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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

Lytic EBV protein expression in NOKs-Akata cells is restricted to the more differentiated cell layers.Two different independently generated NOKs-Akata cell lines (panels A and B) were grown in organotypic air-interface raft culture, and in situ hybridization or immunohistochemistry was performed to detect expression of the EBV EBERs or lytic EBV proteins (Z and BMRF1) as indicated. Examples of Z and BMRF1 stained cells are indicated by red arrows. C) NOKs-Akata cells grown in organotypic air-interface raft culture were examined by immunofluorescence using both anti-K10 (red) and anti-Z (green) antibodies. An example of a Z and K10 co-staining cell is shown in the left panel, and a Z-positive/K10 negative cell is shown in the right panel.
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ppat.1005195.g002: Lytic EBV protein expression in NOKs-Akata cells is restricted to the more differentiated cell layers.Two different independently generated NOKs-Akata cell lines (panels A and B) were grown in organotypic air-interface raft culture, and in situ hybridization or immunohistochemistry was performed to detect expression of the EBV EBERs or lytic EBV proteins (Z and BMRF1) as indicated. Examples of Z and BMRF1 stained cells are indicated by red arrows. C) NOKs-Akata cells grown in organotypic air-interface raft culture were examined by immunofluorescence using both anti-K10 (red) and anti-Z (green) antibodies. An example of a Z and K10 co-staining cell is shown in the left panel, and a Z-positive/K10 negative cell is shown in the right panel.

Mentions: We next examined whether signs of lytic EBV reactivation arise within raft cultures of two independently isolated populations of EBV-infected NOKs (Fig 2A and 2B). First we stained for EBV-encoded small nuclear non-coding RNAs (EBERs), which are highly expressed in latently EBV-infected cells, to confirm that the raft cultures retained EBV. EBERs were detected by in situ hybridization throughout the raft cultures of EBV-infected NOKs, while uninfected cells had no detectable EBERs (S1 Fig). Next we stained for two markers of lytic reactivation, the EBV immediate-early BZLF1 gene encoding the Z protein and the early lytic BMRF1 gene encoding the viral DNA polymerase processivity factor. Cells positive for these markers were exclusively detected within the suprabasal compartment of the raft culture. Uninfected NOKs cells did not stain positively (S1 Fig). Immunofluorescence co-staining using antibodies directed against K10 and Z showed that while expression of Z can be associated with expression of differentiation marker K10 (Fig 2C, left panel), some Z-positive cells do not express K10 (Fig 2C, right panel).


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)

Lytic EBV protein expression in NOKs-Akata cells is restricted to the more differentiated cell layers.Two different independently generated NOKs-Akata cell lines (panels A and B) were grown in organotypic air-interface raft culture, and in situ hybridization or immunohistochemistry was performed to detect expression of the EBV EBERs or lytic EBV proteins (Z and BMRF1) as indicated. Examples of Z and BMRF1 stained cells are indicated by red arrows. C) NOKs-Akata cells grown in organotypic air-interface raft culture were examined by immunofluorescence using both anti-K10 (red) and anti-Z (green) antibodies. An example of a Z and K10 co-staining cell is shown in the left panel, and a Z-positive/K10 negative cell is shown in the right panel.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005195.g002: Lytic EBV protein expression in NOKs-Akata cells is restricted to the more differentiated cell layers.Two different independently generated NOKs-Akata cell lines (panels A and B) were grown in organotypic air-interface raft culture, and in situ hybridization or immunohistochemistry was performed to detect expression of the EBV EBERs or lytic EBV proteins (Z and BMRF1) as indicated. Examples of Z and BMRF1 stained cells are indicated by red arrows. C) NOKs-Akata cells grown in organotypic air-interface raft culture were examined by immunofluorescence using both anti-K10 (red) and anti-Z (green) antibodies. An example of a Z and K10 co-staining cell is shown in the left panel, and a Z-positive/K10 negative cell is shown in the right panel.
Mentions: We next examined whether signs of lytic EBV reactivation arise within raft cultures of two independently isolated populations of EBV-infected NOKs (Fig 2A and 2B). First we stained for EBV-encoded small nuclear non-coding RNAs (EBERs), which are highly expressed in latently EBV-infected cells, to confirm that the raft cultures retained EBV. EBERs were detected by in situ hybridization throughout the raft cultures of EBV-infected NOKs, while uninfected cells had no detectable EBERs (S1 Fig). Next we stained for two markers of lytic reactivation, the EBV immediate-early BZLF1 gene encoding the Z protein and the early lytic BMRF1 gene encoding the viral DNA polymerase processivity factor. Cells positive for these markers were exclusively detected within the suprabasal compartment of the raft culture. Uninfected NOKs cells did not stain positively (S1 Fig). Immunofluorescence co-staining using antibodies directed against K10 and Z showed that while expression of Z can be associated with expression of differentiation marker K10 (Fig 2C, left panel), some Z-positive cells do not express K10 (Fig 2C, right panel).

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