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.
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.
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.
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.
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.
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.
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.
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.
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.