The Epstein-Barr Virus Glycoprotein BDLF2 Is Essential for Efficient Viral Spread in Stratified Epithelium

Abstract

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The DBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium.

Original language	English (US)
Journal	Journal of virology
Volume	97
Issue number	2
DOIs	https://doi.org/10.1128/jvi.01528-22
State	Published - Feb 2023

All Science Journal Classification (ASJC) codes

Microbiology
Immunology
Insect Science
Virology

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This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1128/jvi.01528-22

Cite this

@article{d0c1e203b5824a11b4ec3d0414cd8f03,

title = "The Epstein-Barr Virus Glycoprotein BDLF2 Is Essential for Efficient Viral Spread in Stratified Epithelium",

abstract = "Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The DBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium.",

author = "Walston, {Joshua J.} and Hayman, {Ian R.} and Mindy Gore and Mary Ferguson and Temple, {Rachel M.} and Jason Liao and Samina Alam and Craig Meyers and Tugizov, {Sharof M.} and Lindsey Hutt-Fletcher and Sample, {Clare E.}",

note = "Funding Information: We thank Nate Sheaffer and Jade Vogel from the Penn State Cancer Institute Flow Cytometry Shared Resource for assistance with flow cytometry analysis and the Penn State Cancer Institute Biostatistics and Bioinformatics Shared Resource for statistical analysis. We also thank Lynn Budgeon and Debra Shearer for tissue processing. We thank J. Hearing, R. Longnecker, and G. Miller for providing antibodies, T. Kanda for the EBV Akata BAC, and J. Sample for critical reading of the manuscript. Publisher Copyright: Copyright {\textcopyright} 2023 American Society for Microbiology. All Rights Reserved.",

year = "2023",

month = feb,

doi = "10.1128/jvi.01528-22",

language = "English (US)",

volume = "97",

journal = "Journal of virology",

issn = "0022-538X",

publisher = "American Society for Microbiology",

number = "2",

}

TY - JOUR

T1 - The Epstein-Barr Virus Glycoprotein BDLF2 Is Essential for Efficient Viral Spread in Stratified Epithelium

AU - Walston, Joshua J.

AU - Hayman, Ian R.

AU - Gore, Mindy

AU - Ferguson, Mary

AU - Temple, Rachel M.

AU - Liao, Jason

AU - Alam, Samina

AU - Meyers, Craig

AU - Tugizov, Sharof M.

AU - Hutt-Fletcher, Lindsey

AU - Sample, Clare E.

N1 - Funding Information: We thank Nate Sheaffer and Jade Vogel from the Penn State Cancer Institute Flow Cytometry Shared Resource for assistance with flow cytometry analysis and the Penn State Cancer Institute Biostatistics and Bioinformatics Shared Resource for statistical analysis. We also thank Lynn Budgeon and Debra Shearer for tissue processing. We thank J. Hearing, R. Longnecker, and G. Miller for providing antibodies, T. Kanda for the EBV Akata BAC, and J. Sample for critical reading of the manuscript. Publisher Copyright: Copyright © 2023 American Society for Microbiology. All Rights Reserved.

PY - 2023/2

Y1 - 2023/2

N2 - Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The DBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium.

AB - Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The DBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium.

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