Human papillomavirus and Epstein-Barr virus co-infection in cervical carcinoma in Algerian women.

BACKGROUND: Despite the fact that the implication of human papillomavirus (HPV) in the carcinogenesis and prognosis of cervical cancer is well established, the impact of a co-infection with high risk HPV (HR-HPV) and Epstein-Barr virus (EBV) is still not fully understood. METHODS: Fifty eight randomly selected cases of squamous cell carcinomas (SCC) of the uterine cervix, 14 normal cervices specimens, 21 CIN-2/3 and 16 CIN-1 cases were examined for EBV and HPV infections. Detection of HR-HPV specific sequences was carried out by PCR amplification using consensus primers of Manos and by Digene Hybrid Capture. The presence of EBV was revealed by amplifying a 660 bp specific EBV sequence of BALF1. mRNA expression of LMP-1 in one hand and protein levels of BARF-1, LMP-1 and EBNA-1 in the other hand were assessed by RT-PCR and immunoblotting and/or immunohischemistry respectively. RESULTS: HR-HPV infection was found in patients with SCC (88%), low-grade (75%) and high grade (95%) lesions compared to only 14% of normal cervix cases. However, 69%, 12.5%, 38.1%, and 14% of SCC, CIN-1, CIN-2/3 and normal cervix tissues, respectively, were EBV infected. The highest co-infection (HR-HPV and EBV) was found in squamous cell carcinoma cases (67%). The latter cases showed 27% and 29% expression of EBV BARF-1 and LMP-1 oncogenes respectively. CONCLUSION: The high rate of HR-HPV and EBV co-infection in SCC suggests that EBV infection is incriminated in cervical cancer progression. This could be taken into account as bad prognosis in this type of cancer. However, the mode of action in dual infection in cervical oncogenesis needs further investigation.

Cellular localization of BARF1 oncoprotein and its cell stimulating activity in human epithelial cell.

BARF1 gene encoded by Epstein-Barr virus is capable of immortalizing the primary monkey epithelial cells and of inducing malignant transformation in human EBV-negative B cell lines as well as rodent fibroblast. This oncoprotein is a secreted protein capable of acting as a powerful mitogen. We have studied the effect of BARF1 protein in transfected or BARF1 protein treated human HaCaT epithelial cells. In BARF1-transfected cells, cell growth was activated and its protein was found both in culture medium and cellular compartment (membrane, cytoplasm and nuclei). When purified BARF1 protein was exogenously added in the cell culture medium of HaCaT cells in absence of fetal calf serum led to its entrance into cells and its intracellular localization in cytoplasm, nuclear periphery and nuclei at 14h treatment, determined by confocal and immunoelectron microscopy. Cell fractionation confirmed its nuclear localization. Nuclear localization was observed in both systems. More interestingly, purified BARF1 protein p29 exogenously added in the cell culture medium activated cell passage of G1 to S phase. S phase activation by its autocrine activity and its tumorigenic activity would be associated with the development of EBV-associated carcinomas.

Synergism of BARF1 with Ras induces malignant transformation in primary primate epithelial cells and human nasopharyngeal epithelial cells.

Although it is well known that nasopharyngeal carcinoma (NPC) is closely related with Epstein-Barr virus (EBV), few data are available about which and how EBV-expressed gene is involved in the carcinogenesis of human nasopharyngeal epithelial cells. EBV-encoded BARF1 (BamH I-A right frame 1) gene has been shown to be oncogenic and capable of inducing malignant transformation in BALB/c3T3 and NIH3T3 cells as well as in human B-cell lines Louckes and Akata. It remains unclear, however, whether BARF1 can transform primate or human epithelial cells. Here, we have shown that overexpression of H-Ras gene transformed BARF1-immortalized PATAS cells into malignant cell line. Furthermore, we found that cooperation of BARF1 with H-Ras and SV40 T antigens was sufficient to transform nonmalignant human nasopharyngeal epithelial NP69 cells when serially introduced BARF1 and H-Ras into the SV40 T antigens-immortalized NP69 cells. Taken together, these results demonstrated that the cooperation of BARF1 with Ras suffices to transform primary primate epithelial cell PATAS. Similarly, BARF1 together with H-Ras and SV40 T can transform human epithelial cell NP69, thereby indicating that BARF1 could be involved in the NPC pathogenesis in combination with additional genetic changes.

Spontaneous T cell responses to Epstein-Barr virus-encoded BARF1 protein and derived peptides in patients with nasopharyngeal carcinoma: bases for improved immunotherapy.

Immunotherapy approaches targeting Epstein-Barr virus (EBV)-encoded antigens induce objective clinical responses only in a fraction of patients with undifferentiated nasopharyngeal carcinoma (UNPC). In the present study, we have characterized the immunogenicity of the EBV-encoded BARF1 oncogene with the aim to assess whether this protein could constitute a new target antigen for immunotherapy in this setting. Spontaneous CD4+ and CD8+ T cell responses specific for the recombinant p29 BARF1 protein were detected by IFNgamma-ELISPOT in both EBV-seropositive donors and UNPC patients, but not in EBV-seronegative individuals. Using immunoinformatic prediction tools, we have selected 5 different candidate BARF1 T cell epitopes presented by HLA-A*0201. Although only one of these peptides was able to bind HLA-A2 with low affinity in the T2 stabilization assay, all 5 BARF1 nonamers readily elicited specific CD8+ T cell responses in EBV-seropositive HLA-A*0201+ donors and UNPC patients. Notably, the magnitude of CD8+ T cell responses to the whole BARF1 protein and derived A*0201 peptides was significantly higher in UNPC patients than in healthy donors. Moreover, cytotoxic T lymphocytes specific for the p2-10, p23-31, or p49-57 BARF1 peptides were easily obtained from HLA-A*0201+ donors. These cultures were not only able to lyse autologous targets loaded with the antigenic peptide, but also recognized tumor cells endogenously expressing BARF1 in an antigen-specific and HLA-A2-restricted manner. These findings, indicate that BARF1 is a particularly attractive antigen with immunogenic properties in most UNPC patients and provide valuable information to develop new strategies to improve the efficacy of EBV-targeting immunotherapy of UNPC patients.

Secretion of Epstein-Barr virus-encoded BARF1 oncoprotein from latently infected B cells.

Epstein-Barr virus (EBV) encodes two oncogenes, LMP1(Latent Membrane Protein-1) and BARF1 (BamH1-A Reading Frame-1). LMP1 belongs to latent gene family and BARF1 is considered so far as one of early gene family. However BARF1 oncogene was expressed highly in Nasopharyngeal (NPC) and gastric (GC) carcinoma as a type II latency, and in EBV-positive Akata cell and primary epithelial cell infected in vitro by EBV as type I latency. Its expression was also reported in Burkitt's lymphoma's biopsy frequent in Malawi in Africa as well as in nasal NK/T-cell lymphoma. We recently observed a massive secretion of BARF1 protein in serum and saliva of NPC patients. NPC-derived c666-1 epithelial cells also expressed and secreted BARF1 protein without other lytic genes expression. We asked whether this oncogene belongs to latent gene family. To investigate, we examined its transcriptional and translational expression in IB4 and Akata B cells where both cell lines belong to latent cell family. Transcriptional expression was analyzed by RT-PCR. As BARF1 protein is one of secreted proteins, its translational expression was analyzed by immunoblot after concentration of culture medium. Secreted BARF1 protein was futher purified by concanavalin A affinity column. BARF1 was transcribed in both EBV-positive AKATA and IB4 cells, and BARF1 protein was secreted from these latently infected human B cells. Its secretion does not depend EBV genome form in infected cells. Both episomal and integrated form of EBV genome were capable of expressing BARF1 gene. These results suggests that BARF1 is expressed in latent stage and increases its expression during lytic stage.

Cyclin D1 expression is induced by viral BARF1 and is overexpressed in EBV-associated gastric cancer.

Approximately 10% of gastric carcinomas (GC) worldwide are associated with Epstein-Barr virus (EBV). GC is one of the most frequent human malignancies associated with EBV. The latent expression of the EBV-oncogene BARF1 is restricted to epithelial malignancies. To investigate the underlying BARF1-related mechanisms of oncogenic epithelial transformation, we analyzed gene expression profiles of a BARF1-transfected epithelial (HaCaT+) and the corresponding BARF1-negative (HaCaT-) cell line by cDNA microarray analysis. Real-time PCR was performed to confirm the cDNA microarray results. In addition, immunohistochemistry and fluorescence in situ hybridization were performed on a tissue microarray of 181 GC including 11 EBV-associated GC. Among other genes cyclin D1 expression was significantly upregulated in HaCaT+ on the transcriptional and protein level. Cyclin D1 protein expression in GC revealed a significant overexpression of cyclin D1 in EBV-associated GC (p<0.012) but not in EBV-negative GC. Cyclin D1 FISH showed that cyclin D1 overexpression was not due to gene amplification in EBV-associated GC. Cyclin D1 is induced in HaCaT+ by BARF1 and is overexpressed in EBV-associated GC indicating an interaction of viral BARF1 and cyclin D1.

Genotypic detection of acyclovir-resistant HSV-1: characterization of 67 ACV-sensitive and 14 ACV-resistant viruses.

Infections due to herpes simplex virus (HSV) resistant to acyclovir (ACV) represent an important clinical concern in immunocompromised patients. In order to switch promptly to an appropriate treatment, rapid viral susceptibility assays are required. We developed herein a genotyping analysis focusing on thymidine kinase gene (TK) mutations in order to detect acyclovir-resistant HSV in clinical specimens. A total of 85 HSV-1 positive specimens collected from 69 patients were analyzed. TK gene could be sequenced directly for 81 clinical specimens (95%) and 68 HSV-1 specimens could be characterized as sensitive or resistant by genotyping (84%). Genetic characterization of 67 susceptible HSV-1 specimens revealed 10 polymorphisms never previously described. Genetic characterization of 14 resistant HSV-1 revealed 12 HSV-1 with either TK gene additions/deletions (8 strains) or substitutions (4 strains) and 2 HSV-1 with no mutation in the TK gene. DNA polymerase gene was afterwards explored. With this rapid PCR-based assay, ACV-resistant HSV could be detected directly in clinical specimens within 24 h.

Reconstitution of nasopharyngeal carcinoma-type EBV infection induces tumorigenicity.

Several reports have shown that the EBV-encoded BARF1 gene has oncogenic activity. We have recently reported that BARF1 is expressed as a latent gene in most nasopharyngeal carcinomas (NPC), suggesting that BARF1 may have an important role in NPC oncogenesis. However, we found that when the NPC-derived EBV-negative cell lines, HONE-1 and CNE-1, were infected with EBV in vitro, BARF1 was not expressed, although the expression of other latent genes was identical to that of NPC tumors. Therefore, we generated a recombinant EBV (rEBV) carrying the BARF1 gene (BARF1-rEBV) under the SV40 promoter to reconstitute the NPC-type EBV infection. NPC-derived EBV-negative cell lines were stably infected with either a wild-type rEBV (wild-rEBV) or BARF1-rEBV. The resultant BARF1-rEBV-infected NPC cell clones represented NPC-type EBV expression, and BARF1 expression was similar to that observed in NPC tissues. BARF1-rEBV-infected cell clones grew to a higher cell density and were more resistant to apoptosis than wild-rEBV-infected counterparts. BARF1 protein was quickly secreted into the culture medium, and secreted BARF1 contributed to the increase of cell densities in NPC cells, but it had no effect on resistance to apoptosis. Furthermore, BARF1-rEBV-infected cell clones became tumorigenic in nude mice. These results suggest that BARF1 plays an important role in NPC development.

Post-translational modifications of Epstein Barr virus BARF1 oncogene-encoded polypeptide.

Epstein-Barr virus is associated with several human lymphomas and carcinomas, and its BARF1 oncogene encodes a protein that is thought to play an important role in carcinogenesis. A BARF1 recombinant adenovirus expression system, which led us to discover the macromolecular size of the cleaved and secreted form of the BARF1 protein in the native state and its mitogenic capacity on various cell lines in culture, was used further to investigate the structure and maturation of the BARF1 protein. We recently reported biophysical studies that showed dimer-based oligomerization of the BARF1 polypeptide. Here, new data are presented that confirm post-translational modifications predicted from the BARF1 sequence: phosphorylation on serine and threonine, and N- and O-glycosylation. The N- and O-glycans were partially characterized and it was demonstrated that both modifications are required for active secretion of the BARF1 protein via the classical pathway.

A new diagnostic marker for secreted Epstein-Barr virus encoded LMP1 and BARF1 oncoproteins in the serum and saliva of patients with nasopharyngeal carcinoma.

PURPOSE: EBV has been associated with nasopharyngeal carcinomas (NPC). In North Africa, the incidence is bimodal-the first peak occurring at approximately 20 years of age and the second peak occurring at approximately 50 years. Standard diagnostic tests based on immunofluorescence using anti-IgA EBV have shown that young North African patients have a negative serology compared with older patients. We are interested in two EBV-encoded oncoproteins, LMP1 and BARF1, which have thus far not been studied in terms of their potential as diagnostic markers for NPC. These two viral oncoproteins have been detected in cell culture media, so we tested whether they could be detected in the serum and saliva of patients with NPC. EXPERIMENTAL DESIGN: LMP1 and BARF1 proteins were analyzed in the sera and saliva of young patients and adult patients with NPC from North Africa and China. We then examined whether the secreted proteins had biological activity by analyzing their mitogenic activity. RESULTS: Both LMP1 and BARF1 were present in the serum and saliva from North African and Chinese patients with NPC. All young North African patients secreted both proteins, whereas 62% and 100% of adult patients secreted LMP1 and BARF1, respectively. From animal studies, the secreted LMP1 was associated with exosome-like vesicles. These secreted EBV oncoproteins showed a powerful mitogenic activity in B cells. CONCLUSION: Both proteins will be a good diagnostic marker for NPC whereas BARF1 is a particularly promising marker for all ages of patients with NPC. Their mitogenic activity suggests their implication in the oncogenic development of NPC.

Functional advantage of NPC-related V-val subtype of Epstein-Barr virus nuclear antigen 1 compared with prototype in epithelial cell line.

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC) and the viral nuclear antigen 1 (EBNA1) plays a crucial role in viral latency. Three EBNA1 subtypes, P-ala, V-thr and V-val have been detected from healthy carriers in Guangzhou area. A close relation of V-val EBNA1 with NPC was suggested by its preference to infect NPC cells. We therefore investigated the functional difference among these three EBNA1 subtypes in human epithelial cell line. The three coding sequences of the EBNA1 subtypes were cloned into the pGFP-C2 vector, and transfected into 293 cells, respectively. Effect of EBNA1 expression on cell proliferation was examined. The maintenance activity and expression level of EBNA1-plasmid in 293 cells were evaluated by using GFP as a reporter. The expression of P-ala, V-thr or V-val EBNA1 had no effect on 293 cell growth, while the relative average intensity of fluorescence after 14-day selection in V-val-EBNA1/293 cells was statistically higher than P-ala-EBNA1/293 (P<0.05, t test). We suggest that V-val EBNA1 with the functional advantage compared with prototype shown in this study might contribute to the tumorigenesis of NPC by increasing the expression of itself or other viral or cellular genes.

Resistance of herpes simplex virus type 1 to acyclovir: thymidine kinase gene mutagenesis study.

By site-directed mutagenesis, we investigate the role of six mutations of herpes simplex virus type 1 thymidine kinase (TK) gene in the acquisition of resistance to acyclovir (ACV). TK activity was not impaired by substitutions located at codons 17, 161 and 374 and these mutations were thus related to TK gene polymorphism. Mutations His105Pro, Leu364Pro and Asp162Ala lead to the loss of TK activity that could result in ACV-resistance.

Structure of the Epstein-Barr virus oncogene BARF1.

The Epstein-Barr virus is a human gamma-herpesvirus that persistently infects more than 90% of the human population. It is associated with numerous epithelial cancers, principally undifferentiated nasopharyngeal carcinoma and gastric carcinoma. The BARF1 gene is expressed in a high proportion of these cancers. An oncogenic, mitogenic and immortalizing activity of the BARF1 protein has been shown. We solved the structure of the secreted BARF1 glycoprotein expressed in a human cell line by X-ray crystallography at a resolution of 2.3A. The BARF1 protein consists of two immunoglobulin (Ig)-like domains. The N-terminal domain belongs to the subfamily of variable domains whereas the C-terminal one is related to a constant Ig-domain. BARF1 shows an unusual hexamerisation involving two principal contacts, one between the C-terminal domains and one between the N-terminal domains. The C-terminal contact with an uncommonly large contact surface extends the beta-sandwich of the Ig-domain through the second molecule. The N-terminal contact involves Ig-domains with an unusual relative orientation but with a more classical contact surface with a size in the range of dimer interactions of Ig-domains. The structure of BARF1 is most closely related to CD80 or B7-1, a co-stimulatory molecule present on antigen presenting cells, from which BARF1 must have been derived during evolution. Still, domain orientation and oligomerization differ between BARF1 and CD80. It had been shown that BARF1 binds to hCSF-1, the human colony-stimulating factor 1, but this interaction has to be principally different from the one between CSF-1 and CSF-1 receptor.

Anti-apoptotic role of BARF1 in gastric cancer cells.

Epstein-Barr virus (EBV) infection has been implicated in the carcinogenesis of several types of human cancer, including gastric cancer. In contrast to two other EBV-related malingancies, nasopharyngeal carcinoma and Hodgkins Lympomain which the latent membrane protein (LMP)-1 is often detected, in gastric cancer, BARF1, one of the early EBV genes, is frequently expressed in EBV-positive specimens. This indicates that expression of BARF1 may play a positive role in the development of gastric cancer. The aim of this study was to investigate the effect of BARF1 expression in gastric cancer cells. First, a retroviral vector containing the full length BARF1 gene was transfected into an EBV negative gastric cancer cell line, BGC823, and stable transfectants expressing ectopic BARF1 were generated. Microarray analysis was then performed and gene expression profiles were analysed and compared between the cells expressing ectopic BARF1 and the vector control. In addition, the effect of BARF1 on gastric cancer cell proliferation and apoptosis was investigated by MTT assay, DAPI staining, flow cytometry as well as Western blotting. We found that expression of BARF1 in gastric cancer cells led to significant alterations of gene expression, especially genes related to proliferation and apoptosis. In addition, the BARF1 expressing cells were more resistant to apoptosis induced by a commonly used anticancer drug, taxol. This chemo-protective effect of BARF1 was associated with increased Bcl-2 and Bax ratio and decreased expression of cleaved PARP, but not alterations in cell proliferation. Our results suggest that BARF1 expression in gastric cancer cells may provide a protective role against apoptosis through an increased Bcl-2 to Bax ratio, thus promoting cancer cell survival.

Epstein-Barr virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression.

The BARF1 gene is located in the BamHI-A fragment of the Epstein-Barr virus (EBV) genome, encodes 221 amino acids, and has activity as an oncogene. Several reports have demonstrated that BARF1 is expressed in the tissues of various EBV-associated epithelioid malignancies. However,BARF1 is thought to be a lytic gene, since its expression is induced upon induction of the lytic cycle in Burkitt's lymphoma cell lines. Therefore, the possibility cannot be excluded that BARF1 expression in EBV-associated epithelioid malignancies reflects spontaneous induction of the lytic cycle in carcinoma cells. The present study aimed to clarify whether BARF1 was expressed as a latent gene or a lytic gene in epithelioid malignancies. Quantitative real-time RT-PCR assay revealed that BARF1 was highly expressed in nasopharyngeal carcinoma (NPC) and EBV-positive gastric carcinoma tissues in the absence of expression of lytic genes. On the other hand, BARF1 protein was detectable only in two of seven NPC tissue samples by immunoblot analysis. Analysis of BARF1-transfected CNE1 cells revealed that BARF1 was quickly secreted into culture medium and was hardly detectable in the cell lysate, which would account for why some NPC tissues were negative for BARF1 protein expression even though they were strongly positive forBARF1 expression at the transcriptional level. The present findings indicate that BARF1 is expressed in NPC and EBV-positive gastric carcinoma tissues as a latent gene and suggest that BARF1 plays a role in the pathogenesis of these malignancies.

BAMHI DNA fragment H-polymorphism of Epstein-Barr virus is associated with the mutations present in an 89 BP sequence localized in EBNA2 gene.

To characterize the genotypes of Epstein-Barr virus (EBV) isolate present in North Africa, viruses were isolated from B-lymphoblastoid cell lines established from the saliva of both Algerian Nasopharyngeal Carcinoma (NPC) patients and EBV-positive normal individuals, Algerian Burkitt's lymphoma cell lines, and NPC biopsies. By nucleotide sequence analysis, we showed that there were two specific missense mutations in an 89 bp region of EBNA2 gene at position 49390-49479 of the EBV genome: a mutation at 49449 (C-->A) and another mutation at 49444 (T-->C), changing their amino acid sequence. The first mutation was found in all B cell lines established from the saliva and 50% of BL cell lines, as well as the W91 cell line, while the second mutation was found in EBV isolates from NPC biopsies, BL cell lines and the M-ABA isolate. A PCR-RFLP analysis on the BamHI DNA fragment H showed that the Hl-H2-polymorphism was specifically associated with M-ABA-like mutation, while H-polymorphism was linked with W91-like mutation. The latter was not identified in NPC biopsies, but was found rather in saliva from NPC patients, normal individuals and BL cell lines. The M-ABA-like mutation, on the other hand, was found in 100% of NPC biopsies and some BL cell lines. This suggests that EBV with H1-H2-polymorphism is tightly implicated in NPC development in North Africa rather than EBV with H-polymorphism.

Mitogenic activity of Epstein-Barr virus-encoded BARF1 protein.

We previously reported that BARF1 gene has either an immortalizing effect, when expressed in primary primate epithelial cells, or a malignant transforming activity, when expressed in established and nontumoral rodent fibroblast or human B-cell lines. As predicted from sequence analysis, we found that BARF1 coded protein can be secreted from different cell lines, among them BARF1-transfected Balb/c3T3 rodent fibroblasts. Thus, as an initial step to clarify BARF1 oncogenic functions, we investigated whether the secreted form of BARF1 protein can activate the cell cycle as a growth factor. Since efficient BARF1 expression could be obtained from 293-tTA cells infected with a tetracycline-regulatable recombinant adenovirus, secreted BARF1 product could be purified from the culture medium of such cells by ammonium sulfate precipitation, ion exchange chromotography and sucrose gradient sedimentation. We describe in this paper that addition of a purified product of secreted BARF1 protein to serum-free culture medium of Balb/c3T3 rodent fibroblasts, human Louckes B-cell line and primary monkey kidney epithelial cells resulted in a cell cycle activation that was inhibited by affinity-purified anti-BARF1 antibody. Our demonstration of a specific stimulation of cell cycle in vitro by BARF1 secreted product suggests that this EBV-encoded BARF1 protein could act as a growth factor in vivo.

Malignant transformation of Epstein-Barr virus-negative Akata cells by introduction of the BARF1 gene carried by Epstein-Barr virus.

Spontaneous loss of the Epstein-Barr virus (EBV) genome in the BL cell line Akata led to loss of tumorigenicity in SCID mice, suggesting an important oncogenic activity of EBV in B cells. We previously showed that introduction of the BARF1 gene into the human B-cell line Louckes induced a malignant transformation in newborn rats (M. X. Wei, J. C. Moulin, G. Decaussin, F. Berger, and T. Ooka, Cancer Res. 54:1843-1848, 1994). Since 1 to 2% of Akata cells expressed lytic antigens and expressed the BARF1 gene, we investigated whether introduction of the BARF1 gene into EBV-negative Akata cells can induce malignant transformation. Here we show that BARF1-transfected, EBV-negative Akata cells activated Bcl2 expression and induced tumor formation when they were injected into SCID mice. In addition, when EBV-positive Akata cells expressing a low level of BARF1 protein were injected into SCID mice, the expression of BARF1, as well as several lytic proteins, such as EA-D, ZEBRA, and a 135-kDa DNA binding protein, increased in tumor cells while no latent LMP1 and late gp220-320 expression was observed in tumor cells. These observations suggest that the BARF1 gene may be involved in the conferral of tumorigenicity by EBV.