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

Transmission electron microscopy (TEM) has been used extensively to visualise HSV-1 capsids in infected cells [34–36]. However, TEM and cryoSXT have different strategies for introducing contrast in imaging. In TEM, signal is produced by adding a contrast agent, whereas cryoSXT is label-free and contrast is generated via the differential local density of carbon and oxygen in the material. Although cryoSXT has been used previously to image virus particles in infected cells, it was unclear whether individual ‘naked’ HSV-1 capsids, which are approximately 125 nm in diameter [32,37–40], 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 perforated 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 ring [41]. An objective zone plate with 25 nm outer zone was used for our experiments here, affording image resolution of up to 30 nm. To produce a 3D imaging volume (tomogram), a series of X-ray projection images (tilt series) 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. For each tomogram the projections spanned up to 120° of rotation with increments of 0.2° or 0.5° per image. To correct for small inaccuracies in the tilting of the microscope stage during imaging, the projections in the series were aligned together in the program IMOD [42] using gold fiducials or lipid droplets as landmarks for registration. We collected 19 tilt series that were processed into 3D tomograms. We found that uninfected cell nuclei lacked distinctive internal features other than a difference in average intensity when compared with the cytosol (Fig 1A).

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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, 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. (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. Gold fiducials are indicated with stars. (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 nm) [38,106]. 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 nm) [51]. Due to unequal variance, a Mann-Whitney U 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.

https://doi.org/10.1371/journal.ppat.1010629.g001

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 perforated-carbon EM grids, infected with HSV-1 at a multiplicity of infection (MOI) of 2 and plunge cryocooled at 16 hours post-infection (hpi). Infected cells were imaged via cryoSXT using a 40 nm zone plate objective, illuminating a 15.14×15.14 μm field of view, using the image acquisition and analysis workflow detailed above (Fig 1B). These samples were prepared and cryopreserved on three separate 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 imaging [29].

During virus assembly, capsids enter the perinuclear space by budding at the inner nuclear membrane (primary envelopment), forming a membrane-wrapped perinuclear virus particle that rapidly fuses with the outer nuclear membrane en route to the cytoplasm [11]. These enveloped virions in the perinuclear space are infrequently observed by EM [5,43–46] because they are short-lived and the thin sectioning required for imaging using electrons decreases the probability that such structures will be present within the cellular volume being examined. The penetrating power of soft X-rays in unstained cryopreserved samples (> 10 μm in depth) removes the requirement for sectioning, allowing the entire depth of the cell to be imaged for any given field of view. This increases the likelihood of observing short-lived structures such as primary enveloped virus 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 the nuclear envelope, as shown in a segmented image (Fig 1E). The width of the nuclear envelope at putative sites of primary envelopment (190.5 ± 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 that substantial deformation of the nuclear envelope must occur to accommodate the presence of perinuclear virus particles.

Dark puncta representing viral capsids were also observed in the cytoplasm in close proximity to vesicles, highlighting potential 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 tetherin [47,48]. In addition, we expected to see HSV-1 particles between cells because virions are targeted to cell junctions to promote cell-cell spread [49]. Linear arrays of dark puncta were observed on the cell surface and between cells (Fig 1H and 1I) and likely represent released virus particles (extracellular virions). Virus particles increase in size during the assembly process as they accumulate their tegument and become enveloped in the cytoplasm 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 capsids (~125 nm) [38] and of capsids inside infected-cell nuclei [50]. 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 nm) [37,51]. These differences were found to be significant with a Mann-Whitney U test for unequal variance (W = 126, p-value<2.2×10⁻¹⁶).

Fluorescently tagged ICP0 and gC can be used to monitor the progression of HSV-1 infection 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 infection [16,52]. 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, respectively [16]. 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 cells using cryoSXT, we sought to obtain higher-resolution temporal information on the morphological changes that occur over the course of HSV-1 infection by using the timestamp virus. Preliminary experiments performed using infected HFF-hTERT cells were unsuccessful as they proved sensitive to prolonged exposure to the soft X-ray beam when collecting data with a 25 nm zone plate, the objective available at the time on the microscope at the synchrotron beamline used for these experiments, leading to localized sample heating and low-quality tomograms. We thus turned instead to U2OS osteosarcoma cells, which have been used previously for HSV-1 ultrastructural analysis [53,54] and have been shown previously to be robust imaging subjects that yield consistently high-quality tomograms when exposed to high doses of soft X-rays [32,33].

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 the fluorescently tagged proteins between the two cell types. Cells were infected at an MOI of 1–3 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 expressed [55]. In both cell lines, eYFP-ICP0 was expressed throughout the course of infection. However, the spatial localization of eYFP-ICP0 differed somewhat between HFF-hTERT and U2OS cells. In HFF-hTERT cells, eYFP-ICP0 was observed in the nucleus in stage 1 whereas it became more concentrated in the cytoplasm with relatively weaker signal in the nucleus in stages 2–4. However, in U2OS cells, eYFP-ICP0 expression displayed a high signal in the nucleus throughout infection while also becoming more concentrated in the cytoplasm as infection progressed, suggesting U2OS cells retain more eYFP-ICP0 in the nucleus at later stages of infection than is observed for HFF-hTERT cells (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 cells [56]. The continued high signal levels of eYFP-ICP0 in the nucleus complicated the distinction between the early stages (stages 1+2) of infection in U2OS cells.

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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 stages of infection based on the expression and localization of the early protein eYFP-ICP0 and the late protein gC-mCherry [16]. The spatiotemporal expression of these fusion proteins was similar in HFF-hTERT and U2OS cells, except for increased retention of eYFP-ICP0 in the nucleus of U2OS cells during all stages. Outlines show the nuclei and arrows indicate juxtanuclear compartments rich in gC-mCherry. Scale bars = 50 μm. Boxes show a sample of the eYFP-ICP0 intensity from the nucleus (N) and cytoplasm (C). (B) The proportion of infected cells in each stage was determined using widefield imaging at 2, 4, 6, 9, 12, and 24 hpi following infection (MOI 3) of HFF-hTERT and U2OS cells with timestamp HSV-1.

https://doi.org/10.1371/journal.ppat.1010629.g002

The spatial expression of gC-mCherry was broadly similar between HFF-hTERT and U2OS cells. gC is a viral glycoprotein expressed at late stages of virus replication [57] that is incorporated into nascent virus particles at sites of virus envelopment [58]. In HFF-hTERT cells, gC-mCherry was enriched at a juxtanuclear site in stage 3 but became fragmented and dispersed throughout the cytoplasm and at the plasma membrane by stage 4 (Fig 2A). A similar spatial expression was observed for late stage U2OS cells, with redistribution of gC from juxtanuclear sites to the periphery likely representing progressively later stages of infection. However, there existed a continuum of gC distribution between juxtanuclear and dispersed in late stages of infection in U2OS cells. This, combined with the difficulties in differentiating between early infection stages due to nuclear retention of eYFP-ICP0, led us to group U2OS infection stages into two broader categories (“early” and “late”). The designation of early or late-stage infection was determined by the absence or presence of gC-mCherry signal in eYFP-ICP0 positive cells, respectively.

Next, we probed whether progression through the replication cycle follows the same timecourse in HFF-hTERT and U2OS cells. Both cell types were inoculated with timestamp virus (MOI 3) for one hour, at which time unabsorbed viruses were inactivated with a citric acid wash, and cells were fixed at various time points over the course of 24 hrs before imaging (Fig 2B). We observed that the infection proceeds at a similar pace in both cells types, with a similar proportion of cells in equivalent stages of infection (1+2/early and 3+4/late) at each time point.

CryoSXT following infection with ‘timestamp’ HSV-1 allows temporal correlation of ultrastructural changes during infection

To characterize the changes in morphology of cellular compartments that accompany different stages of virus infection, U2OS cells were grown on perforated-carbon EM grids before being infected (or mock infected) with timestamp HSV-1 and cryogenically preserved by plunge cryocooling in liquid nitrogen-cooled liquid ethane (Fig 3A). Vitrified samples were analysed by cryo-widefield 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). Cells were infected at MOI 1 and the samples were plunge cryocooled at 9 hpi in an attempt to evenly sample the different stages of infection (Fig 2B). In total, 139 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 (S1A–S1E Fig). The observed width of nuclear capsids in U2OS cells imaged using the 25 nm zone plate (S1F Fig) is similar to those observed in infected HFF-hTERT cells imaged using the 40 nm zone plate (Fig 1J). 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.

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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 perforated 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 cryocooling 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 expression of 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 IMOD [42]. 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 with tools like Contour [60] facilitates quantitation and visualization in three dimensions of the observed cellular structures.

https://doi.org/10.1371/journal.ppat.1010629.g003

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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.

https://doi.org/10.1371/journal.ppat.1010629.t001

We observed that HSV-1 infection does not dramatically affect the morphology of the nucleus or integrity of the nuclear envelope, despite the continuous budding and fusion of capsids that occur at the inner and outer nuclear membranes, respectively, during infection. We occasionally observed bulging of the nuclear envelope into the cytoplasm without separation of the inner and outer nuclear membranes (S1G Fig). This bulging could be seen in both uninfected and infected cells. It was distinct from the separation of the inner and outer nuclear membranes caused by the expansion of the perinuclear space, which we observed in the presence of perinuclear viral particles (Fig 1D–1F). It also differed from the expansion of the perinuclear space observed previously in uninfected murine adenocarcinoma cells imaged using cryoSXT [59]. The cryopreservation protocol used for preparing our cryoSXT samples does not result in dehydration artefacts that can alter the apparent morphology of the nuclear membrane in TEM samples [59], suggesting that these bulges are not artefacts of the sample preparation, but the biological relevance of this observation remains unclear.

Striking changes were observed in the size and dispersal of vesicles during HSV-1 infection (Fig 4A and S1 Video). 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 envelopment [13]. Infected cells had a greater number of vesicles in juxtanuclear regions when compared with uninfected cells (Fig 4A and S2 Video). 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 [60]. 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 = 654) 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). Segmented vesicles (Fig 4A) were open-ended because the contrast of membranes in the sample differs based on their orientation with respect to incident X-ray beam. This arises because the sample can only be rotated by 120° during cryoSXT tilt series image acquisition, rather than 180° as would be required for isotropic data collection, due to geometric constraints between microscope and sample holder components. The edges of vesicles that lie parallel to the incident X-ray beam (i.e. the ‘sides’ of the vesicle with respect to the XY projection plane) produce high contrast, since the X-rays pass tangentially through the carbon-rich membrane of the vesicle and thus traverse a large volume of material that strongly absorbs X-rays. The ‘front’ and ‘back’ edges of the vesicle with respect to the XY projection plane yield less contrast because the X-rays pass radially through these membranes, traversing a shorter path through this carbon-rich X-ray absorbing material. The lower contrast for the front and back edges of vesicles prevented their reliable segmentation, yielding gaps in the resultant volumes.

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

CryoSXT tomograms were recorded from uninfected cells, or cells at an early or late stage of infection with timestamp HSV-1, as determined via wide field fluorescence cryo-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 [60]. Width was measured instead of volume because the segmented vesicles were open-ended owing to reduced contrast in the tomograms of membranes normal to the incident X-ray beam. 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. (C) Lipid droplets were segmented and measured using Contour [60] and their distributions were plotted on a logarithmic scale. Median volumes ± SD (hollow circles plus error bars) are shown for each group because median values are less affected than mean values by non-normal distributions. The median volume was highest in cells at early stages of infection in all three replicates. A linear plot of the distributions and significance tests for the lipid droplet volumes are shown in S2 Fig.

https://doi.org/10.1371/journal.ppat.1010629.g004

Lipid droplets are carbon-dense organelles that produce high contrast in cryoSXT and were clearly visible as dark solid spheres in the tomograms. These lipid droplets could be readily segmented using Contour [60], allowing measurement of their volumes. We observed an increase in the volume of these droplets in cells during the early stage of infection when compared with uninfected cells or to cells at the late stage of infection (Fig 4C). A small number of extremely large lipid droplets (>5×10⁷ nm³) were observed in tomograms from each of the three replicate infections, but the presence of these large droplets was not correlated with progression through the infection (S2A and S2B Fig). The distribution of lipid droplet sizes was non-Gaussian (positively skewed; S2C Fig) and a non-parametric Mann-Whitney U test confirmed that, in all three replicate experiments, the lipid droplets were significantly larger in cells at early stages of infection than in uninfected cells (S2D Fig). There is not a consistent difference in the size of lipid droplets between uninfected cells and those at late stages of infection, suggesting that lipid droplets undergo a transient change in size during infection.

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 (S3A Fig), similar to observations of mitochondria made by EM [61–63] and cryoSXT [59]. This swollen morphology can be associated with release of cytochrome c from porous mitochondria during apoptosis [61]. Swollen mitochondria were observed in each of the three independent sets of cell growth, infection and plunge cryocooling experiments performed, but these swollen mitochondria were most prevalent in the uninfected cells of replicate 3 (S3B Fig). In uninfected cells, non-swollen mitochondria were heterogeneous in shape, with numerous being small and spherical or long and curved in the same cell (Fig 5A). 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–5D and S3C Fig, and S3 Video), in line with previous observations made using super-resolution fluorescence microscopy of HFF-hTERT cells infected with the timestamp virus [16]. The number of points where mitochondria branch into two or more arms (branching nodes) was significantly increased (p<0.05) in cells at late stages of infection (20.5 ± 5.45 nodes SD; n = 15) compared with uninfected cells (7.0 ± 4.02 nodes SD; n = 15) according to a one-way ANOVA and Tukey tests performed on each replicate (Fig 5D). In some cases, the mitochondria fused into a single, branched network (Fig 5B and S3 Video), 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. Confocal microscopy qualitatively confirmed the observations made with cryoSXT that mitochondria appear more elongated in infected cells and that mitochondrial morphology in peripheral areas of the cell was more heterogenous in uninfected cells than in infected cells (S4 Fig.). However, the limited resolution of confocal imaging made it difficult to differentiate between highly-branched and separate-but-overlapping mitochondria, particularly at crowded juxtanuclear locations.

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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) Examples of spherical, short, and long mitochondria are indicated with white arrows. (B) A shift towards elongated and branched mitochondria was observed during infection. Mitochondria were segmented and differentiated using Contour [60] to highlight the abundance and 3D geometry of individual mitochondria. (C) Venn diagrams showing the percentage of tomograms at each stage of infection with Spherical, Short or Long mitochondria, or a combination of these phenotypes. The percentages of tomograms with long mitochondria were greater for cells at early- or late-stages of infection than for uninfected cells. Mitochondrial morphology was more heterogenous in uninfected cells. Combined percentages from all replicates are shown here and Venn diagrams for each replicate are shown in S2C Fig. (D) 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.

https://doi.org/10.1371/journal.ppat.1010629.g005

Golgi membranes and the microtubule network are disrupted during HSV-1 infection of U2OS cells

HSV-1 infection is known to be accompanied by dispersal of the Golgi complex and fragmentation of the trans-Golgi network [16,64,65]. However, Golgi-related compartments can be difficult to distinguish from other vesicular compartments by cryoSXT and are infrequently observed [59]. We therefore used SIM super-resolution fluorescence microscopy to monitor the changes in Golgi organisation that accompany HSV-1 infection. Fixed U2OS cells that had been infected (MOI 3) with the timestamp reporter HSV-1 for 6 hours were immunostained with the cis-Golgi marker GM130, demonstrating that the GM130⁺ Golgi membranes are clustered with a tubular morphology at early stages of infection (Fig 6A). In cells at late stages of infection the distribution of GM130 was more punctate and more widely distributed throughout the cell, consistent with fragmentation of the Golgi (Fig 6B). The gC-mCherry signal was also present at multiple sites throughout the cell, often adjacent to the GM130 signal (Fig 6B). SIM imaging of infected U2OS cells stained with the trans-Golgi network marker TGN46 also demonstrated increasing dispersion of TGN46⁺ membranes at late- versus early-stages of infection (Fig 7) and again we observed that the TGN46 signal was adjacent to, or partially overlapping with, gC-mCherry signal in cells at late stages of infection (Fig 7B).

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Fig 6. Fragmentation and dispersal of cis-Golgi membranes during HSV-1 infection.

U2OS cells infected (MOI 3) with timestamp HSV-1 were fixed at 6 hpi and imaged by SIM and deconvolution microscopy. GM130 immunolabelling was used to identify cis-Golgi membranes [107]. Dotted outlines denote the cell boundaries. (A) Cells at early stages of infection were identified by the presence of eYFP-ICP0 signal using deconvolution microscopy and by the absence of high gC-mCherry signal using SIM. GM130⁺ membranes, which appeared clustered at early stages of infection, are outlined. (B) Cells at late stages of infection were identified by the presence of high gC-mCherry signal. GM130⁺ membranes were dispersed and fragmented in these cells. Boxes (i–vii) and corresponding insets showing adjacent localization of GM130 and gC-mCherry.

https://doi.org/10.1371/journal.ppat.1010629.g006

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Fig 7. Fragmentation and dispersal of trans-Golgi membranes during HSV-1 infection.

U2OS cells infected (MOI 3) with timestamp HSV-1 were fixed 6 hpi and imaged by SIM and deconvolution microscopy. TGN46 immunolabelling was used to identify trans-Golgi network membranes [108]. Dotted outlines denote the cell boundaries. (A) Cells at early stages of infection were identified by the presence of eYFP-ICP0 signal using deconvolution microscopy and by the absence of high gC-mCherry signal using SIM. TGN46⁺ membranes, which appeared both clustered and dispersed at early stages of infection, are outlined. (B) Cells at late stages of infection were identified by the presence of high gC-mCherry signal using SIM and TGN46⁺ membranes were widely dispersed in these cells. Boxes (i–vii) and corresponding insets indicate sites of colocalization and adjacent signal between TGN46 and gC-mCherry.

https://doi.org/10.1371/journal.ppat.1010629.g007

As demonstrated above, HSV-1 infection of U2OS cells changes the morphology of lipid droplets, mitochondria, vesicles and Golgi membranes, all of which interact with the microtubule network [66–69]. While cytoskeletal filaments can occasionally be observed using cryoSXT (S1C Fig), microtubules are too thin (25 nm width) [70] to be reliably detected using this technique. We therefore used confocal fluorescence microscopy to study microtubule morphology in uninfected U2OS cells or cells infected with timestamp HSV-1 (MOI 3) at early and late stages of infection, captured at 6 and 16 hpi, respectively. Microtubules were monitored by immunolabelling β-tubulin. In uninfected cells microtubules formed long filaments that radiated out of a prominent microtubule organising centre (MTOC) (Fig 8A). By early stages of infection, MTOCs were less pronounced and microtubules no longer had a well-dispersed, radial distribution (Fig 8B). At late stages of infection, MTOCs could not be detected in most cells, the microtubule network became more compact, and fewer long filaments were observed (Fig 8C).

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Fig 8. Remodelling of microtubules during HSV-1 infection.

U2OS cells infected with timestamp HSV-1 were fixed at indicated times and imaged by confocal microscopy. β-tubulin immunolabelling was used to identify microtubules. Scale bars = 10 μm. Putative microtubule organising centres (MTOCs) are indicated with asterisks (*). (A) Uninfected cells exhibited an outspread microtubule network with long filaments, largely radiating from a putative MTOC. (B) The microtubule network was closely packed in cells at early stages of infection (6 hpi). (C) In cells at late stages of infection (16 hpi), fewer long filaments were observed, the cells lacked noticeable MTOCs, and the network became very closely packed.

https://doi.org/10.1371/journal.ppat.1010629.g008

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 Hanks’ Balanced Salt Solution (HBSS; Thermo Fisher). The fiducials were sonicated at 80 kHz (100% power) and 6°C to prevent aggregation. 3 mm gold EM finder grids with a perforated carbon film (R 2/2, 200 mesh) were purchased from Quantifoil (AU G200F1 finder, batches Q45352 & Q45353). Poly-l-lysine was purchased from Sigma Aldrich.

Cell lines

Mycoplasma-free U2OS cells (ATCC HTB-96; RRID CVCL_0042) and human foreskin fibroblast cells immortalized with human telomerase reverse transcriptase (HFF-hTERT cells) [96] were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher) supplemented with 10% (v/v) foetal bovine serum (FBS; Capricorn), 2 mM l-glutamine (Thermo Fisher), and 100 U/mL penicillin/streptomycin (Thermo Fisher). HBSS and 0.25% Trypsin-EDTA (Thermo Fisher) were used to wash and detach adherent cells, respectively. Cells were maintained in a humidified 5% CO₂ atmosphere at 37°C.

Biosafety measures

All cells and viruses were handled according to containment level 2 (CL2) guidelines and a risk assessment was carried out and approved by the Rutherford Appleton Laboratory (RAL) Health and Safety Committee. EM grids containing cells and viruses were handled in appropriate microbiology safety cabinets and forceps were regularly washed in 70% (v/v) ethanol. Personal protective equipment in the form of lab coats, lab gloves, and goggles were worn to protect experimenters. All tissue-culture, cryocooling, and imaging equipment was stored in CL2 laboratories.

Recombinant viruses

Infections were performed using recombinant HSV-1 strain KOS expressing either the endogenously tagged viral proteins eYFP-VP26 and gM-mCherry (Fig 1) or the endogenously tagged viral proteins eYFP-ICP0 and gC-mCherry (timestamp HSV-1, Figs 2–8 and S1–S4 Figs) [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% amplitude 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 [97].

Infection assays

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 [33]. 3×10⁵ U2OS or HFF-hTERT cells per well were seeded in 6-well plates containing the treated EM grids and were incubated overnight. Subsequently, the cells were infected with timestamp HSV-1 at an MOI of 1–3. For widefield microscopy of timestamp HSV-1 to measure the progression of replication over time, U2OS and HFF-hTERT cells were allowed to grow overnight following seeding in 6-well plates at 2×10⁵ cells per well (Fig 2A) or on borosilicate coverslips in 12-well plates at 1×10⁵ cells per well (Fig 2B). The cells were infected with the recombinant HSV-1 with an MOI of 1–3. For SIM and confocal microscopy, U2OS cells were seeded on borosilicate coverslips in 12-well plates overnight at 1×10⁵ cells per well. The cells were infected with the recombinant HSV-1 with an MOI of 3. The time of inoculation was designated the start time of infection. For all infections, to maximize adsorption of virus, cells were incubated in a low volume of medium (250 μL/well in 12-well plates and 500 μL/well in 6-well plates) for 1 hour in a humidified 5% CO₂ atmosphere at 37°C and the plates were swirled every 15 minutes. For widefield imaging under cryogenic conditions and cryoSXT, the medium was topped up to 2 mL and the samples were incubated for 9 hours alongside uninfected controls, except for the samples in Fig 1, which were incubated for 16 hours. The EM grids were overlayed with 2 μL of the gold fiducial working mixture as 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 then plunged into liquid nitrogen-cooled liquid ethane and transferred into liquid nitrogen storage before imaging. For the timestamp HSV-1 images (Fig 2A), the inoculum was diluted to 2 mL with medium for the remainder of the incubation (9–24 hours). For the experiments measuring the progression of timestamp HSV-1 replication over time (Fig 2B), media was aspirated after the 1 hour of incubation and the cells were treated for 1 min with citric acid (40 mM citric acid pH 3, 135 mM NaCl, 10 mM KCl) to inactivate unabsorbed virus. Cells were then washed thrice with PBS and then overlain with 500 μL of fresh medium before incubation for a further 2–24 hours. For confocal microscopy and SIM, the media were topped up to 1 mL for the remainder of the incubation (see figure legends for varied time points). For cells stained with MitoTracker Deep Red FM (Thermo Fisher), the media was aspirated 30 minutes before fixation and washed twice with serum-free media. 50 nM MitoTracker in serum-free media was added to the cells for 30 minutes. For all samples not prepared on EM grids, the cells were washed twice with HBSS or PBS and were fixed with 4% (v/v) formaldehyde for 20 minutes, followed by three HBSS/PBS washes. For confocal microscopy and SIM, the cells were washed twice with PBS, permeabilised with 0.1% saponin in PBS for 30 minutes at room temperature on a rocking platform and were blocked for 30 minutes with a PBS solution of 0.1% saponin and 5% (v/v) FBS at room temperature on a rocking platform. The samples were stained with either 2.5 μg/mL mouse anti-human GM130 (Clone 35/GM130 (RUO), BD Biosciences, RRID: AB_398142), 10 μg/mL mouse anti-human TGN46 (SAB4200355, Merck), or a 1 in 20 dilution of rat anti-β-tubulin (clone YL1/2) hybridoma supernatant [98] in blocking solution, washed, then stained with 4 μg/mL goat anti-mouse Alexa Fluor 647 antibody (A-21235, ThermoFisher) in blocking solution, washed and mounted using ProLong Gold containing DAPI (P36930, Thermo Fisher).

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) 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 as described above. A liquid nitrogen cryostage (Linkam Scientific) was used to maintain the samples at 77 K. Each grid was mapped in its entirety in the brightfield and fluorescent channels (as above) using LINK (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). Only one zone plate can be installed in the microscope and the zone plate is not user changeable. The installed zone plate differed across the beam time allocations use for this study, with all images being collected using the 25 nm zone plate except for Fig 1C, 1D and 1G–1I, for which the 40 nm zone plate was used. 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 scans 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 140° in increments of 0.2° or 0.5° per image, with maximum tilt angles of −60°/+60° and −70°/+70° for the 25nm and 40nm objective, respectively. SXT tilt series were processed using IMOD (version 4.9.2) [42]. 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.

Structured illumination microscopy and deconvolution

A custom, three-colour SIM microscope [99] was used to collect images. A ferroelectric binary Spatial Light Modulator (SLM) (SXGA-3DM, Forth Dimension Displays) was used to pattern the light with a grating structure (3 angles and 3 phases). Light was collected by a 60× water immersion objective with a NA of 1.2 (UPLSAPO60XW, Olympus) and a sCMOS camera (C11440, Hamamatsu). eYFP-ICP0 fluorescent emission was captured using a 488 nm laser (iBEAM-SMART-488, Toptica) and an BA510-550 (Olympus) emission filter. gC-mCherry fluorescence was captured using a 561 nm laser (OBIS561, Coherent) and a BrightLineFF01-600/37 filter (Semrock). AF647 fluorescence was captured using a 640 nm laser (MLD640, Cobolt) and a BrightLineFF01-676/29 filter (Semrock). Background-reduced and resolution-enhanced images were reconstructed from raw SIM data using FairSIM [100]. Deconvolution was performed alongside using a Richardson-Lucy algorithm with 5 iterations. 100 nm beads (TetraSpeck Microspheres, Thermo Fisher) were used to determine the shifts in X and Y for channel alignment. The channels were aligned using the TransformJ plugin in Fiji [101].

Confocal microscopy

A Zeiss LSM700 inverted confocal AxioObserver.Z1 microscope was used at room temperature to capture images with ZEN software (Zeiss). A 100× apochromatic objective with oil immersion and pinhole set to 1 airy unit was used to collect images. Z stacks containing 1024×1024 pixels at 400 nm increments were captured within a 16-bit unsigned range using 8-fold line averaging. Maximum Z projections were generated using Fiji [101].

Segmentation

Mitochondria were segmented using Contour, a bespoke semi-automated segmentation and quantitation tool developed with Python 3 (full details on Contour can be found in [60]). 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 elements [101]. 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 plugin [101] 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 ImageJ [101]. Cytoplasmic vesicles were segmented from 12 well-reconstructed X-ray tomograms lacking X-ray damage. Lipid droplets were automatically segmented from 94 tomograms using Contour from 8-bit tomograms. Tomograms were excluded from segmentation if they were poorly reconstructed, were subjected to X-ray damage, or were from thick sections and contained out-of-focus lipid droplets in some projection planes. Given that gold fiducials and lipid droplets have similar projection intensities in 8-bit images, gold fiducials were also included in the automatic segmentation. During curation of the segmented volumes, gold fiducials were manually erased. This was possible because they are easy to distinguish from lipid droplets based on the higher intensity of their missing wedge artefacts. The projection intensities of lipid droplets and gold fiducials are lower than other material in the tomograms, including noise, and they could thus be segmented based purely on threshold values without applying a width restriction. This prevented the exclusion of very small lipid droplets. Volumes were calculated in Contour for each lipid droplet. Lipid droplets that could not be individually resolved or were cut off at the edges of tomograms were excluded from the analysis.

Graphs and statistics

Distributions of capsid and virion widths were illustrated using a Violin SuperPlot [102], 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 package [103] in R studio [104]. The distribution of vesicle widths were illustrated using a SuperPlot [105], with data grouped by source tomograms. The numbers of mitochondrial branch points (branching nodes) were illustrated using a Violin SuperPlot [102], 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). The t-test was two-tailed because we observed a normal distribution of widths and it was paired because the two data points (width of nuclear envelope at perinuclear viral particle and in nearby region) were collected from the same tomogram. The variance in the width of the extracellular virions was greater than four times the variance in the width of nuclear capsids. As a result, a Mann-Whitney U test for unequal variance was used to assess the significance of the difference in width of capsids and virions using R Studio [104]. To assess significance of differences in mean vesicle widths, a one-way ANOVA and Tukey tests were performed using Prism version 8.2.1 (GraphPad Software). These tests were used instead of t tests to avoid the higher risk of type I errors associated with performing multiple t tests on more than two conditions. For the same reason, one-way ANOVA and Tukey tests were used to assess significant differences in the number of mitochondrial branching nodes (using R Studio [104]). The data distributions for the lipid droplet volumes were positively skewed and median volumes were chosen for further analysis because they are less affected by extreme values than means. Owing to the skew, Mann-Whitney U tests were used to determine significance of differences between conditions for the lipid droplet volumes. The tomograms used to quantitate the number of branching nodes were selected according to the following criteria. Firstly, we excluded tomograms that were poorly reconstructed (e.g. due to displacement of the rotational axis during data collection or due to a lack of fiducials or lipid droplets for image alignment). Next, we only included tomograms in which the mitochondria were well dispersed throughout the field of view to ensure that we didn’t systematically underestimate the number of branching nodes by including tomograms where only small fragments of individual mitochondria were visible, for example in the corners of the field of view. Finally, we excluded tomograms that contained swollen mitochondria because this was taken to indicate that the cells might be undergoing apoptosis, a process known to cause significant alteration to mitochondrial morphology [61]. We assessed an equal number of tomograms for each replicate and condition to avoid introducing unequal variance into our ANOVA test.