Antibodies to gp350/220 enhance the ability of Epstein-Barr virus to infect epithelial cells

Epstein-Barr virus (EBV) is a persistent, orally transmitted herpesvirus that replicates in B cells and epithelial cells and is associated with lymphoid and epithelial malignancies. The virus binds to CD21 on B cells via glycoprotein gp350/220 and infects efficiently. Infection of cultured epithelial cells has not typically been efficient but can occur in the absence of gp350/220 and CD21 and in vivo is thought to be important to the development of nasopharyngeal carcinoma. We report here that antibodies to gp350/220, which inhibit EBV infection of B cells, enhance infection of epithelial cells. The effect is not mediated by Fc receptor binding but is further enhanced by antibody cross-linking, which may patch gp350/220 in the virus envelope. Saliva from EBV-seropositive individuals has similar effects that can be reversed by depletion of antibody. The results are consistent with a model in which gp350/220 interferes with the access of other important players to the epithelial cell surface. The results may have implications for the development of nasopharyngeal carcinoma in high-risk populations in which elevated titers of antibody to EBV lytic cycle proteins are prognostic.     

EB病毒潜伏膜蛋白1诱导人鼻咽上皮细胞端粒酶的表达

Telomerase activation has been linked to cell immortalization in vitro and tumorigenicity in vivo. In this study, for the first, we reported that Epstein-Barr virus activated the telomerase activity of human nasopharyngeal epithelial cells in the early stage of immortalization as tested by the PCR-ELISA. The telomerase activity in nasopharyngeal epithelial cells was only observed in presenescent cells. It was implicated that Epstein-Barr virus induced the escape of nasopharyngeal epithelial cells from senescence via the activation of telomerase. We further showed that telomerase activation in infected cells was dependent on the protein level of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus using a Tetracycline regulatory cell line expressing LMP1, pTet-on-LMP1-HNE2. The activity of telomerase in nasopharyngeal cells was decreased when the protein level of LMP1 was blocked by antisense LMP1 plasmid DNA. And the activity of telmerase was also related to the carboxyl terminus of LMP1. It was implicated that the ability of Epstein-Barr virus to suppress senescence is associated with telomerase activation by LMP1.     

Observation of the biological characterizations of nasopharyngeal epithelial cells by EB virus infection in early phase of immortalization]

The multi-stage cell model of the nasopharyngeal carcinoma development in vitro by Epstein-Barr virus transformation is beneficial for the elucidation of the mechanism of nasopharyngeal cancer. To observe the biological changes of primary human nasopharyngeal epithelial cells in early phase of immortalization, in this study, we have detected the morphological changes and the expression profile of senescence-associated beta-galactosidase (SA-beta-Gal) in primary culture. In addition, the expression of EB virus latent membrane protein 1 (LMP1) and the growth curve of primary cells were also detected. Our results showed a low percentage of cells infected with EB virus expressing SA-beta-Gal activity at the late primary culture. In morphology, the cells also formed multilayer foci, and the cell population doubling time was showed. These results demonstrated that the nasopharyngeal epithelial cells by EB virus infection have passed through the senescence and entered the early phase of immortalization. These cells have some of the transformed characteristics. Our results provided the data for further study on the mechanism of immortalization and the establishment of human nasopharyngeal epithelial cell line.     

EB病毒感染人胎鼻咽上皮细胞逃避老化期过程中抑制p16^INK4a表达

细胞周期调控紊乱是细胞永生化进程中一个重要的分子事件,本文利用Western blotting和S-P法分别检测p16^INK4a、p53、p21^WAF1／CIP1和E2F1的蛋白表达,试图从细胞周期调控的角度,探讨EB病毒诱导人胎鼻咽上皮细胞逃避老化期的分子机制。结果表明,EB病毒通过抑制p16^INK4a表达而阻断p16^INK4a／Rb途径,上调转录因子E2F1,而对p53、p21^WAF1／CIP表达无明显的影响。结果初步揭示,EB病毒介导的p16^INK4a／Rb／E2F1细胞周期调控紊乱参与了人胎鼻咽上皮细胞逃避老化期过程,为进一步探讨鼻咽癌发病机制提供了科学依据。     

EB病毒感染人胎鼻咽上皮细胞逃避老化期过程中抑制p16INK4a表达

细胞周期调控紊乱是细胞永生化进程中一个重要的分子事件,本文利用Western blotting和S-P法分别检测p16~(INK4a)、p53、p21~(WAF1/CIP1)和E2F1的蛋白表达,试图从细胞周期调控的角度,探讨EB病毒诱导人胎鼻咽上皮细胞逃避老化期的分子机制。结果表明,EB病毒通过抑制p16~(INK4a)表达而阻断p16~(INK4a)/Rb途径,上调转录因子E2F1,而对p53、p21~(WAF1/CIP1)表达无明显的影响。结果初步揭示,EB病毒介导的p16~(INK4a)/Rb/E2F1细胞周期调控紊乱参与了人胎鼻咽上皮细胞逃避老化期过程,为进一步探讨鼻咽癌发病机制提供了科学依据。     

Efficient Immortalization of Primary Nasopharyngeal Epithelial Cells for EBV Infection Study

Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma.     

EB病毒潜伏膜蛋白1诱导人鼻咽上皮细胞端粒酶的表达

上皮细胞逃避老化期是细胞永生化过程中一个重要分子事件,端粒酶活性表达是维持人染色体端粒长度、抑制细胞进入老化期的关键因素之一.我们利用最新的端粒酶PCR-ELISA半定量技术,检测永生化早期阶段人胚鼻咽上皮细胞中端粒酶表达的情况,探讨EB病毒在人胚鼻咽上皮细胞永生化过程中的分子机制.结果表明,EB病毒诱导老化前期人胚鼻咽上皮细胞端粒酶表达,从而促使人胚鼻咽上皮细胞逃避老化期、进入永生化早期阶段.此外,我们还首次发现,人胚鼻咽上皮细胞表达端粒酶活性依赖于EB病毒LMP1蛋白的表达水平和LMP1分子的完整性,LMP1可能通过诱导端粒酶活性表达促进人鼻咽上皮细胞永生化.我们的实验为进一步探讨EB病毒诱导人胚鼻咽上皮细胞永生化的作用机制提供了实验基础.     

Telomerase Activity Impacts on Epstein-Barr Virus Infection of AGS Cells

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva.     

Requirement for Cell-to-Cell Contact in Epstein-Barr Virus Infection of Nasopharyngeal Carcinoma Cells and Keratinocytes

Two nasopharyngeal carcinoma (NPC) cell lines and one keratinocyte cell line could be infected with Epstein-Barr virus (EBV) by cocultivation with virus-producing cells but not by cell-free virus. Using porous culture inserts to manipulate the cell-to-cell contact, we demonstrated that contact between EBV donor B cells and EBV recipient epithelial cells was required for the infection. Cell-to-cell contact not only provided a CR2-independent route of infection but also enhanced CR2-mediated infection in a synergistic manner. Activity of two EBV promoters (Cp and Wp) and expression of EBNA2 were detected in the infected population. A small proportion of the infected cells spontaneously entered an EBV lytic state, which could be induced prominently by chemical treatment. This study provides information on how EBV may infect epithelial cells in vivo, such as at the onset of NPC development.     

Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma

Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.     

Detection of Epstein-Barr virus-associated antigen in fine needle aspiration smears from cervical lymph nodes in the diagnosis of nasopharyngeal carcinoma

Epstein-Barr virus (EBV) has consistently been shown to be associated with undifferentiated nasopharyngeal carcinoma, and an EBV-associated nuclear antigen (EBNA) has been detected in the cells of nasopharyngeal carcinoma. A study on the applicability of EBNA detection in fine needle aspiration (FNA) smears from cervical lymph nodes in the diagnosis of metastatic nasopharyngeal carcinoma was performed. All 11 cases (100%) with metastatic nasopharyngeal carcinoma showed EBNA-positive tumor cells, characterized by bright, granulated nuclear fluorescence. Three (50%) of six cases with other metastatic head and neck carcinomas also showed EBNA-positive tumor cells. These findings suggest that the presence of EBNA-positive tumor cells in FNA smears from cervical lymph nodes is not specific for metastatic nasopharyngeal carcinoma. On the other hand, a negative result in the presence of tumor cells may help to exclude it. A larger study is required to verify these preliminary findings.     

Epstein-Barr virus microRNA BART10-3p promotes dedifferentiation and proliferation of nasopharyngeal carcinoma by targeting ALK7

Non-keratinizing nasopharyngeal carcinoma, the major subtype of nasopharyngeal carcinoma, is characterized by low differentiation and a close relation to Epstein-Barr virus infection, which indicates a link between Epstein-Barr virus oncogenesis and loss of differentiation, and raises our interest in investigating the involvement of Epstein-Barr virus in nasopharyngeal carcinoma dedifferentiation. Our previous study showed abundant expression of an Epstein-Barr virus-encoded microRNA, BART10-3p, in nasopharyngeal carcinoma tissues, but the association between BART10-3p and nasopharyngeal carcinoma differentiation remains unknown. Here, we examined the expression and prognostic value of BART10-3p, and undertook bioinformatics analysis and functional assays to investigate the influence of BART10-3p on nasopharyngeal carcinoma differentiation and proliferation and the underpinning mechanism. Microarray analysis identified BART10-3p as the most significantly upregulated Epstein-Barr virus-encoded microRNA in nasopharyngeal carcinoma tissues and the upregulation was confirmed in two public datasets. The expression of BART10-3p was an independent unfavorable prognosticator in nasopharyngeal carcinoma and its integration with the clinical stage showed improved prognosis predictive performance. Bioinformatics analysis suggested a potential role of BART10-3p in tumor differentiation and progression. Functional assays demonstrated that BART10-3p could promote nasopharyngeal carcinoma cell dedifferentiation, epithelial-mesenchymal transition, and proliferation in vitro, and tumorigenicity in vivo. Mechanistically, BART10-3p directly targeted the 3′UTR of ALK7 and suppressed its expression. Reconstitution of ALK7 rescued BART10-3p-induced malignant phenotypes. Overall, our study demonstrates that BART10-3p promotes dedifferentiation and proliferation of nasopharyngeal carcinoma by targeting ALK7, suggesting a promising therapeutic opportunity to reverse the malignant phenotypes of nasopharyngeal carcinoma.     

激活Raji细胞EB病毒早期抗原植物的研究

以前材料证明Raji细胞EB病毒早期抗原对于诊断鼻咽癌有很高的特异性。我们研究了1693种植物,试验发现52种植物具有激活EB病毒作用。本文报道了这些植物并讨论它们与鼻咽癌关系。     

Proteome analysis of the transformation potential of the Epstein–Barr virus-encoded latent membrane protein 1 in nasopharyngeal epithelial cells NP69

Latent membrane protein 1 (LMP1) of Epstein-Barr virus has been identified to be crucial in inducing cell transformation. However, the mechanism of LMP1-mediated epithelial cell transformation remains unclear. In this study, nasopharyngeal epithelial cells NP69 were infected with retrovirus with gene encoding wild type LMP1 or mutational LMP1 defective in binding to tumor necrosis factor receptor-associated death domain (TRADD). The NP69-LMP1(TRADD) lost some malignant phenotypes compared with the NP69-LMP1(WT). We performed proteomic approach to gain the differential protein expression profile associated with LMP1-mediated epithelial cell transformation. Furthermore, the differential expressional levels of partial identified proteins were confirmed by Western blot and real-time RT-PCR. Some were known to be related to the development of LMP1-induced transformation, and some were new LMP1-associated proteins. These data are valuable for further study of the mechanism of LMP1 in human nasopharyngeal carcinoma and provide some new clues for investigating other LMP1-associated tumors.     

鼻咽癌Epstein-Barr病毒的原位分子杂交研究

应用Epstein-Barr病毒编码的小RNA－1（Epstein-Barr virus encoded small RNA-1,EBER-1)分子探针进行原位分子杂交,检测沈阳地区鼻咽癌的Epstein-Barr病毒存在状况及意义。结果发现：EBER－1杂交信号定位于鼻咽癌的癌细胞核,在正常的鼻咽粘膜上皮细胞、间质细胞、粘液腺及淋巴细胞均为阴性。在53例鼻咽癌病例中,37例阳性,阳性率为69.8%。EBER－1的阳性表达与鼻咽癌的分化程度呈负相关,与患的生存期未见明显关系。提示：应用EBER－1分子探针可进行回顾性研究：EB病毒的存在与鼻咽癌的分化程度成负相关。     

Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers

Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo. Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo. In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo. Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation. Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia. A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus. We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection. We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant. Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo. This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection.