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Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

Nawandar DM, Wang A, Makielski K, Lee D, Ma S, Barlow E, Reusch J, Jiang R, Wille CK, Greenspan D, Greenspan JS, Mertz JE, Hutt-Fletcher L, Johannsen EC, Lambert PF, Kenney SC - PLoS Pathog. (2015)

Bottom Line: Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies.We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture.In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America; Cellular and Molecular Biology Graduate Program, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

ABSTRACT
Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

No MeSH data available.


Related in: MedlinePlus

EBV-infected NOKs cells retain ability to be partially differentiated.Uninfected NOKs (left panel) and EBV-infected NOKs-Akata (right panel) cells were grown in organotypic raft culture, samples formalin fixed, embedded in paraffin and 5-micron thick sections analyzed. A). Images from hematoxylin and eosin (H&E) stained sections are shown. Note the presence of squames at the top of the NOKs-Akata raft indicative of terminal differentiation. Also note that the basal compartment of the NOKs-Akata raft is much less well organized with signs of migration/invasion of the epithelial cells into the underlying dermal equivalent (poorly stained portion of the image) at the bottom of the image. Immunofluorescent images from sections of rafts stained with antibody against the epithelial cell differentiation markers, cytokeratin 10 (B), and involucrin (C) are shown. Cytokeratin 10 (K10)- and involucrin- positive cells are stained red. Blue nuclear counter stain is DAPI. Note the paucity of K10- and involucrin- positive cells in the NOKs-Akata raft indicating a perturbation of normal differentiation by EBV, even though morphologically terminal differentiation does occur as evidenced by the presence of squames.
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ppat.1005195.g001: EBV-infected NOKs cells retain ability to be partially differentiated.Uninfected NOKs (left panel) and EBV-infected NOKs-Akata (right panel) cells were grown in organotypic raft culture, samples formalin fixed, embedded in paraffin and 5-micron thick sections analyzed. A). Images from hematoxylin and eosin (H&E) stained sections are shown. Note the presence of squames at the top of the NOKs-Akata raft indicative of terminal differentiation. Also note that the basal compartment of the NOKs-Akata raft is much less well organized with signs of migration/invasion of the epithelial cells into the underlying dermal equivalent (poorly stained portion of the image) at the bottom of the image. Immunofluorescent images from sections of rafts stained with antibody against the epithelial cell differentiation markers, cytokeratin 10 (B), and involucrin (C) are shown. Cytokeratin 10 (K10)- and involucrin- positive cells are stained red. Blue nuclear counter stain is DAPI. Note the paucity of K10- and involucrin- positive cells in the NOKs-Akata raft indicating a perturbation of normal differentiation by EBV, even though morphologically terminal differentiation does occur as evidenced by the presence of squames.

Mentions: The differentiation program of stratified epithelia can be recapitulated in vitro using the organotypic culture technique in which epithelial cells are plated on a dermal equivalent composed of human fibroblasts embedded in collagen, allowed to grow to confluence, then raised to the air/liquid interface (thereby commonly referred to as 'raft' cultures) and cultured for approximately two weeks’ time during which the epithelial cells proliferate, stratify, and daughter cells that lose contact with the dermal equivalent undergo terminal differentiation. Rafting is considered the gold standard method for recapitulating the normal differentiation program of stratified epithelial in vitro. To determine if EBV-infected NOKs cells (NOKs-Akata) retain the ability to be differentiated, uninfected and EBV-infected NOKs were grown in raft cultures. As shown in Fig 1A, the EBV-infected NOKs cells undergo stratification as does the uninfected parental cell population, with classical morphological signs of differentiation including the presence of terminally differentiated squames at the top surface, clearly visible in the EBV-infected NOKs. Closer examination of the EBV infected cells, however, showed evidence of a much less well organized basal layer of epithelial cells (the layer of cells directly in contact with the underlying dermal equivalent), with signs of epithelial migration/invasion into the underlying dermal equivalent, suggestive of a partial disruption in the normal program of epithelial cell differentiation. Consistent with these morphological changes, the pattern of expression of cytokeratin 10 (K10) and involucrin, cellular proteins whose expression is induced in suprabasal cells, was less uniform in the EBV-infected raft culture (Fig 1B and Fig 1C). These observations validate recent findings made using other approaches for inducing epithelial cell differentiation that less well recapitulate the in vivo process: suspension of epithelial cells in methylcellulose or growth of epithelial cells in high concentration of calcium chloride [42].


Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

Nawandar DM, Wang A, Makielski K, Lee D, Ma S, Barlow E, Reusch J, Jiang R, Wille CK, Greenspan D, Greenspan JS, Mertz JE, Hutt-Fletcher L, Johannsen EC, Lambert PF, Kenney SC - PLoS Pathog. (2015)

EBV-infected NOKs cells retain ability to be partially differentiated.Uninfected NOKs (left panel) and EBV-infected NOKs-Akata (right panel) cells were grown in organotypic raft culture, samples formalin fixed, embedded in paraffin and 5-micron thick sections analyzed. A). Images from hematoxylin and eosin (H&E) stained sections are shown. Note the presence of squames at the top of the NOKs-Akata raft indicative of terminal differentiation. Also note that the basal compartment of the NOKs-Akata raft is much less well organized with signs of migration/invasion of the epithelial cells into the underlying dermal equivalent (poorly stained portion of the image) at the bottom of the image. Immunofluorescent images from sections of rafts stained with antibody against the epithelial cell differentiation markers, cytokeratin 10 (B), and involucrin (C) are shown. Cytokeratin 10 (K10)- and involucrin- positive cells are stained red. Blue nuclear counter stain is DAPI. Note the paucity of K10- and involucrin- positive cells in the NOKs-Akata raft indicating a perturbation of normal differentiation by EBV, even though morphologically terminal differentiation does occur as evidenced by the presence of squames.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592227&req=5

ppat.1005195.g001: EBV-infected NOKs cells retain ability to be partially differentiated.Uninfected NOKs (left panel) and EBV-infected NOKs-Akata (right panel) cells were grown in organotypic raft culture, samples formalin fixed, embedded in paraffin and 5-micron thick sections analyzed. A). Images from hematoxylin and eosin (H&E) stained sections are shown. Note the presence of squames at the top of the NOKs-Akata raft indicative of terminal differentiation. Also note that the basal compartment of the NOKs-Akata raft is much less well organized with signs of migration/invasion of the epithelial cells into the underlying dermal equivalent (poorly stained portion of the image) at the bottom of the image. Immunofluorescent images from sections of rafts stained with antibody against the epithelial cell differentiation markers, cytokeratin 10 (B), and involucrin (C) are shown. Cytokeratin 10 (K10)- and involucrin- positive cells are stained red. Blue nuclear counter stain is DAPI. Note the paucity of K10- and involucrin- positive cells in the NOKs-Akata raft indicating a perturbation of normal differentiation by EBV, even though morphologically terminal differentiation does occur as evidenced by the presence of squames.
Mentions: The differentiation program of stratified epithelia can be recapitulated in vitro using the organotypic culture technique in which epithelial cells are plated on a dermal equivalent composed of human fibroblasts embedded in collagen, allowed to grow to confluence, then raised to the air/liquid interface (thereby commonly referred to as 'raft' cultures) and cultured for approximately two weeks’ time during which the epithelial cells proliferate, stratify, and daughter cells that lose contact with the dermal equivalent undergo terminal differentiation. Rafting is considered the gold standard method for recapitulating the normal differentiation program of stratified epithelial in vitro. To determine if EBV-infected NOKs cells (NOKs-Akata) retain the ability to be differentiated, uninfected and EBV-infected NOKs were grown in raft cultures. As shown in Fig 1A, the EBV-infected NOKs cells undergo stratification as does the uninfected parental cell population, with classical morphological signs of differentiation including the presence of terminally differentiated squames at the top surface, clearly visible in the EBV-infected NOKs. Closer examination of the EBV infected cells, however, showed evidence of a much less well organized basal layer of epithelial cells (the layer of cells directly in contact with the underlying dermal equivalent), with signs of epithelial migration/invasion into the underlying dermal equivalent, suggestive of a partial disruption in the normal program of epithelial cell differentiation. Consistent with these morphological changes, the pattern of expression of cytokeratin 10 (K10) and involucrin, cellular proteins whose expression is induced in suprabasal cells, was less uniform in the EBV-infected raft culture (Fig 1B and Fig 1C). These observations validate recent findings made using other approaches for inducing epithelial cell differentiation that less well recapitulate the in vivo process: suspension of epithelial cells in methylcellulose or growth of epithelial cells in high concentration of calcium chloride [42].

Bottom Line: Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies.We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture.In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America; Cellular and Molecular Biology Graduate Program, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

ABSTRACT
Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

No MeSH data available.


Related in: MedlinePlus