Differentiation-Dependent LMP1 Expression Is Required for Efficient Lytic Epstein-Barr Virus Reactivation in Epithelial Cells

Abstract

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--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. The 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.

Human Cytomegalovirus Productively Replicates In Vitro in Undifferentiated Oral Epithelial Cells

“…Prior to rafting, oral epithelial cells can be maintained in vitro as monolayers in their undifferentiated state by subconfluent culture in serum-free medium. As monolayers, they can be differentiated either through treatment with fetal bovine serum (FBS) and calcium or by the addition of methylcellulose to the growth medium (25,36,37). Our NOKs and hGETs maintained as monolayers in serum-free media did not express involucrin, but involucrin expression (differentiation) could be induced either by FBS and calcium or by methylcellulose ( Fig.…”

(-)-Epigallocatechin‑3‑gallate inhibition of Epstein‑Barr virus spontaneous lytic infection involves downregulation of latent membrane protein 1

“…The latent form of infection allows the virus to persist for the lifetime of the host, whereas the lytic form of infection enables infectious virion production and transmission from cell to cell and from host to host. Both forms of infection are essential for the long-term success of the virus (45). It is also speculated that once EBV is reactivated into the lytic cycle, the induced expression of LMP1 is considered to be critical for efficient virus release and infection of new host cells.…”

“…A number of studies have demonstrated that LMP1 serves an important role in the EBV lytic cycle (24,45,46). While LMP1 may be expressed in some states of EBV latency, significant induction of full-length LMP1 is frequently observed during virus reactivation into the lytic cycle (13,47,48).…”

“…Therefore, EBV lytic infection may be a notable factor to consider in malignant transformation, as EBV infection results in changes to the infected host cells or nearby cells (45), and the biological characteristics of these cells may be altered in a way that may increase the degree of malignancy and promote the occurrence of metastasis (51). Furthermore, LMP1 is an important viral protein required for the EBV lytic life cycle (45). Therefore, selection of this protein for augmenting virus release may be a critical evolutionary step for EBV.…”

Detection of Epstein-Barr Virus in Saliva and Gen LMP1 among HIVInfected Patients HIVInfected Patients

“…The granular epithelial layer contains apoptotic cells, and the stratum corneum is characterized by keratin cells of fibrils which form an extracellular barrier. 14 After lytic replication in epithelial cells, the virus spreads to all lymphoid tissues and infects naive B cells through binding of glycoprotein gp350 virus to the CD21 receptor on the B cell's surface. The infected B cells trigger specific primary cytotoxic cell responses that ultimately control EBV infection.…”