Differentiation-Dependent LMP1 Expression Is Required for Efficient Lytic Epstein-Barr Virus Reactivation in Epithelial Cells.
Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors that have latent infection, such as nasopharyngeal carcinoma (NPC), and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically infected cells, LMP1 expression is activated by the BRLF1 (R) immediate early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors KLF4 and BLIMP1 induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we found that differentiation of NOKs-Akata cells by either methylcellulose suspension or organotypic culture induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic infection-inducing effects of epithelial cell differentiation, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression and then cooperate with LMP1 to activate Z and R expression.IMPORTANCEThe EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia) or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.|Cellular and Molecular Biology Graduate Program, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Dhananjay M
DM
Nawandar
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Makoto
M
Ohashi
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Reza
R
Djavadian
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Elizabeth
E
Barlow
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Kathleen
K
Makielski
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.|National Center for Research, Khartoum, Sudan.
Ahmed
A
Ali
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Denis
D
Lee
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Paul F
PF
Lambert
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.|Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Eric
E
Johannsen
Department of Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA skenney@wisc.edu.|Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Shannon C
SC
Kenney
D002454:Cell Differentiation; D004847:Epithelial Cells / Q000502:physiology* / Q000821:virology*; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D054884:Host-Pathogen Interactions; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D000074462:Positive Regulatory Domain I-Binding Factor 1; D012097:Repressor Proteins / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000378:metabolism*; D014775:Virus Activation
C037662:EBV-associated membrane antigen, Epstein-Barr virus; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D012097:Repressor Proteins; D014763:Viral Matrix Proteins; C087042:PRDM1 protein, human; D000074462:Positive Regulatory Domain I-Binding Factor 1
Epstein-Barr virus