Near-native state imaging by cryo-soft-X-ray tomography of uninfected and herpes simplex virus (HSV)-1 infected mammalian cells
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Nahas, K., Connor, V., Harkiolaki, M., Crump, C., & Graham, S. (2021). Near-native state imaging by cryo-soft-X-ray tomography of uninfected and herpes simplex virus (HSV)-1 infected mammalian cells [Dataset]. https://doi.org/10.17863/CAM.78593
This dataset consists of 3D tomographic data collected using cryo-soft-X-ray tomography (cryoSXT) and cryo wide field fluorescence microscopy data. These data were collected for a study designed to assess (a) if herpes simplex virus (HSV)-1 could be detected by cryoSXT and (b) how the morphology and organisation of cytoplasmic vesicles and mitochondria change during herpes simplex virus-1 (HSV-1) infection. Cryo-soft-X-ray tomography collection parameters === An UltraXRM-S/L220c X-ray microscope (Carl Zeiss Xray microscopy) was used to collect the datasets at Beamline B24 at Diamond Light Source. The raw data, known as tilt series, consist of a collation of images collected from the same field of view at different angles. These were collected within a maximum range of -70° and +70° degrees and at increments of 0.2° or 0.5° and with an exposure time of 0.5 seconds or 1 second. 500 eV X rays were focused with a zone plate objective capable of a nominal resolution of 25 nm or 40 nm. Full details on parameters used for individual tilt series can be found in the attached tomographic_collection_parameters.csv file. Tomograms were reconstructed in Imod version 4.9.2. For each field of view, a tilt series and a reconstructed tomogram are provided. Detection of HSV-1 by cryo-soft-X-ray tomography === Tomograms were collected from human foreskin fibroblast cells that had been immortalised with hTERT (HFF-hTERT). The ultrastructure of uninfected cells was compared with that of HSV-1-infected cells to determine if HSV-1 particles could be detected with cryoSXT. In this dataset, we note capsids in the nuclei, viral particles in the nuclear envelope and cytoplasm, and virions at the exposed cell surface and at cell junctions. Changes to cytoplasmic vesicles and mitochondria during HSV-1 infection === We used human osteosarcoma cells (U2OS) to study changes to cytoplasmic vesicles and mitochondria. A population of synchronously infected cells progress through infection at different rates and this could affect the state of these cellular compartments. To account for this, we used a recombinant of HSV-1, known as the timestamp virus, which contains two fusion proteins with different temporal expression. This allowed us to distinguish between early stages of infection (using the immediate early protein eYFP-ICP0) and late stages (using the late protein gC-mCherry). First we collected fluorescence data from infected cells to identify early-stage and late-stage cells for subsequent imaging by cryoSXT. To collect fluorescence data, we used 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) with the following filters: 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). This fluorescence data are supplied here in the form of a map of the whole sample grid. Second, we imaged these early-stage and late-stage cells in addition to uninfected cells on the X-ray microscope as described above. Data were collected from three independent replicates.
Tomograms were reconstructed from tilt series using Imod version 4.9.2 (https://bio3d.colorado.edu/imod/). Images in the tilt series were coarsely aligned with a high frequency cut-off radius of 0.1 and were finely aligned using fiducial tracking. Boundary models were generated to reorient the dataset in 3D and a final alignment was performed using linear interpolation. The back projection strategy was used to reconstruct the tomogram and radial filtering was applied to reduce noise by running 20 iterations of a simultaneous iterations reconstruction technique (SIRT)-like filter. Fluorescent images from the widefield Zeiss AxioImager2 microscope were collected using LINK software from Linkam Scientific (https://www.mccrone.com/product/linkam-link-system-control-and-image-capture-software/). All tomogram tilt series (.st) and reconstruction (.rec) files have been compressed using xz ("xz -T4 -9 <filename>") and can be uncompressed using "unxz <filename.xz>". The file F48_tomogram.rec.xz was split into two chunks using the Unix "split" command to reduce the file size below 2 Gb. The file can be reconstituted using the Unix "cat" command, e.g. "cat F48_tomogram.rec.xz.1 F48_tomogram.rec.xz.2 > F48_tomogram.rec.xz" The .st and .rec files can be opened using image viewing programs such as Fiji (https://imagej.net/software/fiji/). The .rec files can also be opened with ChimeraX (https://www.rbvi.ucsf.edu/chimerax/).
cryoSXT, Herpesvirus, virus:host interactions
Wellcome Trust (098406/Z/12/B)
This record's DOI: https://doi.org/10.17863/CAM.78593
Attribution 4.0 International (CC BY 4.0)
Licence URL: https://creativecommons.org/licenses/by/4.0/
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