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July 17th, 2025
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University of Cambridge
microbiology
biorxiv

Applying 3D correlative structured illumination microscopy and X-ray tomography to characterise herpes simplex virus-1 morphogenesis

Nahas, K. L.Open in Google Scholar•Connor, V.Open in Google Scholar•Wijesinghe, K. J.Open in Google Scholar•Barrow, H. G.Open in Google Scholar•Dobbie, I. M.Open in Google Scholar•Harkiolaki, M.Open in Google Scholar•Graham, S. C.Open in Google Scholar•Crump, C. M.Open in Google Scholar

To date, the characterisation of HSV-1 mutants in the study of virus assembly has been limited to transmission electron microscopy (TEM) without the addition of correlative light microscopy to identify fluorescently labelled viral proteins. In addition, only a small number of mutants are typically used in each study. Herein, a comparative analysis of nine HSV-1 mutants lacking specific structural proteins was performed using correlative fluorescence microscopy and X-ray tomography for the first time, revealing the relative roles of each viral protein in virus assembly. pUL11, pUL51, gK, and gE were shown to have important roles in cytoplasmic envelopment, with the loss of their functions leading to various stalled cytoplasmic envelopment phenotypes involving polarised arrays of capsids at one side of cytoplasmic vesicles that to our knowledge have never been seen with TEM. This correlative imaging approach enabled the study of cytoplasmic envelopment in 3D, revealing an envelopment mechanism driven by capsid budding rather than membrane wrapping. Further cryoSIM experiments revealed that pUL16 and pUL21 are important in nuclear egress of HSV-1 and that VP16 promotes nuclear egress and delivery of capsids to cytoplasmic envelopment compartments. By providing novel and comparative insights into the roles of different viral proteins in various stages of HSV-1 assembly, these findings highlight the utility of correlative cryo-fluorescence plus cryo-soft-X-ray tomography for probing trajectories of intracellular pathogen assembly.

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