Soft X-ray Tomography Reveals HSV-1-Induced Remodeling of Human B Cells

Chen, Jian-Hua; Vanslembrouck, Bieke; Ekman, Axel; Aho, Vesa; Larabell, Carolyn A.; Gros, Mark A. Le; Vihinen‐Ranta, Maija; Weinhardt, Venera

doi:10.3390/v14122651

Cited by 9 publications

(4 citation statements)

References 53 publications

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“…After generating and characterising the mutants, defects in virus assembly were explored with high- resolution imaging techniques under cryogenic conditions. Cryogenic imaging using soft-X-ray tomography captures the ultrastructure of the cell in 3D and has the advantage of allowing the study of samples in a near-native state without the need for chemical fixation 26,50 . U2OS osteosarcoma cells were used in this study because they have been used for ultrastructural HSV-1 research and have been demonstrated to be durable under X-ray exposure ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Nahas,

Connor,

Wijesinghe

et al. 2024

Preprint

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Numerous viral genes are involved in assembly of herpes simplex virus-1 (HSV-1), but their relative importance and function remain poorly characterised. Transmission electron microscopy has been used to study viral protein function in cells infected with HSV-1 mutants; however, these studies were usually conducted without correlative light microscopy to identify specific viral components. In this study, fluorescent capsid (eYFP-VP26) and envelope (gM-mCherry) proteins were imaged by structured illumination microscopy under cryogenic conditions (cryoSIM) and cellular ultrastructure was captured from the same infected cells using cryo-soft-X-ray tomography (cryoSXT). Nine fluorescent HSV-1 mutants, each lacking a different viral protein, were compared to assess the importance of viral proteins in different stages of HSV-1 morphogenesis. The relative importance of five viral proteins to nuclear egress were ranked (pUL34 > pUL21 > VP16 > pUL16 > pUS3) according to the levels of attenuation observed for each virus. Correlative imaging also revealed the roles of five viral proteins in cytoplasmic envelopment. VP16 was found to be important in capsid delivery to envelopment compartments, while cytoplasmic clusters of virus particles plus features of stalled envelopment not previously described were observed in the absence of pUL11, pUL51, gK, and gE. Finally, this 3D imaging approach was used to capture different assembly stages during cytoplasmic envelopment and to determine that envelopment occurs by particle budding rather than wrapping. The findings demonstrate that tomographic 3D correlative imaging is an emerging technology that sheds new light on viral protein functions and virion morphogenesis.ImportanceTo 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. pUL16 and pUL21 were shown to be important in nuclear egress of HSV-1, and we found that VP16 promotes nuclear egress and delivery of capsids to cytoplasmic envelopment compartments. pUL11, pUL51, gK, and gE were also 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. 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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Section: Resultsmentioning

confidence: 99%

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

Nahas,

Connor,

Wijesinghe

et al. 2024

Preprint

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“…In addition to T cells, HSV-1 infection in human B cells has been investigated in a previous study. The research found that several cellular structures of B cells, such as cytoplasmic stress granules and multivesicular structures, were changed during HSV-1 infection (Chen et al, 2022). Nevertheless, there was no data about HSV-1 inducing actin polymerization and filopodia formation in B cells.…”

Section: Hsv Entry Into T Lymphocytesmentioning

confidence: 98%

Impact of actin polymerization and filopodia formation on herpes simplex virus entry in epithelial, neuronal, and T lymphocyte cells

Sasivimolrattana,

Bhattarakosol

2023

Front. Cell. Infect. Microbiol.

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Herpes simplex virus type 1 (HSV-1) has been known as a common viral pathogen that can infect several parts of the body, leading to various clinical manifestations. According to this diverse manifestation, HSV-1 infection in many cell types was demonstrated. Besides the HSV-1 cell tropism, e.g., fibroblast, epithelial, mucosal cells, and neurons, HSV-1 infections can occur in human T lymphocyte cells, especially in activated T cells. In addition, several studies found that actin polymerization and filopodia formation support HSV-1 infection in diverse cell types. Hence, the goal of this review is to explore the mechanism of HSV-1 infection in various types of cells involving filopodia formation and highlight potential future directions for HSV-1 entry-related research. Moreover, this review covers several strategies for possible anti-HSV drugs focused on the entry step, offering insights into potential therapeutic interventions.

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“…[ 4–10 ] Because of these unique characteristics, SXT has been recently used in a wide range of applications, from phenotyping of cells such as bacteria, yeast, and advanced eukaryotes [ 11–16 ] to investigating cell–nanoparticle, [ 17,18 ] cell–parasite, [ 19–21 ] and cell–virus interactions. [ 22–28 ]…”

Section: Introductionmentioning

confidence: 99%

“…[4][5][6][7][8][9][10] Because of these unique characteristics, SXT has been recently used in a wide range of applications, from phenotyping of cells such as bacteria, yeast, and advanced eukaryotes [11][12][13][14][15][16] to investigating cellnanoparticle, [17,18] cell-parasite, [19][20][21] and cell-virus interactions. [22][23][24][25][26][27][28] SXT is suitable for imaging of a wide variety of cell types. However, some investigators have reported that soft X-ray imaging is limited to specimens less than 10 μm thick.…”

Section: Introductionmentioning

confidence: 99%