De novo Epstein-Barr virus infection of pseudostratified nasopharynx epithelial cells differentiated at the air-liquid interface (ALI)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE157243 | GEO | 2021/04/06
REPOSITORIES: GEO
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Project description:Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is only activated when expression of the EBV latency-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and together with some of those gene products ensures completion of the lytic cycle. However, not every latently-infected cell exposed to a lytic trigger turns expression of ZEBRA on, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to variable degrees in latently-infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a pro-lytic state, such upregulation also runs counter to the well-known mechanism of viral nuclease-mediated host shut-off that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed downstream of ZEBRA, indicating an additional mode of virus-mediated host shut-off through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a pro-lytic state that allows a subpopulation of cells to support the EBV lytic cycle.
2020-01-13 | GSE141220 | GEO
Project description:The oral cavity has previously been identified as the major site for transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered epithelial differentiation and morphogenesis. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host and viral gene expression compared to latency or lytic replication. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression.
2022-07-28 | GSE184257 | GEO
Project description:Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes a latent reservoir in peripheral B-lymphocytes with sporadic reactivation. EBV also infects epithelial cells, predominantly resulting in a lytic infection, which may contribute to EBV transmission from saliva. In the nasopharynx, EBV infection can lead to the clonal expansion of a latently infected cell and the development of nasopharyngeal carcinoma (NPC). The mechanisms governing EBV pathogenesis in nasopharyngeal epithelium are largely unknown. An advanced understanding would depend on a physiologically relevant culture model of polarized airway epithelium. The recent application of the organotypic raft culture in keratinocytes has demonstrated great promise for the use of polarized cultures in the study of EBV permissive replication. In this study, the adaptation of an air-liquid interface (ALI) culture method using transwell membranes was explored in an EBV-infected NPC cell line. In the EBV-infected NPC HK1 cell line, ALI culture resulted in the completion of EBV reactivation, with global induction of the lytic cascade, replication of EBV genomes, and production of infectious progeny virus. We propose that the ALI culture method can be widely adopted as a physiologically relevant model to study EBV pathogenesis in polarized nasal epithelial cells. IMPORTANCE:Lifting adherent cells to the air-liquid interface (ALI) is a method conventionally used to culture airway epithelial cells into polarized apical and basolateral surfaces. Reactivation of Epstein-Barr virus (EBV) from monolayer epithelial cultures is sometimes abortive, which may be attributed to the lack of authentic reactivation triggers that occur in stratified epithelium in vivo In the present work, the ALI culture method was applied to study EBV reactivation in nasopharyngeal epithelial cells. The ALI culture of an EBV-infected cell line yielded high titers and can be dissected by a variety of molecular virology assays that measure induction of the EBV lytic cascade and EBV genome replication and assembly. EBV infection of polarized cultures of primary epithelial cells can be challenging and can have variable efficiencies. However, the use of the ALI method with established EBV-infected cell lines offers a readily available and reproducible approach for the study of EBV permissive replication in polarized epithelia.
| S-EPMC6052337 | biostudies-literature
Project description:Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced ERK1/2 and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126 or autophagy initiation inhibitor 3-MA suppressed C6-Cer-induced EBV lytic replication. In contrast, the autophagosome-lysosome fusion inhibitor chloroquine induced BZLF1 expression. Transfection with siCREB reduced ERK1/2 phosphorylation and C6-Cer-induced BZLF1 expression. On the other hand, siJUN transfection did not affect BZLF1 expression. Our results show that increased endogenous ceramide and glycosyl ceramide (GlyCer) following C6-Cer treatment induce EBV lytic replication in gastric cancer cells via ERK1/2 and CREB phosphorylation and autophagosome accumulation.
2023-12-20 | MTBLS7649 | MetaboLights
Project description:De novo Epstein-Barr virus infection of pseudostratified nasopharynx epithelial cells differentiated at the air-liquid interface (ALI)
| PRJNA660622 | ENA
Project description:Mouse tracheal epithelial cells were cultured at air-liquid interface (ALI), RNA was harvested at days 0, 2, and 7 post-ALI, and hybridized to two-channel MEEBO arrays. The experiment was designed to allow investigators to identify genes differentially expressed during airway epithelial cell differentiation and development, including ciliogenesis.
2014-08-12 | GSE60364 | GEO
Project description:The Air Liquid Interface Model of Gastric Mucosa allows to provide a fully polarized epithelium accessible to infection experiments with H.pylori. The influence of several factors on immune response has been studied.
2018-02-15 | GSE94032 | GEO
Project description:Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells
| PRJNA794826 | ENA
Project description:Viral infection leads to heterogeneous cellular outcomes ranging from resistance to virion amplification and cell death. In this study, we apply single-cell techniques to analyze diverse fate trajectories during Epstein-Barr virus (EBV) lytic reactivation in three B cell models. We expand upon the previous finding that c-Myc regulates lytic reactivation and determine that MYC downregulation during the lytic phase is correlated with a loss of germinal center (GC) B cell expression signatures. Consistent with prior work, lytic induction yields both abortive and complete reactivation. Abortive lytic cells, which express high STAT3 and initiate but do not complete the lytic cycle, also upregulate NF-kB and IRF3 pathway target genes. Cells that proceeded through the full lytic cycle exhibited unexpected and striking expression of genes associated with cellular reprogramming, developmental progenitor phenotypes, and cytokine upregulation. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis.
2024-10-31 | GSE272763 | GEO
Project description:Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs, and hundreds of new polyadenylated lytic transcripts. Here we report an additional layer to the EBV transcriptome through the identification of a repertoire of latent and lytic viral circRNAs. Utilizing RNase R-sequencing with cell models representing latency types I, II, and III, we identified circRNAs expressed from the latency Cp promoter involving backsplicing from the W1 and W2 exons to the C1 exon, from the EBNA BamHI U exon, and from the latency long-non-coding RPMS1 locus. We also identified circRNAs expressed during reactivation including an exon 8-to-2 backspliced LMP2 transcript and a highly expressed circRNA derived from the BHLF1 gene. Altogether we identified over 30 EBV circRNA candidates and validated and determined the structural features, expression profiles and nuclear-cytoplasmic distributions of several prominent viral circRNAs. Further, we show that two RPMS1 circRNAs are expressed in stomach cancer specimens. This study increases the known EBV latency and lytic transcriptome repertoires to include viral circRNAs and provides an essential foundation for investigations into the functions of this new class of EBV transcripts in EBV biology and disease.
2018-07-12 | GSE116675 | GEO