RESUMEN
Epstein-Barr virus (EBV) is best known for infection of B cells, in which it usually establishes an asymptomatic lifelong infection, but is also associated with the development of multiple B cell lymphomas. EBV also infects epithelial cells and is associated with all cases of undifferentiated nasopharyngeal carcinoma (NPC). EBV is etiologically linked with at least 8% of gastric cancer (EBVaGC) that comprises a genetically and epigenetically distinct subset of GC. Although we have a very good understanding of B cell entry and lymphomagenesis, the sequence of events leading to EBVaGC remains poorly understood. Recently, ephrin receptor A2 (EPHA2) was proposed as the epithelial cell receptor on human cancer cell lines. Although we confirm some of these results, we demonstrate that EBV does not infect healthy adult stem cell-derived gastric organoids. In matched pairs of normal and cancer-derived organoids from the same patient, EBV only reproducibly infected the cancer organoids. While there was no clear pattern of differential expression between normal and cancer organoids for EPHA2 at the RNA and protein level, the subcellular location of the protein differed markedly. Confocal microscopy showed EPHA2 localization at the cell-cell junctions in primary cells, but not in cancer cell lines. Furthermore, histologic analysis of patient tissue revealed the absence of EBV in healthy epithelium and presence of EBV in epithelial cells from inflamed tissue. These data suggest that the EPHA2 receptor is not accessible to EBV on healthy gastric epithelial cells with intact cell-cell contacts, but either this or another, yet to be identified receptor may become accessible following cellular changes induced by inflammation or transformation, rendering changes in the cellular architecture an essential prerequisite to EBV infection.
Asunto(s)
Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Organoides/virología , Receptor EphA2/metabolismo , Estómago/virología , Internalización del Virus , Células Epiteliales/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Humanos , Organoides/metabolismo , Estómago/fisiología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virologíaRESUMEN
Ctf18-RFC is an alternative PCNA loader which plays important but poorly understood roles in multiple DNA replication-associated processes. To fulfill its specialist roles, the Ctf18-RFC clamp loader contains a unique module in which the Dcc1-Ctf8 complex is bound to the C terminus of Ctf18 (the Ctf18-1-8 module). Here, we report the structural and functional characterization of the heterotetrameric complex formed between Ctf18-1-8 and a 63 kDa fragment of DNA polymerase É. Our data reveal that Ctf18-1-8 binds stably to the polymerase and far from its other functional sites, suggesting that Ctf18-RFC could be associated with Pol É throughout normal replication as the leading strand clamp loader. We also show that Pol É and double-stranded DNA compete to bind the same winged-helix domain on Dcc1, with Pol É being the preferred binding partner, thus suggesting that there are two alternative pathways to recruit Ctf18-RFC to sites of replication.