HIV-1 Proteins gp120 and Tat Promote Epithelial-Mesenchymal Transition and Invasiveness of HPV-Positive and HPV-Negative Neoplastic Genital and Oral Epithelial Cells.

The incidence of human papillomavirus (HPV)-associated anogenital and oropharyngeal cancer in human immunodeficiency virus (HIV)-infected individuals is substantially higher than in HIV-uninfected individuals. HIV may also be a risk factor for the development of HPV-negative head and neck, liver, lung, and kidney cancer. However, the molecular mechanisms underlying HIV-1-associated increase of epithelial malignancies are not fully understood. Here, we showed that HPV-16-immortalized anal AKC-2 and cervical CaSki epithelial cells that undergo prolonged exposure to cell-free HIV-1 virions or HIV-1 viral proteins gp120 and tat respond with the epithelial-mesenchymal transition (EMT) and increased invasiveness. Similar responses were observed in HPV-16-infected SCC-47 and HPV-16-negative HSC-3 oral epithelial cancer cells that were cultured with these viral proteins. EMT induced by gp120 and tat led to detachment of poorly adherent cells from the culture substratum; these cells remained capable of reattachment, upon which they coexpressed both E-cadherin and vimentin, indicative of an intermediate stage of EMT. The reattached cells also expressed stem cell markers CD133 and CD44, which may play a critical role in cancer cell invasion and metastasis. Inhibition of transforming growth factor (TGF)-ß1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasive activity of HPV-16-immortalized anal and cervical epithelial cells. Collectively, our results suggest that these approaches along with HIV viral suppression with antiretroviral therapy (ART) might be useful to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia. IMPORTANCE HPV-16-immortalized genital and oral epithelial cells and HPV-negative oral cancer cells that undergo prolonged contact with cell-free HIV-1 virions or with viral proteins gp120 and tat respond by becoming more invasive. EMT cells induced by HIV-1 in cultures of HPV-16-immortalized anal and cervical epithelial cells express the stem cell markers CD133 and CD44. These results suggest that the interaction of HIV-1 with neoplastic epithelial cells may lead to their de-differentiation into cancer stem cells that are resistant to apoptosis and anti-cancer drugs. Thus, this pathway may play a critical role in the development of invasive cancer. Inhibition of TGF-ß1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasiveness of HPV-16-immortalized anal and cervical epithelial cells. Taken together, these results suggest that these approaches might be exploited to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia.

Inactivation of HIV-1 in Polarized Infant Tonsil Epithelial Cells by Human Beta-Defensins 2 and 3 Tagged with the Protein Transduction Domain of HIV-1 Tat.

Mother-to-child transmission (MTCT) of HIV-1 may occur during pregnancy, labor, and breastfeeding; however, the molecular mechanism of MTCT of virus remains poorly understood. Infant tonsil mucosal epithelium may sequester HIV-1, serving as a transient reservoir, and may play a critical role in MTCT. Innate immune proteins human beta-defensins 2 (hBD-2) and -3 may inactivate intravesicular virions. To establish delivery of hBD-2 and -3 into vesicles containing HIV-1, we tagged hBDs with the protein transduction domain (PTD) of HIV-1 Tat, which facilitates an efficient translocation of proteins across cell membranes. Our new findings showed that hBD-2 and -3 proteins tagged with PTD efficiently penetrated polarized tonsil epithelial cells by endocytosis and direct penetration. PTD-initiated internalization of hBD-2 and -3 proteins into epithelial cells led to their subsequent penetration of multivesicular bodies (MVB) and vacuoles containing HIV-1. Furthermore, PTD played a role in the fusion of vesicles containing HIV-1 with lysosomes, where virus was inactivated. PTD-initiated internalization of hBD-2 and -3 proteins into ex vivo tonsil tissue explants reduced the spread of virus from epithelial cells to CD4+ T lymphocytes, CD68+ macrophages, and CD1c+ dendritic cells, suggesting that this approach may serve as an antiviral strategy for inactivating intraepithelial HIV-1 and reducing viral MTCT.

Human Immunodeficiency Virus (HIV) and Human Cytomegalovirus (HCMV) Coinfection of Infant Tonsil Epithelium May Synergistically Promote both HIV-1 and HCMV Spread and Infection.

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) and human cytomegalovirus (HCMV) may occur during pregnancy, labor, or breastfeeding. These viruses from amniotic fluid, cervicovaginal secretions, and breast milk may simultaneously interact with oropharyngeal and tonsil epithelia; however, the molecular mechanism of HIV-1 and HCMV cotransmission through the oral mucosa and its role in MTCT are poorly understood. To study the molecular mechanism of HIV-1 and HCMV MTCT via oral epithelium, we established polarized infant tonsil epithelial cells and polarized-oriented ex vivo tonsil tissue explants. Using these models, we showed that cell-free HIV-1 and its proteins gp120 and tat induce the disruption of tonsil epithelial tight junctions and increase paracellular permeability, which facilitates HCMV spread within the tonsil mucosa. Inhibition of HIV-1 gp120-induced upregulation of mitogen-activated protein kinase (MAPK) and NF-κB signaling in tonsil epithelial cells, reduces HCMV infection, indicating that HIV-1-activated MAPK and NF-κB signaling may play a critical role in HCMV infection of tonsil epithelium. HCMV infection of tonsil epithelial cells also leads to the disruption of tight junctions and increases paracellular permeability, facilitating HIV-1 paracellular spread into tonsil mucosa. HCMV-promoted paracellular spread of HIV-1 increases its accessibility to tonsil CD4 T lymphocytes, macrophages, and dendritic cells. HIV-1-enhanced HCMV paracellular spread and infection of epithelial cells subsequently leads to the spread of HCMV to tonsil macrophages and dendritic cells. Our findings revealed that HIV-1- and HCMV-induced disruption of infant tonsil epithelial tight junctions promotes MTCT of these viruses through tonsil mucosal epithelium, and therapeutic intervention for both HIV-1 and HCMV infection may substantially reduce their MTCT. IMPORTANCE Most HIV-1 and HCMV MTCT occurs in infancy, and the cotransmission of these viruses may occur via infant oropharyngeal and tonsil epithelia, which are the first biological barriers for viral pathogens. We have shown that HIV-1 and HCMV disrupt epithelial junctions, reducing the barrier functions of epithelia and thus allowing paracellular penetration of both viruses via mucosal epithelia. Subsequently, HCMV infects epithelial cells, macrophages, and dendritic cells, and HIV-1 infects CD4+ lymphocytes, macrophages, and dendritic cells. Infection of these cells in HCMV- and HIV-1-coinfected tonsil tissues is much higher than that by HCMV or HIV-1 infection alone, promoting their MTCT at its initial stages via infant oropharyngeal and tonsil epithelia.

HIV-1 proteins gp120 and tat induce the epithelial-mesenchymal transition in oral and genital mucosal epithelial cells.

The oral, cervical, and genital mucosa, covered by stratified squamous epithelia with polarized organization and strong tight and adherens junctions, play a critical role in preventing transmission of viral pathogens, including human immunodeficiency virus (HIV). HIV-1 interaction with mucosal epithelial cells may depolarize epithelia and disrupt their tight and adherens junctions; however, the molecular mechanism of HIV-induced epithelial disruption has not been completely understood. We showed that prolonged interaction of cell-free HIV-1 virions, and viral envelope and transactivator proteins gp120 and tat, respectively, with tonsil, cervical, and foreskin epithelial cells induces an epithelial-mesenchymal transition (EMT). EMT is an epigenetic process leading to the disruption of mucosal epithelia and allowing the paracellular spread of viral and other pathogens. Interaction of cell-free virions and gp120 and tat proteins with epithelial cells substantially reduced E-cadherin expression and activated vimentin and N-cadherin expression, which are well-known mesenchymal markers. HIV gp120- and tat-induced EMT was mediated by SMAD2 phosphorylation and activation of transcription factors Slug, Snail, Twist1 and ZEB1. Activation of TGF-ß and MAPK signaling by gp120, tat, and cell-free HIV virions revealed the critical roles of these signaling pathways in EMT induction. gp120- and tat-induced EMT cells were highly migratory via collagen-coated membranes, which is one of the main features of mesenchymal cells. Inhibitors of TGF-ß1 and MAPK signaling reduced HIV-induced EMT, suggesting that inactivation of these signaling pathways may restore the normal barrier function of mucosal epithelia.

E5 can be expressed in anal cancer and leads to epidermal growth factor receptor-induced invasion in a human papillomavirus 16-transformed anal epithelial cell line.

We created the first human papillomavirus (HPV)-16-immortalized anal epithelial cell line, known as AKC2 cells to establish an in vitro model of HPV-16-induced anal carcinogenesis. Consistent with detection of E6, E7 and E5 expression in anal cancer biopsies, AKC2 cells expressed high levels of all three HPV oncogenes. Also, similar to findings in anal cancer biopsies, epidermal growth factor receptor (EGFR) was overexpressed in AKC2 cells. AKC2 cells exhibited a poorly differentiated and invasive phenotype in three-dimensional raft culture and inhibition of EGFR function abrogated AKC2 invasion. Reducing E5 expression using E5-targeted siRNAs in AKC2 cells led to knockdown of E5 expression, but also HPV-16 E2, E6 and E7 expression. AKC2 cells treated with E5-targeted siRNA had reduced levels of total and phosphorylated EGFR, and reduced invasion. Rescue of E6/E7 expression with simultaneous E5 knockdown confirmed that E5 plays a key role in EGFR overexpression and EGFR-induced invasion.

HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles.

Recently, we showed that HIV-1 is sequestered, i.e., trapped, in the intracellular vesicles of oral and genital epithelial cells. Here, we investigated the mechanisms of HIV-1 sequestration in vesicles of polarized tonsil, foreskin and cervical epithelial cells. HIV-1 internalization into epithelial cells is initiated by multiple entry pathways, including clathrin-, caveolin/lipid raft-associated endocytosis and macropinocytosis. Inhibition of HIV-1 attachment to galactosylceramide and heparan sulfate proteoglycans, and virus endocytosis and macropinocytosis reduced HIV-1 sequestration by 30-40%. T-cell immunoglobulin and mucin domain 1 (TIM-1) were expressed on the apical surface of polarized tonsil, cervical and foreskin epithelial cells. However, TIM-1-associated HIV-1 macropinocytosis and sequestration were detected mostly in tonsil epithelial cells. Sequestered HIV-1 was resistant to trypsin, pronase, and soluble CD4, indicating that the sequestered virus was intracellular. Inhibition of HIV-1 intraepithelial sequestration and elimination of vesicles containing virus in the mucosal epithelium may help in the prevention of HIV-1 mucosal transmission.

Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction.

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60-70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission.

Human beta-defensins 2 and -3 cointernalize with human immunodeficiency virus via heparan sulfate proteoglycans and reduce infectivity of intracellular virions in tonsil epithelial cells.

We previously showed that expression of the anti-HIV innate proteins human beta-defensin 2 (hBD2) and hBD3 in adult oral epithelial cells reduces HIV transepithelial transmission by inactivation of virus. However, fetal/infant oral epithelia lack beta-defensin expression, leading to transmission of HIV. The mechanisms of hBD2- and hBD3-mediated HIV inactivation in adult oral epithelial cells are poorly understood. Here we found that heparan sulfate proteoglycans (HSPGs) on the apical surfaces of epithelial cells facilitate simultaneous binding of hBDs and HIV gp120 to the cell surface. HSPG-facilitated binding of hBDs and HIV gp120 to the cell surface did not affect viral attachment. HBD2 or -3 cointernalized with virions in endosomes, formed oligomers, and reduced infectivity of HIV. The anti-HIV effect of combining hBD2 and hBD3 was substantially higher than that of the individual peptides. These findings advance our understanding of the mechanisms of anti-HIV resistance in adult oral epithelium.

HIV-associated disruption of mucosal epithelium facilitates paracellular penetration by human papillomavirus.

The incidence of human papillomavirus (HPV)-associated epithelial lesions is substantially higher in human immunodeficiency virus (HIV)-infected individuals than in HIV-uninfected individuals. The molecular mechanisms underlying the increased risk of HPV infection in HIV-infected individuals are poorly understood. We found that HIV proteins tat and gp120 were expressed within the oral and anal mucosal epithelial microenvironment of HIV-infected individuals. Expression of HIV proteins in the mucosal epithelium was correlated with the disruption of epithelial tight junctions (TJ). Treatment of polarized oral, cervical and anal epithelial cells, and oral tissue explants with tat and gp120 led to disruption of epithelial TJ and increased HPV pseudovirion (PsV) paracellular penetration in to the epithelium. PsV entry was observed in the basal/parabasal cells, the cells in which the HPV life cycle is initiated. Our data suggest that HIV-associated TJ disruption of mucosal epithelia may potentiate HPV infection and subsequent development of HPV-associated neoplasia.

Epstein-Barr virus transcytosis through polarized oral epithelial cells.

Although Epstein-Barr virus (EBV) is an orally transmitted virus, viral transmission through the oropharyngeal mucosal epithelium is not well understood. In this study, we investigated how EBV traverses polarized human oral epithelial cells without causing productive infection. We found that EBV may be transcytosed through oral epithelial cells bidirectionally, from both the apical to the basolateral membranes and the basolateral to the apical membranes. Apical to basolateral EBV transcytosis was substantially reduced by amiloride, an inhibitor of macropinocytosis. Electron microscopy showed that virions were surrounded by apical surface protrusions and that virus was present in subapical vesicles. Inactivation of signaling molecules critical for macropinocytosis, including phosphatidylinositol 3-kinases, myosin light-chain kinase, Ras-related C3 botulinum toxin substrate 1, p21-activated kinase 1, ADP-ribosylation factor 6, and cell division control protein 42 homolog, led to significant reduction in EBV apical to basolateral transcytosis. In contrast, basolateral to apical EBV transcytosis was substantially reduced by nystatin, an inhibitor of caveolin-mediated virus entry. Caveolae were detected in the basolateral membranes of polarized human oral epithelial cells, and virions were detected in caveosome-like endosomes. Methyl ß-cyclodextrin, an inhibitor of caveola formation, reduced EBV basolateral entry. EBV virions transcytosed in either direction were able to infect B lymphocytes. Together, these data show that EBV transmigrates across oral epithelial cells by (i) apical to basolateral transcytosis, potentially contributing to initial EBV penetration that leads to systemic infection, and (ii) basolateral to apical transcytosis, which may enable EBV secretion into saliva in EBV-infected individuals.

Differential transmission of HIV traversing fetal oral/intestinal epithelia and adult oral epithelia.

While human immunodeficiency virus (HIV) transmission through the adult oral route is rare, mother-to-child transmission (MTCT) through the neonatal/infant oral and/or gastrointestinal route is common. To study the mechanisms of cell-free and cell-associated HIV transmission across adult oral and neonatal/infant oral/intestinal epithelia, we established ex vivo organ tissue model systems of adult and fetal origin. Given the similarity of neonatal and fetal oral epithelia with respect to epithelial stratification and density of HIV-susceptible immune cells, we used fetal oral the epithelium as a model for neonatal/infant oral epithelium. We found that cell-free HIV traversed fetal oral and intestinal epithelia and infected HIV-susceptible CD4(+) T lymphocytes, Langerhans/dendritic cells, and macrophages. To study the penetration of cell-associated virus into fetal oral and intestinal epithelia, HIV-infected macrophages and lymphocytes were added to the surfaces of fetal oral and intestinal epithelia. HIV-infected macrophages, but not lymphocytes, transmigrated across fetal oral epithelia. HIV-infected macrophages and, to a lesser extent, lymphocytes transmigrated across fetal intestinal epithelia. In contrast to the fetal oral/intestinal epithelia, cell-free HIV transmigration through adult oral epithelia was inefficient and virions did not infect intraepithelial and subepithelial HIV-susceptible cells. In addition, HIV-infected macrophages and lymphocytes did not transmigrate through intact adult oral epithelia. Transmigration of cell-free and cell-associated HIV across the fetal oral/intestinal mucosal epithelium may serve as an initial mechanism for HIV MTCT.

HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells.

Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells - and not those that passed through the adult cells - remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission.

EBV-positive human sera contain antibodies against the EBV BMRF-2 protein.

We previously showed that the EBV glycoprotein BMRF-2 contains a functional integrin-binding Arg-Gly-Asp (RGD) domain that plays an important role in viral infection and cell-to-cell spread of progeny virions in oral epithelial cells. In this study, we found that EBV-seropositive human sera contain antibodies against BMRF-2. The inhibitory effect of EBV-positive sera on EBV infection of oral epithelial cells was substantially reduced by pre-incubation of serum samples with the BMRF-2 RGD peptide, suggesting that anti-BMRF-2 human antibodies possess neutralizing activity. EBV-specific sera reacted strongly with the BMRF-2 extracellular domain (170-213 aa) containing the RGD motif, whereas they reacted only weakly or not at all with a mutated form of the BMRF-2 extracellular domain containing AAA instead of RGD. These data indicate that RGD motif of BMRF-2 is part of an immunodominant antigenic determinant within the extracellular domain of BMRF-2 that may contribute to EBV neutralization during EBV reactivation.

EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells.

We previously reported that the Epstein-Barr virus (EBV) BMRF-2 protein plays an important role in EBV infection of polarized oral epithelial cells by interacting with beta1 and alphav family integrins. Here we show that infection of polarized oral epithelial cells with B27-BMRF-2(low) recombinant virus, expressing a low level of BMRF-2, resulted in significantly smaller plaques compared with infection by parental B95-8 virus. BMRF-2 localized in the trans-Golgi network (TGN) and basolateral sorting vesicles and was transported to the basolateral membranes of polarized epithelial cells. Mutation of the tyrosine- and dileucine-containing basolateral sorting signal, YLLV, in the cytoplasmic domain of BMRF-2 led to the failure of its accumulation in the TGN and its basolateral transport. These data show that BMRF-2 may play an important role in promoting the spread of EBV progeny virions through lateral membranes of oral epithelial cells.

Programa de especialistas para la atención primaria: experiencia y visión de académicos de la Universidad de Chile al primer año de marcha / Specialists for primary care: experience and academic opinions at the University of Chile after the program´s first year

El año 2008, los Departamentos de Medicina, Obstetricia, Ginecología y Pediatría y Cirugía Infantil, del Campus Occidente de la Facultad de Medicina de la Universidad de Chile, iniciaron un programa de 6 años para formar Especialistas para la Atención Primaria. PROPÓSITO: Relatar la experiencia, a 12 meses de iniciada. El Programa alterna rotaciones de 6 meses en la Facultad y en el sitio original de trabajo –en atención primaria– y surgió de un acuerdo con el Ministerio de Salud. En la Facultad la idea fue acogida con interés, pero no sin reservas. El artículo relata las reservas iniciales, con su evolución respectiva, y muestra la forma en que se ha desarrollado la experiencia durante su primer año. MATERIAL Y MÉTODO: Se presentan las opiniones del grupo de académicos ejecutores directos en el Campus Occidente. RESULTADOS: Se diseñó un programa de igual exigencia que el programa tradicionales de tres años pero de doble duración para dejar espacio a las rotaciones por Atención Primaria. Esto ha obligado a implementar muchas medidas (sistema de registro riguroso de experiencias, calificaciones, decisiones, permisos, etc.; reuniones de seguimiento y evaluación de las partes involucradas), recurrir a la creatividad de los académicos y flexibilizar la metodología del programa tradicional, de tres años. CONCLUSIONES: Hasta el momento, la experiencia es altamente satisfactoria y estimulante para la Facultad. No se ha sufrido dificultades mayores y los alumnos están satisfechos. Entre algunas de las múltiples ventajas adicionales que parece mostrar este programa, estos becados han servido de nexo entre sus consultorios de base y nuestros servicios hospitalarios, con los consecuentes beneficios para la población beneficiaria, lo que representa una fortaleza no menor porque lograría dar consistencia a la red de salud. Naturalmente, se mantendrá vigilancia sobre los aspectos...

In 2008 the three Departments of Medicine, Obstetrics and Gynecology and Pediatrics and Infantile Surgery of the Western Campus of the University of Chile began a new 6 years program to train specialist physicians for Primary Care in Internal Medicine, Obstetrics and Gynecology and Pediatrics. The Program alternates 6 months shifts at the Faculty and at original working places in primary care and emerged after an agreement between the Faculty and the Ministry of Health. AIM: To document the first 12 months of this experience. At a Faculty the idea was welcomed with a combination of interest and doubts. This article documents the evolution of these doubts and the way the experience has evolved along its first year. MATERIAL AND METHOD: Academic opinions of directly involved leading Professors of the Program are summarized and presented. RESULTS: Program design kept same objectives than the original 3 years program but doubled to 6 years to give place to primary care shifts. This requirement led to the implementation of several measures (strict records for academic experiences, grades, decisions, permissions; follow up and regular evaluation meetings with all involved people), academic creativity as well as the development of higher flexibility than the traditional three years program. CONCLUSIONS: Up to this moment experience has shown highly satisfactory and stimulant characteristics for the Faculty. No major difficulties have occurred and students declare satisfaction. Among several additional observed advantages students have linked their original working places and hospital with clear benefits for served population. This has become a major strength providing better consistency to the health network. Naturally, all aspects to make a sound evaluation of the experience will be kept under close surveillance. There is a plain belief that by the end of the training period specialist level will be comparable to the traditional specialization program.

The Epstein-Barr virus BMRF-2 protein facilitates virus attachment to oral epithelial cells.

We previously reported that BMRF-2, an Epstein-Barr virus (EBV) glycoprotein, binds to beta1 family integrins and is important for EBV infection of polarized oral epithelial cells. To further study the functions of BMRF-2, we constructed a recombinant EBV that lacks BMRF-2 expression by homologous recombination in B95-8 cells. We found that lack of BMRF-2 resulted in about 50% reduction of EBV attachment to oral epithelial cells, but not to B lymphocytes, suggesting that BMRF-2 is critical for EBV infection in oral epithelial cells, but not in B lymphocytes. In polarized oral epithelial cells, infection rate of the recombinant EBV virus was about 4- to 8-fold lower than the wild-type B95-8 virus. Cell adhesion assays using the BMRF-2 RGD peptide and its RGE and AAA mutants showed that the RGD motif is critical for BMRF-2 binding to integrins. These data are consistent with our previous observation that interactions between EBV BMRF-2 and integrins are critical for infection of oral epithelial cells with EBV.

Epstein-Barr virus (EBV)-infected monocytes facilitate dissemination of EBV within the oral mucosal epithelium.

Epstein-Barr virus (EBV) causes hairy leukoplakia (HL), a benign lesion of oral epithelium that occurs primarily in the setting of human immunodeficiency virus (HIV)-associated immunodeficiency. However, the mechanisms of EBV infection of oral epithelium are poorly understood. Analysis of HL tissues shows a small number of EBV-positive intraepithelial macrophages and dendritic/Langerhans cells. To investigate a role for these cells in spreading EBV to epithelial cells, we used tongue and buccal explants infected ex vivo with EBV. We showed that EBV first infects submucosal CD14(+) monocytes, which then migrate into the epithelium and spread virus to oral epithelial cells, initiating productive viral infection within the terminally differentiated spinosum and granulosum layers. Incubation of EBV-infected monocytes and oral explants with antibodies to CCR2 receptor and monocyte chemotactic protein 1 prevented entry of monocytes into the epithelium and inhibited EBV infection of keratinocytes. B lymphocytes played little part in the spread of EBV to keratinocytes in our explant model. However, cocultivation of EBV-infected B lymphocytes with uninfected monocytes in vitro showed that EBV may spread from B lymphocytes to monocytes. Circulating EBV-positive monocytes were detected in most HIV-infected individuals, consistent with a model in which EBV may be spread from B lymphocytes to monocytes, which then enter the epithelium and initiate productive viral infection of keratinocytes.

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.