Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

Characterization of the Epstein-Barr virus glycoprotein BMRF-2.

Epstein-Barr virus (EBV) BMRF-2 protein interaction with the beta1 family of integrins plays an important role in EBV infection of polarized oral epithelial cells. In this work, we characterized BMRF-2 protein expression in EBV-infected B lymphoblastoid and polarized oral epithelial cells, and in hairy leukoplakia (HL) epithelium. BMRF-2 expression in B cells and polarized oral epithelial cells was associated with the EBV lytic infection. In these cells, BMRF-2 is efficiently transported to the cell membrane and its integrin binding Arg-Gly-Asp (RGD) motif is exposed on the cell surface. BMRF-2 is highly expressed in HL epithelium and accumulates at the lateral border of oral keratinocytes. In EBV-infected polarized oral epithelial cells, this protein is transported to the basolateral membranes and co-localized with beta1 integrin. These data suggest that BMRF-2 may play an important role in cell-to-cell spread of EBV within the oral epithelium. BMRF-2 is glycosylated through O-linked oligosaccharides; it forms oligomers and is associated with the virion envelope. Its C-terminal tail is localized in the cytoplasm. We found that beta1, alpha5, and alpha3 integrins are present in purified EBV virions. We show that BMRF-2 is a ligand for beta1, alpha5, alpha3, and alphav integrins and our data are consistent with a role for BMRF-2 in viral lytic infection.

Interferon regulatory factor 7 is negatively regulated by the Epstein-Barr virus immediate-early gene, BZLF-1.

Virus infection stimulates potent antiviral responses; specifically, Epstein-Barr virus (EBV) infection induces and activates interferon regulatory factor 7 (IRF-7), which is essential for production of alpha/beta interferons (IFN-alpha/beta) and upregulates expression of Tap-2. Here we present evidence that during cytolytic viral replication the immediate-early EBV protein BZLF-1 counteracts effects of IRF-7 that are central to host antiviral responses. We initiated these studies by examining IRF-7 protein expression in vivo in lesions of hairy leukoplakia (HLP) in which there is abundant EBV replication but the expected inflammatory infiltrate is absent. This absence might predict that factors involved in the antiviral response are absent or inactive. First, we detected significant levels of IRF-7 in the nucleus, as well as in the cytoplasm, of cells in HLP lesions. IRF-7 activity in cell lines during cytolytic viral replication was examined by assay of the IRF-7-responsive promoters, IFN-alpha4, IFN-beta, and Tap-2, as well as of an IFN-stimulated response element (ISRE)-containing reporter construct. These reporter constructs showed consistent reduction of activity during lytic replication. Both endogenous and transiently expressed IRF-7 and EBV BZLF-1 proteins physically associate in cell culture, although BZLF-1 had no effect on the nuclear localization of IRF-7. However, IRF-7-dependent activity of the IFN-alpha4, IFN-beta, and Tap-2 promoters, as well as an ISRE promoter construct, was inhibited by BZLF-1. This inhibition occurred in the absence of other EBV proteins and was independent of IFN signaling. Expression of BZLF-1 also inhibited activation of IRF-7 by double-stranded RNA, as well as the activity of a constitutively active mutant form of IRF-7. Negative regulation of IRF-7 by BZLF-1 required the activation domain but not the DNA-binding domain of BZLF-1. Thus, EBV may subvert cellular antiviral responses and immune detection by blocking the activation of IFN-alpha4, IFN-beta, and Tap-2 by IRF-7 through the medium of BZLF-1 as a negative regulator.

Intracellular localization of the Epstein-Barr virus BFRF1 gene product in lymphoid cell lines and oral hairy leukoplakia lesions.

A novel protein encoded by the BFRF1 gene of the Epstein-Barr virus was identified recently [Farina et al. (2000) J Virol 74:3235-3244], which is antigenic "in vivo" and expressed early in the viral replicative cycle. In the present study, its subcellular localization was examined in greater detail comparing Epstein-Barr virus (EBV) induced producing and nonproducing cell lines by immunofluorescence: in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced Raji and B95-8 cells, as well as in anti-IgG-stimulated Akata cells, the protein appeared to be localized over the cell nuclear membrane. A similar nuclear membrane localization was observed in epithelial cells of oral hairy leukoplakia, a pathological manifestation of permissive EBV infection. In contrast, upon transfection of BFRF1 in the EBV-negative Burkitt's lymphoma cell line DG75, the protein was localized predominantly over the plasma membrane. The membrane localization was abolished when DG75 cells were transfected with a C-terminal deletion mutant of BFRF1 lacking the transmembrane domain. Because induced Raji cells do not produce virus, the above observations indicate that the nuclear membrane localization is not associated with viral production, but requires the expression of EBV genes, and suggest that additional proteins, expressed early during viral lytic infection, might be necessary to target the protein to the nuclear membrane. Immunogold electron microscopy on ultrathin cryosections of induced B95-8 cells showed that BFRF1 on the nuclear membranes was concentrated over multilayered domains representing areas of active viral replication or at the sites of viral budding, suggesting that BFRF1 is involved in the process of viral assembly.

Integrin expression in oral hairy leukoplakia and normal tongue epithelium.

OBJECTIVE: To describe the expression of integrins in the epithelium of oral hairy leukoplakia (HL) and compare to that of normal lateral tongue epithelium. MATERIALS AND METHODS: Immunohistochemistry to identify integrins (alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1) was performed, using a standard biotin-streptavidin-peroxidase technique on five clinically and histologically confirmed frozen biopsy specimens of HL and five normal lateral tongue control tissues. RESULTS: Expression of integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 was seen both in HL epithelium and in normal control tissue. alpha 5 expression was not seen in HL or in control tissue epithelium. alpha 2 and alpha 3 were expressed mainly in the basal and suprabasal layers; alpha 6 expression was most intense on the basal surface of the basal cells, alpha v was expressed in the basal and suprabasal layers with more expression seen in the higher differentiated cell layers than the other integrins. beta 1 expression was seen in the basal and suprabasal layers only. No apparent difference between HL and normal oral mucosa was noted in the staining pattern of the various integrins. CONCLUSION: Integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 are expressed in HL and the expression pattern is not different from that of normal oral mucosa. alpha 5 is not expressed in HL or in normal oral epithelium.

Bromodeoxyuridine incorporation and Ki 67 expression in oral hairy leukoplakia.

OBJECTIVES: Oral hairy leukoplakia (HL) is an acanthotic, hyperparakeratotic lesion characterised by the presence of a replicative Epstein-Barr virus (EBV) infection in the superficial and adjoining layers of the epithelium. EBV or its gene products are capable of modifying epithelial differentiation. The aim of this study was to establish whether the presence of EBV was associated with an alteration in cell turnover by assessing bromodeoxyuridine (BrdU) incorporation and Ki 67 expression in lesional tissue and control mucosa. METHODS: Biopsies of HL together with age, site and sex matched controls (n = 7 and 8 respectively) were incubated in 200 microM BrdU in vitro, fixed in methacarn and processed to paraffin wax. Following acid hydrolysis, incorporated BrdU and Ki 67 were identified in serial 5 microns sections using a three-stage immunoperoxidase technique and cell density expressed as the number of positive cells per mm basement membrane length. RESULTS: Overall, there was no difference in the number of BrdU positive cells per mm basement membrane length between control and HL tissue. However, within HL alone, the presence of focal EBV replication was associated with a significant reduction in the number of basal cells incorporating BrdU compared to adjacent EBV free areas (P < 0.05). There was no significant difference between Ki 67 positive cells in control and HL tissue and no evidence of a reduction of Ki 67 positive cells in areas associated with EBV replication. CONCLUSIONS: These results suggest that there is no evidence of a generalised alteration of the proliferative capacity of basal cells in HL, although the focal reduction in BrdU incorporation may reflect subtle changes on cell turnover by EBV infection.

Apoptosis-associated proteins in oral hairy leukoplakia.

OBJECTIVE: To test the hypothesis that the anti-apoptotic ability of Epstein-Barr virus (EBV) may result in altered expression of apoptosis-associated proteins in oral hairy leukoplakia (HL), we evaluated HL tissue and normal epithelium for these proteins by immunohistochemistry. MATERIALS AND METHODS: Twenty formalin-fixed, paraffin-embedded specimens of HL lesions and six specimens of normal control mucosa were selected from archived tissue specimens. Bcl-2, Bcl-x, Bax and p53 apoptosis-associated proteins were evaluated in immunohistochemically stained tissue sections according to staining intensity and pattern. The percentage of p53-positive basal cells was estimated in sequential fields. RESULTS: Generally, there were only slight differences in the expression of Bcl-2 and Bcl-x proteins in the epithelium of HL and control tissue. The staining for Bcl-2 was weaker in keratinocytes than in putative melanocytes and Langerhans cells. Equivocal diffuse cytoplasmic staining of prickle cells was also noted. Keratinocytes throughout the epithelium stained positively for Bcl-x protein, although upper layers were more weakly stained. The 'balloon' keratinocytes in HL were infrequently positive for Bcl-x. Bax staining in HL differed from that in control tissue in being more heterogeneous. The staining reaction in HL was weak to negative in upper epithelial levels where 'balloon' keratinocytes were located. Weak to moderate nuclear p53 protein staining was detected in a mean of 25.3% of basal keratinocytes in all but one of the HL specimens; weak staining was seen in only two control specimens. CONCLUSIONS: We found only slight immunohistochemical evidence that expression of the apoptosis-associated proteins is altered in HL. p53 appears to over-expressed in HL; we speculate that this may be related to up-regulation or stabilization of wild-type p53 protein related to EBV infection.

Elevated cortisol levels in whole saliva in HIV infected individuals.

Free cortisol concentrations in unstimulated whole saliva samples, collected at 10.00 to 11.00 h, from 23 unmedicated HIV-positive patients and 14 control subjects were measured by radioimmunoassay. Mean cortisol level (nmol/l +/- SD) was significantly higher in the HIV patients than in control subjects (27.4 +/0 9.3 vs. 10.1 +/- 3.5). Two HIV patients with pseudomembranous candidiasis had the highest saliva cortisol concentrations (mean of 48.5 nmol/l). Two other HIV patients (one with Kaposi's sarcoma and the other with periodontitis) had a mean cortisol value of 29.9 nmol/l. The possibility of plasma contamination of whole saliva in the HIV patients with inflammatory oral mucosal lesions notwithstanding, our findings suggest an increased oral burden of cortisol in both the asymptomatic and symptomatic HIV-infected individuals. Glucocorticoids caused immunosuppression, provide selective growth advantage to various microorganisms including the fungi, and enhance replication or reactivation of latent viruses (e.g. EBV, CMV, Kaposi's sarcoma-associated herpes viruses). Our findings suggest a need to evaluate the relevance of endogenous glucocorticoid excess in blood and saliva to the causation of some major AIDS-associated oral lesions such as candidiasis, Kaposi's sarcoma, oral hairy leukoplakia and necrotizing gingivitis.

Keratinocyte expression of calprotectin in oral inflammatory mucosal diseases.

Oral mucosal inflammation evolves in response to microbial pathogens and non-infectious antigens which activate humoral and cell-mediated immunologic processes. Most of these disease processes invoke a leukocyte response culminating in cellular infiltration of the submucosa and, to some degree, transmigration into the epithelium itself. Calprotectin, a leukocyte-derived dimeric protein complex that has potent antibacterial and antifungal effects, has recently been identified in skin and mucosal keratinocytes implying that epithelium may biochemically contribute to the overall mechanism of host defense. In this study, the upregulation of calprotectin as assessed immunohistochemically is pursued for oral diseases of immunopathologic, fungal and viral origin. In lichen planus, candidiasis, herpes virus stomatitis, and oral hairy leukoplakia, calprotectin was found to be expressed to a significantly higher level than in normal control mucosal samples.