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

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. ImportanceUltrastructural 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.