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. 2023 Feb 28;97(2):e0152822.
doi: 10.1128/jvi.01528-22. Epub 2023 Jan 23.

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

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

Joshua J Walston et al. J Virol. .

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 ΔBDLF2 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. IMPORTANCE The ubiquitous herpesvirus Epstein-Barr virus (EBV) is associated with cancers of B lymphocytes and epithelial cells and is primarily transmitted in saliva. While several models exist for analyzing the life cycle of EBV in B lymphocytes, models of EBV infection in the epithelium have more recently been established. Using an organotypic culture model of epithelium that we previously determined accurately reflects EBV infection in situ, we have ascertained that the loss of the viral envelope protein BDLF2 had little effect on the EBV life cycle in B cells but severely restricted the number of infected cells in organotypic cultures. Loss of BDLF2 has a substantial impact on the size of infected areas, suggesting that BDLF2 plays a specific role in the spread of infection in stratified epithelium.

Keywords: Epstein-Barr virus; organotypic cultures; stratified epithelium; viral spread.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
BDLF2 expression was successfully knocked out in ΔBDLF2 rEBV. (A) To generate ΔBDLF2 rEBV, the BDLF2 gene was replaced with a puromycin resistance gene under the control of the SV40 promoter by homologous recombination. (B) Akata cells latently infected with either BX1 rEBV or ΔBDLF2 recombinant EBV were induced to productively replicate virus by cross-linking surface IgG with anti-human F(ab)2. At 48 h postinduction, expression of BZLF1 (red) and BDLF2 (green) were identified by immunofluorescence. (C) Expression of EBV genes BDLF1, BDLF2, and BDLF3 were assessed through reverse transcription-PCR. GAPDH was used as an endogenous positive control. (D) Expression of the EBV proteins EBNA1, BZLF1, LMP1, and BDLF2 were analyzed by immunoblotting. (E) Membrane fractions were extracted from purified virions, and BMRF2 expression was analyzed by immunoblotting.
FIG 2
FIG 2
BX1 and ΔBDLF2 rEBV are equivalently competent for virus replication and B-cell immortalization. (A) Akata BL cells containing BX1 or ΔBDLF2 rEBV were induced to productively replicate virus by the addition of anti-human F(ab)2 for 24 h. Encapsidated viral genomes released into the cell medium were quantified 4 days later by qPCR. Shown is a standard box-and-whisker Tukey plot of genomes present in cell medium. Data are representative of each induction used for cell infections (N = 3). (B) BX1 and ΔBDLF2 rEBV were used to infect both Raji B and HEK293 cells. At 4 dpi, the number of infected cells was determined by quantifying GFP-positive cells by flow cytometry. Cells exposed to mock-induced Akata cell supernatant fractions were included as negative controls for GFP. Infection of Raji B cells was found to be statistically significant (P = 0.0003), while infection of HEK293 cells was not (P = 0.1). (C) Primary B lymphocytes were infected with rEBV at multiple MOIs and were assessed at 6 weeks postinfection for immortalization. Data are representative of total wells immortalized out of 12 per MOI.
FIG 3
FIG 3
EBV lacking the viral thymidine kinase gene can replicate in organotypic cultures but is less efficient. (A) The size of infected foci generated by wild-type EBV (circles) or (squares) BX1 rEBV within organotypic cultures was measured at different times postinfection. At least 10 different fields of view per sample were used to measure the size of infected foci. Statistical significance was determined by the Dunnett test, and the differences in sizes of infected foci at 4 dpi and 6 dpi were found to be extremely significant (P = 0.003 and 0.0027, respectively). Data are represented as averages and standard deviations from three different donor pools. (B) Impact of acyclovir treatment on the size of infected foci in organotypic cultures infected by wild-type Akata cells. At least 10 different fields of view per sample were used to measure the size of infected foci. Statistical significance was determined by the Dunnett test. The sizes of infected foci were significantly different from vehicle when using 5.0 or 50 μg/mL (P = 0.0004 and <0.0001, respectively). Data are represented as averages and standard deviations from three different donor pools.
FIG 4
FIG 4
The morphology of organotypic cultures is comparable whether uninfected or infected with either BX1 or ΔBDLF2 rEBV. Uninfected organotypic cultures or those infected with either BX1 or ΔBDLF2 rEBV were examined following hematoxylin and eosin staining for overall thickness and stratification from basal cells through cornified cells. This figure is representative of three experiments performed with two different donor pools (1 and 2).
FIG 5
FIG 5
The loss of BDLF2 results in reduced EBV infection of organotypic cultures. Organotypic cultures were infected with BX1 or ΔBDLF2 rEBV, and at 5 dpi EBV-infected keratinocytes were identified by expression of GFP (green, encoded by the rEBV genome) and keratin-5 (red) as a marker of epithelial cells. Representative images are shown from three separate experiments. Scale bars represent 500 μm.
FIG 6
FIG 6
Productive-cycle proteins are present in both BX1- and ΔBDLF2 rEBV-infected organotypic cultures. The EBV proteins LMP1, BMRF1, and gHgL (green) were detected during both BX1 and ΔBDLF2 rEBV infection of keratin 5-expressing epithelial cells (red) at 5 dpi. Representative images are shown from three separate experiments.
FIG 7
FIG 7
The loss of BDLF2 results in fewer and smaller foci of EBV infection. Cells productively replicating EBV were identified by the presence of BDLF2 (green; plasma membrane) and BZLF1 (red; nuclear). (A) Representative images from three separate experiments of uninfected or BX1 or ΔBDLF2 rEBV-infected organotypic cultures showing a BZLF1-positive focus of infection at 5 dpi. The arrowhead indicates a BZLF1-positive cell in the ΔBDLF2 rEBV-infected tissue. (B) BZLF1-positive foci were quantified using multiple sections (n = 100) of organotypic cultures at 5 dpi with either BX-1 or ΔBDLF2 rEBV; the mean and standard deviation are shown for the number of BZLF1-positive foci found in multiple sections of tissue at 5 dpi with the specified virus strains (N = 60 sections). (C) The number of BZLF1-positive nuclei within a given foci were counted at 5 dpi. Means and standard deviations are shown (number of foci: BX1, n = 997; ΔBDLF2, n = 255). Statistical significance was determined using a random effects Poisson regression analysis (P = 2 × 10−16). (D) Encapsidated viral genomes were isolated from an entire organotypic culture infected with BX1 or ΔBDLF2 rEBV at 5 dpi and assessed by qPCR. The values plotted are from three separate experiments using two different donor pools. Statistical significance was assessed by linear regression after cubic root transformation of the data (P = 2 × 10−16). PCR was performed in triplicate, and data are displayed as standard box-and-whisker Tukey plots in log format.

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