Molecular Pathogenesis of Human Immunodeficiency Virus-Associated Disease of Oropharyngeal Mucosal Epithelium.

The oropharyngeal mucosal epithelia have a polarized organization, which is critical for maintaining a highly efficient barrier as well as innate immune functions. In human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) disease, the barrier and innate immune functions of the oral mucosa are impaired via a number of mechanisms. The goal of this review was to discuss the molecular mechanisms of HIV/AIDS-associated changes in the oropharyngeal mucosa and their role in promoting HIV transmission and disease pathogenesis, notably the development of opportunistic infections, including human cytomegalovirus, herpes simplex virus, and Epstein-Barr virus. In addition, the significance of adult and newborn/infant oral mucosa in HIV resistance and transmission was analyzed. HIV/AIDS-associated changes in the oropharyngeal mucosal epithelium and their role in promoting human papillomavirus-positive and negative neoplastic malignancy are also discussed.

The Epstein-Barr Virus Glycoprotein BDLF2 Is Essential for Efficient Viral Spread in Stratified Epithelium.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The ΔBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium. IMPORTANCE The ubiquitous herpesvirus Epstein-Barr virus (EBV) is associated with cancers of B lymphocytes and epithelial cells and is primarily transmitted in saliva. While several models exist for analyzing the life cycle of EBV in B lymphocytes, models of EBV infection in the epithelium have more recently been established. Using an organotypic culture model of epithelium that we previously determined accurately reflects EBV infection in situ, we have ascertained that the loss of the viral envelope protein BDLF2 had little effect on the EBV life cycle in B cells but severely restricted the number of infected cells in organotypic cultures. Loss of BDLF2 has a substantial impact on the size of infected areas, suggesting that BDLF2 plays a specific role in the spread of infection in stratified epithelium.

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.

Human immunodeficiency virus interaction with oral and genital mucosal epithelia may lead to epithelial-mesenchymal transition and sequestration of virions in the endosomal compartments.

Oral and genital mucosal epithelia are multistratified epithelial barriers with well-developed tight and adherens junctions. These barriers serve as the first line of defense against many pathogens, including human immunodeficiency virus (HIV). HIV interaction with the surface of mucosal epithelial cells, however, may activate transforming growth factor-beta (TGF-ß) and mitogen-activated protein kinase signaling pathways. When activated, these pathways may lead to the disruption of epithelial junctions and epithelial-mesenchymal transition (EMT). HIV-induced impairment of the mucosal barrier may facilitate the spread of pathogenic viral, bacterial, fungal, and other infectious agents. HIV-induced EMT promotes highly motile/migratory cells. In oral and genital mucosa, if EMT occurs within a human papillomavirus (HPV)-infected premalignant or malignant cell environment, the HPV-associated neoplastic process could be accelerated by promoting viral invasion of malignant cells. HIV also internalizes into oral and genital mucosal epithelial cells. The majority (90%) of internalized virions do not cross the epithelium, but are retained in endosomal compartments for several days. These sequestered virions are infectious. Upon interaction with activated peripheral blood mononuclear cells and CD4+ T lymphocytes, epithelial cells containing the virus can be transferred. The induction of HIV-1 release and the cell-to-cell spread of virus from epithelial cells to lymphocytes is mediated by interaction of lymphocyte receptor function-associated antigen-1 with the epithelial cell receptor intercellular adhesion molecule-1. Thus, mucosal epithelial cells may serve as a transient reservoir for HIV, which could play a critical role in viral transmission.

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.

HIV-induced matrix metalloproteinase-9 activation through mitogen-activated protein kinase signalling promotes HSV-1 cell-to-cell spread in oral epithelial cells.

We have shown that cell-free HIV-1 and viral proteins tat and gp120 activate mitogen-activated protein kinases (MAPKs) in tonsil epithelial cells, disrupting their tight and adherens junctions. This causes liberation of the HSV-1 receptor nectin-1 from assembled adherens junctions, leading to promotion of HSV-1 infection and spread. In the present study, we show that HIV-associated activation of MAPK leads to upregulation of transcription factor NF-κB and matrix metalloproteinase-9 (MMP-9). This induces the disruption of tight and adherens junctions, increasing HSV-1 cell-to-cell spread. Inhibition of HIV-associated MAPK activation by U0126 abolishes NF-κB and MMP-9 upregulation and reduces HSV-1 spread. Inactivation of MMP-9 also reduced HIV-promoted HSV-1 spread. These results indicate that HIV-1-activated MAPK/NF-κB and MMP-9 play a critical role in the disruption of oral epithelial junctions and HSV-1 cell-to-cell spread. Inhibition of MMP-9 expression in the oral epithelium of HIV-infected individuals may prevent the development of diseases caused by HSV-1, such as ulcers, necrotic lesions and gingivostomatitis.

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.

HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.

Herpes simplex virus (HSV) types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD). Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals.

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.

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.

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.

Epstein-Barr virus infection of polarized tongue and nasopharyngeal epithelial cells.

Epstein-Barr virus (EBV) initially enters the body through the oropharyngeal mucosa and subsequently infects B lymphocytes through their CD21 (CR2) complement receptor. Mechanisms of EBV entry into and release from epithelial cells are poorly understood. To study EBV infection in mucosal oropharyngeal epithelial cells, we established human polarized tongue and pharyngeal epithelial cells in culture. We show that EBV enters these cells through three CD21-independent pathways: (i) by direct cell-to-cell contact of apical cell membranes with EBV-infected lymphocytes; (ii) by entry of cell-free virions through basolateral membranes, mediated in part through an interaction between beta1 or alpha5beta1 integrins and the EBV BMRF-2 protein; and (iii) after initial infection, by virus spread directly across lateral membranes to adjacent epithelial cells. Release of progeny virions from polarized cells occurs from both their apical and basolateral membranes. These data indicate that multiple approaches to prevention of epithelial infection with EBV will be necessary.