved in a near-native state by plunge freezing in liquid ethane. (B) Multi-modal imaging of infected U2OS cells. A widefield microscope with a cryo stage is used to locate the grid positions of infected cells. The stage of infection for each cell is determined based on the differential expression and localisation of the eYFP-ICP0 and gC-mCherry (as shown in Fig. 2). These grids are then loaded into the cryo-soft-X-ray microscope at Diamond Light Source beamline B24 and are illuminated with soft X-rays at the marked grid positions. X-ray projections of regions of interest (ROIs) are collected at multiple angles and aligned using the gold fiducials and intracellular features, such as lipid droplets (LDs), with the program IMOD31. Tomograms are reconstructed from these projections using IMOD to compare intracellular morphology between uninfected cells and those at early- or late-stages of infection. Segmentation facilitates quantitation and visualization in three dimensions of the observed cellular structures.

In total, 139 SXT tomograms were reconstructed; 76 from uninfected cells alongside 22 and 41 from cells at early- or late-stages of infection, respectively, across three independent replicates (Table 1). Manual inspection of the resultant tomograms revealed that the 25 nm zone plate allows detection of higher resolution features than is possible with the 40 nm zone plate, such as the lumen of the endoplasmic reticulum, cytoskeletal filaments, and small membrane structures (Supp. Fig. 1A-E). The observed width of nuclear capsids is very similar in infected HFF-hTERT cells imaged using the 40 nm zone plate (Fig. 1J) or U2OS cells imaged using the 25 nm zone plate (Supp. Fig. 1F). The tomograms collected from U2OS cells using the 25 nm zone plate were thus deemed suitable for identifying changes to cellular compartments that occur during HSV-1 infection.

View this table:
Table 1. Collection of cryoSXT data to analyse changes in cellular morphology accompanying infection.

CryoSXT data was collected using a 25 nm zone plate from multiple uninfected cells or cells at early and late stages of infection across three independent replicates. Tiled X-ray projections (‘X-ray mosaics’) with a 66.2×66.2 μm field of view were collected at multiple areas on the sample grid to identify cells of interest. Tilt series were collected at perinuclear or peripheral regions of the cytoplasm within these cells and were processed to generate tomograms.

We observed that the HSV-1 infection does not affect the morphology of cellular compartments such as the nucleus and lipid droplets. Despite the continuous budding and fusion of capsids at the inner and outer nuclear membranes, we did not observe any obvious changes to the integrity of the nuclear envelope. We occasionally observed bulging of the nuclear envelope into the cytoplasm (Supp. Fig. 1G) but this could be seen in both uninfected and infected cells. The size and abundance of lipid droplets did not appear to differ between uninfected, cells and those at early- or late-stages of infection. However, striking changes were observed in the size and dispersal of vesicles, and in the length and connectivity of the mitochondrial network, between uninfected and infected cells, with the changes becoming more pronounced in cells at late stages of infection (Fig. 4&5 and Supp. Video 1).

Fig. 4.
Fig. 4. Remodelling of cytoplasmic vesicles during HSV-1 infection.

CryoSXT tomograms were recorded of cells classified as uninfected, early-stage infection (stages 1+2) or late-stage infection (stages 3+4) with timestamp HSV-1 based on fluorescent cryo-wide field microscopy. Data are representative of three independent experiments. Scale bar = 1 μm. (A) A higher concentration of vesicles is observed at the juxtanuclear compartment in cells at early- or late-stages of infection compared with uninfected cells. (B) The maximum width of each vesicle in three-dimensions was measured in Contour (manuscript in preparation). Vesicles with a spherical, ellipsoidal, or dumbbell shape were included in the analysis but vesicles with a shape that didn’t fall into these categories were excluded. Intra-luminal vesicles and vesicles that were not individually resolved by the segmentation were also excluded from the analysis. Significance of differences was assessed with a one-way ANOVA and Tukey tests for the combinations: uninfected-early (p=0.04), uninfected-late (p=0.01), and early-late (p=0.62). Big circles show the mean vesicle width per tomogram (4 tomograms per condition). Error bars show overall mean ± SD.

Fig. 5.
Fig. 5. Remodelling of mitochondria during HSV-1 infection.

Morphological changes to mitochondria were assessed from cryoSXT tomograms collected from uninfected cells and cells at early- or late-stages of infection with timestamp HSV-1. Data are representative of three independent experiments. Scale bars = 1 μm. (A) A shift towards elongated and branched mitochondria was observed during infection. Mitochondria were segmented and differentiated using Contour to highlight the abundance and 3D geometry of individual mitochondria. (B) The percentages of tomograms with long mitochondria were greater for cells at early- or late-stages of infection than for uninfected cells in replicate 2. Mitochondrial morphology was more heterogenous in uninfected cells. See Supp. Fig 2 for equivalent data from replicates 1 and 3. (C) The numbers of branching nodes were calculated for 45 tomograms across all replicates and significant differences in the number of nodes between uninfected cells and those at late stages of infection were determined for each replicate using ANOVA and Tukey tests (p <0.05). Error bars show mean ± SD.

HSV-1 capsids are thought to interact with several types of vesicles in the cytoplasm, including trans-Golgi network vesicles and endosomes, both of which have been implicated in secondary envelopment13. There was a striking difference in the concentration of vesicles at the juxtanuclear compartment between uninfected and infected cells (Fig. 4A and Supp. Video 2). Infected cells had a greater number of vesicles in juxtanuclear regions than uninfected cells. To determine if there was a difference in the size of vesicles between uninfected cells and those at early- or late-stages of infection we developed Contour, a program to segment and quantitate cellular features in 3D volumes (manuscript in preparation). The widest point of each vesicle in three dimensions from 4 tomograms for each condition was measured (Fig. 4B). The mean vesicle width was higher for uninfected cells (802.23 ± 348.47 nm SD, N=96) than for early-stage (688.66 ± 271.76 nm SD, N=184) and late-stage (631.85 ± 270.60 nm SD, N=184) infected cells. The mean vesicle widths for each tomogram were compared using a one-way ANOVA and Tukey test and the vesicle widths of uninfected cells were found to be significantly different from early-stage (p=0.04) and late-stage (p=0.01) infected cells. The vesicle width did not differ significantly between early-stage and late-stage infected cells (p=0.62).

Mitochondria were the most phenotypically diverse organelles monitored in this study. In most cases, they were thin and possessed a dark matrix (Fig. 5A). However, occasionally there were cells that contained swollen mitochondria with a lighter matrix with highly contrasting cristae (Supp. Fig. 2A), similar to observations of mitochondria made by EM44–46. This swollen morphology is associated with release of cytochrome c from porous mitochondria during apoptosis44. Mitochondria are known to interact with lipid droplets, for example to acquire fatty acids for respiration47. Interestingly, we observed that lipid droplets were less frequently in close apposition to swollen mitochondria than to mitochondria possessing dark matrices (Supp. Fig. 2B). Swollen mitochondria were observed in each of the three independent sets of cell growth, infection and plunge freezing experiments performed, but these swollen mitochondria were most prevalent in the uninfected cells of replicate 3 (Supp. Fig. 2C). In uninfected cells, non-swollen mitochondria were heterogeneous in shape, with numerous being small and spherical or long and curved in the same cell. We observed branching in some elongated mitochondria. However, mitochondria appeared less heterogenous in shape in infected cells, and were consistently more elongated and branched (Fig. 5B and C, Supp. Fig. 2D, and Supp. Video 3), in line with previous observations made using super-resolution fluorescence microscopy of HFF-hTERT cells infected with the timestamp virus16. Segmentation of mitochondria using Contour demonstrated that the number of points where mitochondria branch into two or more arms (branching nodes) was significantly increased in cells at late stages of infection (20.5 ± 5.45 nodes SD; n = 15) compared to uninfected cells (7.0 ± 4.02 nodes SD; n = 15) (Fig. 5C). In some cases, the mitochondria fused into a single, branched network (Fig. 5A and Supp. Video 3), providing a dramatic demonstration of the increase in mitochondrial branching and decrease in number of distinct mitochondrial networks that accompanies HSV-1 infection. It was also observed that the number of distinct mitochondria decreased in infected cells, although ambiguity regarding the connectivity of mitochondrial networks that extend beyond the tomogram field-of-view prevented precise quantitation of this effect.

Discussion

In this study, we used cryoSXT to monitor the production of nascent HSV-1 particles and observe changes in the architecture of cellular compartments during infection. The penetrating power of X-rays facilitates cryoSXT imaging throughout the depth of the cell, allowing rare or transient events to be captured such as the transit of nascent capsids through the nuclear envelope. Furthermore, the lack of requirement for contrast-enhancing agents or chemical fixation allows direct imaging of cellular compartments in a near-native state. We exploited these properties of cryoSXT to compare the morphology of cellular compartments between uninfected and infected U2OS cells, using a recombinant strain of HSV-1 expressing fluorescently tagged early and late viral proteins to identify the infection stage of individual cells within the infected population.

HFF-hTERT and U2OS cells are commonly used for the study of HSV-1 infection16,38,39. We had intended to use HFF-hTERT cells for this study, to allow comparison with super-resolution fluorescence microscopy studies16, but found infected HFF-hTERT cells to be less amenable to interrogation by cryoSXT. We therefore explored the differences in the dynamics of viral infection between HFF-hTERT and U2OS cells using the timestamp virus. In general, the modifications to cellular compartments observed in this study largely replicated those observed in HFF-hTERT cells16, suggesting the interactions between viral components and cellular compartments are broadly similar in these two cell types. We observed subtle differences between the infections in these cells, including a U2OS-specific reduction in the dispersion of gC-containing membranes and a change in the nuclear-to-cytoplasmic translocation of the early viral protein ICP0 (Fig. 2). Residues important for the nuclear import/export dynamics of ICP0 have previously been identified: ICP0 possesses a canonical nuclear localization signal at residues 500–506 and deletion of 57 residues from the C terminus abolishes nuclear export of ICP048. Although residues important for trafficking of ICP0 have been mapped, the cellular proteins involved in ICP0 trafficking have yet to be identified. In this study, a higher intensity of eYFP-ICP0 was detected in the nucleus compared with the cytoplasm of infected U2OS cells at every timepoint. In contrast, higher cytoplasmic intensity of ICP0 is observed at late stages of infection in HFF-hTERT cells and other cell lines16,49,50. This suggests that the expression of host proteins that regulate nuclear import and/or export of ICP0 may differ in U2OS cells. Several host proteins have been identified to participate in the nuclear trafficking of EP0, the pseudorabies virus orthologue of ICP0: Ran, Importin α1, α3, α7, β1, and transportin-151. Future work is required to identify whether U2OS cells are depleted or enriched in proteins involved in nuclear import/export of ICP0, which may illuminate the mechanisms regulating subcellular localisation of this important viral E3 ligase during infection.

Compared with uninfected U2OS cells, infected cells had a greater local concentration of detectable vesicles in the juxtanuclear space (Figure 4), consistent with previous research into the distribution of vesicles during HSV-1 infection. For instance, early endosomes and lysosomes have been shown to accumulate at the juxtanuclear region during infection16. This reorganization of vesicle distribution may be related to a change in microtubule dynamics during infection. Previous studies have shown that γ-tubulin and pericentrin, which are components of the microtubule-organizing centre (MTOC), become dispersed during infection, suggesting breakdown of the MTOC52. Thereafter microtubules polymerize at multiple foci in the cytoplasm rather than at a single site and the growth rate, length, and stability of nascent microtubules become reduced compared with uninfected cells52. As the morphology of microtubules changes, the transport of vesicles towards the cell periphery may be obstructed, resulting in the accumulation of vesicles in juxtanuclear regions that may partly explain the increased local concentration of vesicles. An additional source of new vesicles may arise from the fragmentation of the Golgi complex during HSV-1 infection53. Most of the evidence for Golgi fragmentation is based on the dispersion of several Golgi markers (β-COP, Giantin, GM130, 58K protein, and beta-1,4-galactosyltransferase 1) throughout the cytoplasm during HSV-1 infection as assessed by fluorescence microscopy16,17,53. Golgi fragmentation has been studied to a lesser extent by ultrathin section EM, revealing cisternae become swollen and separated during infection17. Golgi fragmentation is thought to be a consequence of disrupted microtubule dynamics and can be induced by treatment with nocodazole, an inhibitor of β-tubulin polymerization53. Although our results are consistent with these observations, the lack of markers for different types of vesicles meant that we could not determine if the vesicles we observed with SXT were Golgi-derived, of endosomal origin, or were unrelated to these cellular compartments. We observed a reduction in the mean size of vesicles as the infection progressed (Figure 4B), which could arise either from fragmentation of the Golgi complex into small vesicles or an inability of small vesicles to be trafficked from the juxtanuclear region to their target organelles via microtubule transport. Future work could focus on the use of fluorescent markers and correlative cryoSIM and cryoSXT imaging to identify which cellular compartments are found with an increased concentration at the juxtanuclear region25,54.

The spatial resolution afforded by cryoSXT allowed us to easily distinguish individual mitochondria in uninfected and infected U2OS cells. Consistent with previous studies of infected HFF-hTERT cells16, we observed that mitochondria became more elongated and branched as infection progresses and form extensive networks in some cases (Figure 5). Branching of mitochondria can either occur via de novo synthesis or by fusion of mitochondria55,56. Our observation that some cells at late stages of infection contain just one single large network of mitochondria, rather than numerous highly branched but disconnected mitochondrial networks, is consistent with fusion predominating over fission as a driver of mitochondrial network formation.

There are several possible explanations for the change in mitochondrial morphology observed during HSV-1 infection. Mitochondrial movement tends to occur along microtubules and this movement influences mitochondrial fusion/fission dynamics. Fission can arise from divergent movement of mitochondrial extensions along microtubules and fusion is supported by convergent movement of mitochondria57. Nocodazole treatment to depolymerize microtubules blocks transport, fusion and fission of mitochondria, and there is evidence that thin microtubule extensions develop when fission is obstructed58. It is possible that fission of existing mitochondrial networks may be obstructed when microtubules depolymerize during HSV-1 infection, and this may prevent the generation of small mitochondria. Such changes to the microtubule network begin at 6 hpi and would thus be expected to have a greater influence on mitochondrial morphology in the late stages of infection52, consistent with our observations. Alternatively, the morphological changes to mitochondria may reflect a cellular response to increased respiratory demand59. An increase in ATP production can be achieved by mitochondrial elongation, for example under conditions of stress such as hypoxia and starvation of glucose metabolism60,61. An increase in respiration, including oxidative phosphorylation, has been observed for the related herpesvirus human cytomegalovirus62. The increased number of elongated mitochondria in cells at late stages of infection could facilitate an increase in ATP production during infection. Increased oxidative stress provides a third plausible explanation for the observed changes in mitochondrial morphology. Increased production of reactive oxygen species (ROS) during respiration appears to be a common feature of viral infection that has been observed for hepatitis C virus, respiratory syncytial virus and the herpesvirus Epstein-Barr virus63–65. One mechanism by which the cell responds to oxidative stress is by fusion of undamaged and ROS-damaged mitochondria to allow for compensatory effects by sharing resources needed for ATP production56. It is possible that the increased fusion events we observed may have arisen in response to increased oxidative stress during infection.

Although a change in energy metabolism may reflect a generalized response by the cell to infection, several HSV-1 proteins have been reported to localize at mitochondria (pUL7, pUL16, pUS3, pUL12.5), suggesting that HSV-1 directly modulates mitochondrial activity66–69. pUS3 inhibits the activity of electron transport chain complexes II and III as early as 6 hpi67 and pUL12.5 functions in the depletion of mitochondrial DNA and downregulation of mitochondrial proteins, including ND6 and COX2, as early as 4–8 hpi68. The functional consequences of pUL16 binding mitochondria are not well characterized, although we note that a pUL16 mutant co-localized with mitochondrial fission sites69. The precise mechanisms by which HSV-1 alters the architecture of mitochondria and the role of specific viral proteins, versus virus-induced metabolic strain, thus remains unclear. Combining metabolic profiling of infected cells with ultrastructural analysis of mitochondrial morphology, using wild-type and mutant (knock-out) viruses, will help illuminate the factors that drive the dramatic remodelling of mitochondria observed during HSV-1 infection and the functional consequences thereof.

In conclusion, cryoSXT allows the detection HSV-1 capsids and virions in different subcellular locations, such as the nucleus, perinuclear space, cytoplasmic vesicles, and cell surface. Use of the timestamp HSV-1 reporter virus facilitated identification of individual cells at early or late stages of infection, and we observed accumulation of vesicles at juxtanuclear assembly compartments plus the elongation and branching of mitochondria in infected cells as infection progresses. The ability of cryoSXT to image the entire depth of infected cells in a near-native state, with minimal sample processing, highlights its utility as a tool for 3D imaging to identify changes in cellular architecture that accompany virus infection.

Materials & Methods

Reagents

250 nm gold colloid fiducials were purchased from BBI Solutions (EM.GC250, batch 026935). The working mixture was prepared via sedimentation of 1 mL of stock solution by centrifugation (12×g, 5 mins, RT) and then resuspending the pellet in 50 μL HBSS. The fiducials were sonicated at 80 kHz (100% power) and 6°C to prevent aggregation. 3 mm gold EM grids with a holey carbon film (R 2/2, 200 mesh) were purchased from Quantifoil (Cat# AU G200F1 finder, batches Q45352 & Q45353). Poly-L-lysine was purchased from Sigma Aldrich (Cat# P4832).

Cell Lines

U2OS cells (ATCC HTB-96; RRID CVCL_0042) and human foreskin fibroblast cells immortalized with human telomerase reverse transcriptase (HFF-hTERT cells)70 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Cat# 450 11590366) supplemented with 10% (v/v) fetal bovine serum (FBS; Capricorn, Cat#: FBS-11A), L-glutamine (Thermo Fisher Scientific, Cat# 25030081), and penicillin/streptomycin (10000 U/ml; Thermo Fisher Scientific, Cat# 15070063). Hanks’ Balanced Salt Solution (HBSS; Thermo Fisher Scientific, Cat# 14175095) and 0.25% Trypsin-EDTA (Thermo Fisher Scientific, Cat# 25200056) were used to wash and detach adherent cells, respectively. Cells were maintained in a humidified 5% CO2 atmosphere at 37°C.

Recombinant Viruses

Infections were performed using recombinant HSV-1 strain KOS expressing either the endogenously tagged viral proteins eYFP-VP26 and gM-mCherry (Figure 1) or the endogenously tagged viral proteins eYFP-ICP0 and gC-mCherry (timestamp HSV-1, Figures 2-5 and Supplementary Figures 1-2)16, to allow distinction between early and late stages of infection in U2OS and HFF-hTERT cells, with the exception of the leftmost panel in Fig. 1I for which a non-fluorescent wild-type HSV-1 strain KOS was used. Virus stocks were prepared by infection of Vero cells at low MOI (0.01) for 3–5 days, until cytopathic effect was evident, before scraping cells into the medium. The cells were frozen at −70°C, thawed and sonicated at 50 amps for 40 seconds. Crude virus stocks were clarified by centrifugation at 3,200×g in a benchtop centrifuge, aliquoted, and viral titers of the aliquots were quantified on Vero and U2OS cells as described previously (71).

Infection Assays

For widefield imaging at ambient temperatures, HFF-hTERT and U2OS cells were seeded directly in 6-well plates overnight at 2×105 cells per well. The cells were infected with the recombinant HSV-1 at 1–3 PFU per cell in a low volume of media (500 μL) and this was designated as the start time of infection. To maximize adsorption of virus, cells were incubated in the low-volume media for 1 hour in a humidified 5% CO2 atmosphere at 37°C and the plates were swirled every 15 minutes. The inoculum was diluted to 2 mL with medium and cells were incubated for 9-, 12-, 16- or 24-hours in total. The cells were washed twice with HBSS and were fixed with 4% (v/v) formaldehyde for 20 minutes. The fixed cells were washed three times with HBSS before imaging.

For widefield imaging under cryogenic conditions and cryoSXT, EM grids were glow discharged and treated with filtered poly-L-lysine for 10 minutes as described previously (26). 3×105 cells per well were seeded in 6-well plates containing the treated EM grids and were incubated overnight. Subsequently, the cells were infected with 1 PFU per cell of timestamp HSV-1 as described above and were incubated for 9 hours alongside uninfected controls. The EM grids were overlayed with 2 μL of the gold fiducial working mixture described in the Reagents section. A Leica EM GP2 plunge freezer was used to blot the grids for 0.5–1 s at 30°C and 80% humidity. The grids were plunged into liquid nitrogen-cooled liquid ethane and then transferred into liquid nitrogen storage before imaging.

Widefield Microscopy

For room temperature samples, a Zeiss AxioImager2 microscope with an achromatic 50× air objective (Zeiss LD EC Epiplan-Neofluar 50x/0.55 DIC M27; NA=0.55; free working distance=9.1 mm) was used to image fixed infected cells grown on plastic 6-well plates. Fluorescent images were collected using the Zeiss 46 HE YFP filter (Excitation 500±25 nm, Emission 535±30 nm) and the Zeiss 64 HE mPlum filter (Excitation 587±25 nm, Emission 647±70 nm).

For cryo-widefield microscopy, cells at early- and late-stages of infection were identified based on the spatiotemporal expression of eYFP-ICP0 and gC-mCherry using a Zeiss AxioImager2 microscope with an achromatic 50× objective (described above) without immersion. The microscope was equipped with a liquid nitrogen cryostage (Linkam) to maintain the sample at 77 K during imaging. Each grid was mapped in its entirety in the brightfield and fluorescent channels (Zeiss 46 HE YFP filter and HE mPlum filter as described above) using the LINK software (Linkam Scientific).

Cryo-Soft-X-Ray Tomography

X-ray images were collected using an UltraXRM-S/L220c X-ray microscope (Carl Zeiss X-ray Microscopy) at beamline B24 at the UK synchrotron Diamond Light Source. Grids were imaged in a liquid nitrogen-cooled vacuum chamber and samples were illuminated with 500 eV X-rays (λ = 2.48 nm) for 0.5 or 1 s per projection. The transmitted light was focused by diffraction using zone plate objectives with nominal resolution limits of either 25 nm or 40 nm. The 25 nm zone plate offers higher resolution but captures a smaller field of view (~10×10 μm) than the 40 nm zone plate (~16×16 μm). Transmitted images were collected using a 1024B Pixis CCD camera (Princeton instruments). X-ray mosaic images (7×7 images capturing 66.2×66.2 μm for the 25 nm objective and 106.0×106.0 μm for the 40 nm objective) were collected from different areas on the grid to assess overall cell morphology. For identification of early and late stages of infection, X-ray mosaics were compared with fluorescent imaged acquired on the cryo-widefield microscope to identify specific infected cells. These mosaics were also used to identify regions of interest for tomography. Tilt series of projections were collected from these regions by rotating the sample around an axis normal to the incident X-ray beam by up to 120° in increments of 0.2° or 0.5° per image, with maximum tilt angles of −60° and +60° and −70° and +70° for the 25nm and 40nm objective respectively. SXT tilt series were processed using IMOD (version 4.9.2)31. The images were aligned along a single axis. A coarse alignment was performed by cross-correlation with a high frequency cut-off radius of 0.1. Coarsely aligned tilt series were further aligned manually using gold fiducials and dark cellular compartments, such as lipid droplets. A boundary model was generated to reorient the 3D data in case the sample was collected at an angle and final alignment was performed using linear interpolation. Tomograms were generated using back projection followed by 20 iterations of a simultaneous iterations reconstruction technique (SIRT)-like filter to reduce noise.

Segmentation

Mitochondria were segmented using Contour, a bespoke semi-automated segmentation and quantitation tool developed with Python 3. Full details on Contour will be described elsewhere but, briefly, Contour automatically segments high contrast features such as mitochondria by thresholding and then applying a restriction on the minimum number of consecutive segmented pixels vertically and horizontally. Next, gaps in the segmented volume can be filled in by running this algorithm in local regions of interest. Separate elements in the segmented volume are differentiated by grouping of neighbouring voxels together. The differentiated elements are colour coded and their volumes are quantitated from the number of voxels. The edges of the segmented elements are smoothened in each image plane by translating the image by one pixel in all eight cardinal and ordinal directions in the XY plane and calculating the median pixel value for all these translations. A 3D Gaussian filter with a sigma of 2 was also added using Fiji to further smoothen the elements72. In Contour, the width of each segmented element was calculated by finding all the coordinates of voxels at the perimeter of segmented elements and calculating the largest modulus between any two coordinates. Segmented volumes of cytoplasmic vesicles were generated manually using the Segmentation Editor 3.0.3 ImageJ plugin72 and these were imported into Contour to differentiate between segmented elements and quantitate the width of the vesicles. Segmented volumes were visualized in 3D using the 3D Viewer plugin in ImageJ72.

Graphs and statistics

Distributions of capsid and virion widths were illustrated using a Violin SuperPlot73, with data grouped by source tomograms. The stacked area plots for the proportion of infected cells at different stages of infection were generated using the ggplot2 package74 in R studio75. The distribution of vesicle widths were illustrated using a SuperPlot76, with data grouped by source tomograms. The numbers of mitochondrial branch points (branching nodes) were illustrated using a Violin SuperPlot73, with data grouped by replicate. A two-tailed paired t-test was used to compare the width of the nuclear envelope at a site of primary envelopment with the width of the nuclear envelope elsewhere on the same tomogram using Excel (Microsoft). A Wilcox test for unequal variance was used to assess a significant difference in the widths of capsids and virions using R Studio75. One-way ANOVA and Tukey tests were used to assess significant differences in the mean vesicle width using Prism version 8.2.1 (GraphPad Software) and in the number of mitochondrial branching nodes (using R Studio75).

Data Availability

Original imaging data for tomograms illustrated in the manuscript is deposited at the BioImage Archive (https://www.ebi.ac.uk/biostudies/BioImages) and EMPIAR (https://www.ebi.ac.uk/pdbe/emdb/empiar/). The accession numbers for the data are EMPIAR: EMPIAR-XXXX, EMPIAR-XXXX,… and BioImage Archive: S-BIADXX, S-BIADXX,…. Additional tilt series and tomograms used for quantitative analysis have been deposited with the University of Cambridge Apollo Repository, available at https://doi.org/10.17863/CAM.XXXX.

Supp. Fig. 1.
Supp. Fig. 1. High resolution structures visible with the 25 nm zone plate objective.

139 CryoSXT tomograms were recorded from 107 cells using a 25 nm zone plate (ZP) objective and several structures that were unrelated to HSV-1 infection were observed, including some that were not visible using the 40 nm zone plate objective. (A) The endoplasmic reticulum (ER) forms a silhouette (arrows) around the mitochondria and the ER lumen is visible with the 25 nm zone plate. Cyto, cytoplasm. (B) Linear structures resembling cytoskeletal filaments are visible with the 25 nm zone plate (arrows). (C) A putative cytoskeletal filament (arrows) is in close apposition to a lipid droplet (LD) and may represent a physical interaction. (D) Small vesicles with widths of 150–300 nm in the peripheral cytoplasm are observed (arrows). (E) Large internalisations of the plasma membrane with depths of 1.6–2.2 μm (cyan) and smaller side extensions (black arrows) are visible and may represent events of clathrin-independent bulk endocytosis77. (F) The width of nuclear capsids was remeasured after imaging with the 25 nm zone plate: 124.5 nm ± 0.96 nm SEM (n=80 from 4 tomograms; 8.55 nm SD). (G) Bulging of the nuclear envelope is observed (arrows). We initially observed these in HSV-1 infection and thought it may represent a virus-directed decrease in the integrity of the nuclear envelope, but we found multiple examples in uninfected cells suggesting that they are a general feature of U2OS cells. Nuc, nucleus.

Supp. Fig. 2.
Supp. Fig. 2. The heterogenous morphology of mitochondria.

Heterogenous mitochondrial morphologies are observed in cryoSXT tomograms collected from uninfected cells and cells at early and late stages of infection with timestamp HSV-1. Scale bars = 1 μm. (A) In some cases, mitochondria have light matrices with highly contrasting cristae (cyan arrows). This “swollen” phenotype has been reported to occur during cytochrome c release from porous mitochondria during apoptosis44. Dark matter is also observed in the matrix (magenta arrows) and may represent vesiculation. Small dark puncta are present in the matrix (white arrows) and could represent vesicles or short cristae. (B) Mitochondria with dark matrices were commonly coupled with lipid droplets (cyan arrows) but most lipid droplets were uncoupled from swollen mitochondria. (C) The percentages of tomograms with swollen mitochondria for uninfected cells and cells at early- or late-stages of infection in three independent replicates. (D) The percentages of tomograms collected from uninfected cells and those at early- or late-stages on infection in replicates 1 and 3 that contain different combinations of mitochondrial morphologies.

Supp. Video 1. Segmentation of vesicles and mitochondria in the cytoplasm of a cell at a late stage of infection. CryoSXT data was collected from U2OS cells infected for 9 hours with 1 plaque-forming unit per cell of the timestamp HSV-1 virus. Cryo-fluorescence microscopy revealed that this cell was at a late stage of infection. The mitochondria were segmented using Contour and separate mitochondria are colour-coded in shades of orange, red, pink and purple. Cytoplasmic vesicles were segmented using Segmentation Editor in ImageJ. The vesicles were later differentiated and color-coded using Contour and are displayed here in shades of blue and green. Field of view is 9.46×9.46 μm.

Supp. Video 2. Segmentation of cytoplasmic vesicles reveals the effect of HSV-1 infection on vesicle concentration at juxtanuclear sites. CryoSXT data was collected from uninfected U2OS cells and U2OS cells infected for 9 hours with 1 plaque-forming unit per cell of the timestamp HSV-1 virus. Cytoplasmic vesicles were segmented using Segmentation Editor in ImageJ. The vesicles were later differentiated and colour-coded using Contour. Fields of view are 9.46×9.46 μm.

Supp. Video 3. 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 1 plaque-forming unit per cell of the timestamp HSV-1 virus. 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.

Acknowledgements

We thank Diamond Light Source for access to beamline B24 (mx18925, mx19958, bi21485 and bi23508) and the experimental hall coordinators for helpful support. We thank members of beamline B24 at the Diamond Light Source (Mohamed Koronfel, Ilias Kounatidis, Chidinma Okalo, and Matt Spink) for technical support with cryoSXT. We thank João Ferreira Fernandes (University of Oxford) and Thomas Fish (Diamond Light Source) for help with the development of Contour. We thank Harriet Groom (University of Cambridge) for reading and advising on the manuscript. This work was supported by a PhD studentship co-funded by Diamond Light Source and the Department of Pathology, University of Cambridge, to KLN, by a Biotechnology and Biological Sciences Research Council (BBSRC) Research Grant to CMC (BB/M021424/1), and by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society, to SCG (098406/Z/12/B).

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Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of cytoplasmic vesicles and mitochondria during HSV-1 infection
Kamal L. Nahas, Viv Connor, Maria Harkiolaki, Colin M. Crump, Stephen C. Graham
bioRxiv 2021.10.11.463900; doi: https://doi.org/10.1101/2021.10.11.463900
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