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Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes.

ABSTRACT: Epstein-Barr virus (EBV) is implicated in the pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC). EBV-associated cancers harbor a latent EBV infection characterized by a lack of viral replication and the expression of viral oncogenes. Cellular changes promoted by HPV are comparable to those shown to facilitate EBV latency, though whether HPV-positive cells support a latent EBV infection has not been demonstrated. Using a model of direct EBV infection into HPV16-immortalized tonsillar cells grown in organotypic raft culture, we showed robust EBV replication in HPV-negative rafts but little to no replication in HPV-immortalized rafts. The reduced EBV replication was independent of immortalization, as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication. Furthermore, we observed reduced EBV lytic gene expression and increased expression of EBER1, a noncoding RNA highly expressed in latently infected cells, in the presence of HPV. The use of human foreskin keratinocyte rafts expressing the HPV16 E6 and/or E7 oncogene(s) (HPV E6 and E7 rafts) showed that E7 was sufficient to reduce EBV replication. EBV replication is dependent upon epithelial differentiation and the differentiation-dependent expression of the transcription factors KLF4 and PRDM1. While KLF4 and PRDM1 levels were unaltered, the expression levels of KLF4 transcriptional targets, including late differentiation markers, were reduced in HPV E6 and E7 rafts compared to their levels in parental rafts. However, the HPV E7-mediated block in EBV replication correlated with delayed expression of early differentiation markers. Overall, this study reveals an HPV16-mediated block in EBV replication, through E7, that may facilitate EBV latency and long-term persistence in the tumor context.IMPORTANCE Using a model examining the establishment of EBV infection in HPV-immortalized tissues, we showed an HPV-induced interruption of the normal EBV life cycle reminiscent of a latent EBV infection. Our data support the notion that a persistent EBV epithelial infection depends upon preexisting cellular alterations and suggest the ability of HPV to promote such changes. More importantly, these findings introduce a model for how EBV coinfection may influence HPV-positive (HPV-pos) OSCC pathogenesis. Latently EBV-infected epithelial cells, as well as other EBV-associated head-and-neck carcinomas, exhibit oncogenic phenotypes commonly seen in HPV-pos OSCC. Therefore, an HPV-induced shift in the EBV life cycle toward latency would not only facilitate EBV persistence but also provide additional viral oncogene expression, which can contribute to the rapid progression of HPV-pos OSCC. These findings provide a step toward defining a role for EBV as a cofactor in HPV-positive oropharyngeal tumors.

SUBMITTER: Guidry JT

PROVIDER: S-EPMC6321917 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes.

Guidry J T JT Myers J E JE Bienkowska-Haba M M Songock W K WK Ma X X Shi M M Nathan C O CO Bodily J M JM Sapp M J MJ Scott R S RS

Journal of virology 20190104 2

Epstein-Barr virus (EBV) is implicated in the pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC). EBV-associated cancers harbor a latent EBV infection characterized by a lack of viral replication and the expression of viral oncogenes. Cellular changes promoted by HPV are comparable to those shown to facilitate EBV latency, though whether HPV-positive cells support a latent EBV infection has not been demonstrated. Using a model of direct EBV infecti ...[more]

PMID: 30381489

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Project description:UnlabelledThe oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involving DNA methylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcellulose-induced differentiation was delayed in EBV-positive clones and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellular DNA methylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells.ImportanceHere, we show that EBV infection of oral keratinocytes led to CpG island hypermethylation as an epigenetic scar of prior EBV infection that was retained after loss of the virus. Such EBV-induced epigenetic modification recapitulated the hypermethylated CpG island methylator phenotype (CIMP) observed in EBV-associated carcinomas. These epigenetic alterations not only impacted gene expression but also resulted in delayed calcium and methylcellulose-induced keratinocyte differentiation. Importantly, these epigenetic changes occurred in cells that were not as genetically unstable as carcinoma cells, indicating that EBV infection induced an epigenetic mutator phenotype. The impact of this work is that we have provided a mechanistic framework for how a tumor virus using the epigenetic machinery can act in a "hit-and-run" fashion, with retention of epigenetic alterations after loss of the virus. Unlike genetic alterations, these virally induced epigenetic changes can be reversed pharmacologically, providing therapeutic interventions to EBV-associated malignancies.

Project description:The oral cavity is the persistent reservoir for EBV with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns that are regulated by epigenetic modifications involving DNA methylation and chromatin structure. Such regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may inadvertently result in long-lasting, oncogenic host epigenetic reprogramming. To test this hypothesis in the context of EBV infection of epithelial cells, we established a transient infection model to identify the epigenetic consequences after EBV infection of immortalized normal oral keratinocytes and subsequent viral loss. With mounting evidence that EBV can induce epigenetic alterations, we developed a transient infection model where a clonal derivative (designated cl1) from a human telomerase immortalized normal oral keratinocyte (NOK) cell line was infected with a recombinant Akata EBV carrying neomycin resistance and GfP cassette in place of the BXLF1 open reading frame. Infected cells were passaged ten times, and then selection pressure was removed for an additional ten passages to allow for viral loss. Three transiently-infected EBV-negative clones were identified by single cell cloning (designated cl1, cl3, cl4). Uninfected parental clone and cells transfected with PTRUF5 plasmid were passaged alongside the transiently-infected clones. The transcription profiles were analyzed using Affymetrix microarray U133Plus2.0 arrays in duplicate. NOK cells were single cell cloned (designated as cl1) and EBV infected by co-culture with a recombinant Akata EBV strain carrying neomcycin resistance and GFP expression cassette in place of EBV BXLF1. Cells were selected with 350 ug/ml G418. As a selection control, cells were transfected a plasmid carrying a neomycin resistance cassette. Cells were passaged 10 times with G418, followed by removal of selection pressure. After an additional 10 passages, cells were single cell cloned and the presence of EBV was determined by various methods. Three clones were identified as being EBV negative and said to be transiently infected. Uninfected parental and vector transfected cells were passaged in the same manner and also single cell cloned.

Dataset Information

Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes.

Publications

Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes.

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