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

Nawandar, Dhananjay M.; Wang, Anqi; Makielski, Kathleen R.; Lee, Denis; Ma, Shidong; Barlow, Elizabeth A.; Reusch, Jessica A.; Jiang, Ru; Wille, Coral K.; Greenspan, Deborah; Greenspan, John S.; Mertz, Janet E.; Hutt-Fletcher, Lindsey; Johannsen, Eric; Lambert, Paul F.; Kenney, Shannon C.

doi:10.1371/journal.ppat.1005195

Cited by 84 publications

(152 citation statements)

References 95 publications

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“…However, no in vitro model has been developed to study EBV and HPV co-infections in stratified epithelium. While it is known that the EBV life cycle is supported in NOKs cells, a tert-immortalized normal oral keratinocyte cell line, [8, 10] it was unknown whether NOKs cells support the HPV life cycle prior to this study. To determine if NOKs cells can stably harbor HPV18, a high-risk HPV subtype associated with human oral infections and human head and neck cancers, we transfected HPV18 genomes into NOKs cells and performed Southern blot analysis.…”

Section: Resultsmentioning

confidence: 99%

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Human papillomavirus promotes Epstein-Barr virus maintenance and lytic reactivation in immortalized oral keratinocytes

et al. 2016

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Epstein-Barr virus and human papillomaviruses are human tumor viruses that infect and replicate in upper aerodigestive tract epithelia and cause head and neck cancers. The productive phases of both viruses are tied to stratified epithelia highlighting the possibility that these viruses may affect each other’s life cycles. Our lab has established an in vitro model system to test the effects of EBV and HPV co-infection in stratified squamous oral epithelial cells. Our results indicate that HPV increases maintenance of the EBV genome in the co-infected cells and promotes lytic reactivation of EBV in upper layers of stratified epithelium. Expression of the HPV oncogenes E6 and E7 were found to be necessary and sufficient to account for HPV-mediated lytic reactivation of EBV. Our findings indicate that HPV increases the capacity of epithelial cells to support the EBV life cycle, which could in turn increase EBV-mediated pathogenesis in the oral cavity.

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Section: Resultsmentioning

confidence: 99%

“…The productive phase of the HPV life cycle, which includes late protein expression and genome amplification, occurs in the suprabasal compartment of stratified epithelium [7]. Similarly, EBV immediate-early protein expression and genome amplification, two hallmarks of EBV lytic reactivation, occur in suprabasal epithelial layers [8, 9]. …”

Section: Introductionmentioning

confidence: 99%

Human papillomavirus promotes Epstein-Barr virus maintenance and lytic reactivation in immortalized oral keratinocytes

et al. 2016

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“…Consistent with this part of the model, NPC tumors commonly have only rare cells expressing lytic viral proteins, despite the high level of antibody titers to lytic viral proteins in NPC patients. Loss of Z expression in NPCs may reflect the absence of differentiation-dependent cellular transcription factors (BLIMP1 and KLF4) that synergistically activate the Z and R promoters (40). In conclusion, our results here suggest that loss of TET function in EBV-infected epithelial cells, or the presence of other mutations that globally inhibit 5hmC, may initiate a series of events that promote EBVinduced NPC not only by enhancing methylation of cellular tumor suppressor genes but also by altering 5mC and 5hmC modification of lytic EBV promoters.…”

Section: Discussionmentioning

confidence: 99%

5-hydroxymethylation of the EBV genome regulates the latent to lytic switch

Wille

Nawandar

Henning

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Latent Epstein-Barr virus (EBV) infection and cellular hypermethylation are hallmarks of undifferentiated nasopharyngeal carcinoma (NPC). However, EBV infection of normal oral epithelial cells is confined to differentiated cells and is lytic. Here we demonstrate that the EBV genome can become 5-hydroxymethylated and that this DNA modification affects EBV lytic reactivation. We show that global 5-hydroxymethylcytosine (5hmC)-modified DNA accumulates during normal epithelial-cell differentiation, whereas EBV+ NPCs have little if any 5hmC-modified DNA. Furthermore, we find that increasing cellular ten-eleven translocation (TET) activity [which converts methylated cytosine (5mC) to 5hmC] decreases methylation, and increases 5hmC modification, of lytic EBV promoters in EBV-infected cell lines containing highly methylated viral genomes. Conversely, inhibition of endogenous TET activity increases lytic EBV promoter methylation in an EBV-infected telomerase-immortalized normal oral keratinocyte (NOKs) cell line where lytic viral promoters are largely unmethylated. We demonstrate that these cytosine modifications differentially affect the ability of the two EBV immediateearly proteins, BZLF1 (Z) and BRLF1 (R), to induce the lytic form of viral infection. Although methylation of lytic EBV promoters increases Z-mediated and inhibits R-mediated lytic reactivation, 5hmC modification of lytic EBV promoters has the opposite effect. We also identify a specific CpG-containing Z-binding site on the BRLF1 promoter that must be methylated for Z-mediated viral reactivation and show that TET-mediated 5hmC modification of this site in NOKs prevents Z-mediated viral reactivation. Decreased 5-hydroxymethylation of cellular and viral genes may contribute to NPC formation.EBV | 5hmC | lytic reactivation | NPC

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“…Expression of BRLF1 is enhanced by B lymphocyte-induced maturation protein 1 (BLIMP1) and KLF4, both of which are expressed in epithelial cells in a differentiation-dependent manner. Thus, when telomerase-immortalized normal oral keratinocytes are latently infected with EBV carrying a drug resistance marker for selection and are differentiated in air-liquid interface organotypic raft cultures, the virus also undergoes lytic replication (22). Thus, the differentiation status of the epithelial cell is critical for its ability to support EBV replication.…”

Section: Now and The Futurementioning

confidence: 99%

“…The basal layers were reported to not contain the virus (25). Revisiting of this question with a more sensitive reverse transcriptase real-time PCR analysis of basal cells isolated from formalin-fixed paraffin-embedded sections by laser capture microdissection challenged this conclusion by demonstrating EBV-encoded RNA (EBER) transcripts in the absence of lytic gene expression (22). The same type of analysis also found evidence for latent infection in basal epithelial cells taken from sections of a healthy tonsil.…”

Section: Now and The Futurementioning

confidence: 99%

The Long and Complicated Relationship between Epstein-Barr Virus and Epithelial Cells

Hutt-Fletcher

2017

J Virol

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The roles of epithelial cells in infection and persistence of the EpsteinBarr virus (EBV) have long been difficult to resolve. However, recent developments have reinforced the conclusion that these cells are a major site of virus replication and raised the possibility that, like papillomaviruses, EBV has evolved to take advantage of epithelial differentiation to ensure survival, persistence, and spread.KEYWORDS Epstein-Barr virus, epithelial cells, tropism E pstein-Barr virus (EBV) is well known as a human lymphotropic herpesvirus that was isolated from a Burkitt's lymphoma in the early 1960s (1). It is a well-described causal agent of infectious mononucleosis and an important player in the development of lymphoid tumors. It is well used as a tool for immortalizing human B cells. Soon after its discovery, an association between EBV and epithelial malignancies was revealed (2) and the presence of the virus was confirmed within epithelial cells of nasopharyngeal carcinoma (3). Yet, as late as 2005, a distinguished speaker at an international herpesvirus meeting declared that epithelial cells were not relevant to the general biology of EBV. How was that possible, and where are we today? EARLY WORKEarly works described EBV DNA and RNA in squamous epithelial cells shed in the oral cavity during acute infectious mononucleosis (4, 5). Thus, the model with B cells as the reservoir of latent EBV and epithelial cells as the site of productive lytic replication in vivo was developed. However, additional support for the routine involvement of epithelial cells in primary and persistent infections was, for many years, quite elusive. In the absence of any obvious lesions, such as those seen in herpes simplex infections, the finding of an infected cell in the oral cavity was much like looking for the proverbial needle in the haystack. Compounding the problem was the difficulty in infecting epithelial cells in vitro. With the advent of the AIDS epidemic, oral hairy leukoplakia appeared, which was the first and remains the only disease caused entirely by productive replication of EBV. Oral hairy leukoplakia, which is an epithelial hyperplasia typically found on the lateral tongue (6), is full of and driven by EBV lytic replication (7,8). This bolstered the case for epithelial cells in the oral cavity being responsible for the production of cell-free virus in saliva, but still, normal epithelial cells were readily found to contain virus only in individuals coinfected with the human immunodeficiency virus (9). For a while, the model of persistence pivoted toward the idea that in the absence of malignancy or other underlying disease, B cells alone are involved in infection (10).Three sets of observations subsequently helped spur a return to the yin-and-yang description of epithelial and B cell infection. Despite the uncertainty about the role of epithelial cells in EBV biology, its presence in epithelial cells of patients with nasopharyngeal carcinoma and gastric cancer indicates that EBV remains important for understanding...

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

Cited by 84 publications

References 95 publications

Human papillomavirus promotes Epstein-Barr virus maintenance and lytic reactivation in immortalized oral keratinocytes

Human papillomavirus promotes Epstein-Barr virus maintenance and lytic reactivation in immortalized oral keratinocytes

5-hydroxymethylation of the EBV genome regulates the latent to lytic switch

The Long and Complicated Relationship between Epstein-Barr Virus and Epithelial Cells

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