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 tat and gp120 activate MAPK in polarized oral epithelial cells.

<p>(<b>A</b>) Polarized cells were treated with active or inactive tat and gp120, alone or in combination. In parallel experiments, cells were exposed to UV-inactivated or active HIV-1_SF33. Culture medium was changed daily to add fresh proteins and virus, and at day 5 the TER was measured. (<b>B</b>) After measurement of TER, the same cells were used for evaluation of MAPK activation. Cells were extracted, and total and phosphorylated ERK1/2 were detected by Western blot assay. (<b>C</b>) Polarized cells were treated with active forms of tat or gp120 in the presence or absence of MAPK inhibitor U0126. Tat -and gp120 -untreated cells with or without U0126 served as controls. At day 5 TER of polarized cells was measured. (<b>D</b>) The same cells from panel C after measurement of TER were extracted and used for evaluation of phosphorylated and total ERK1/2. The mean densities of pixels in the protein bands were measured by Image J software, and the results for each gel are shown as a bar graph under each blot. <b>A and C:</b> Error bars indicate SEM (n = 3).</p

HIV tat/gp120-disrupted epithelial junctions facilitate HSV-1 cell-to-cell spread through junctional areas of polarized oral epithelial cells.

<p><b>A.</b> Polarized tonsil cells were treated with active or inactive tat/gp120 and HIV virions for 5 days. Disrupted cells were infected with HSV-1 at 0.01 PFU per cell from basolateral membranes of polarized cells. After 3 days, cells were fixed and immunostained using goat anti-HSV immune serum (green). Cell nuclei are stained in red. Yellow represents colocalization of HSV proteins and nuclei. <b>B.</b> (upper panel) Plaque numbers were counted from 3 independent filter inserts and data are presented as the average number of HSV-infected plaques per insert. (lower panel) Cell-to-cell spread of HSV-1 was quantitatively evaluated by counting HSV-infected cells in the plaques. Results are presented as the average number of HSV-infected cells per plaque. Error bars indicate SEM. <b>C.</b> Polarized cells were infected with HSV-1. After 4 h, antibodies to nectin-1 and gD were added separately and in combination. Cell medium was changed daily to add fresh antibodies. Cells were fixed and immunostained for HSV-1, and the plaque numbers (upper panel) and the number of HSV-1-positive cells in plaques were counted (lower panel). Error bars indicate SEM. *P<0.05, *P<0.01, **P<0.001, all compared with the control group.</p

HIV cell-free virion-associated disruption of epithelial tight junctions facilitates HSV paracellular spread.

<p><b>A.</b> Polarized tonsil epithelial cells were incubated with dual X4- and R5-tropic HIV-1_SF33 for 5 days. One set of cells was exposed to UV-inactivated virions. Culture medium was changed daily to add fresh virus, and TER was measured. <b>B.</b> HSV-1 was added to the apical surface of polarized cells upon complete disruption of TJs at 5 days. HSV paracellular spread at 1, 2, and 4 h after incubation was examined in Vero cells grown in the basolateral chamber of filter inserts by immunostaining of ICP4 protein. HSV-1 paracellular spread was quantified by counting HSV-1-infected Vero cells, and the percentage of cells positive for ICP4 was determined. <b>A, B:</b> Error bars indicate SEM (n = 3).</p

HIV tat- and gp120-induced disruption of TJs of oral epithelial cells facilitates the paracellular spread of HSV-1.

<p><b>A</b> (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. <b>A</b> (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’)₂ from the apical chamber to the basolateral chamber. OD, optical density. <b>B.</b> The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. <b>C.</b> HSV-1 at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. <b>D.</b> HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. <b>A, </b><b>D:</b> Error bars indicate SEM (n = 3).</p

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 β-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

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

HIV-disrupted epithelial junctions facilitate HSV-1 infection.

<p><b>A.</b> Polarized tonsil cells were treated with HIV tat/gp120 or HIV virions for 5 days and infected with HSV-1. After 24 h, cells were fixed and immunostained using anti-HSV-1 gB antibodies (red). Cell nuclei are stained in blue. <b>B.</b> HSV-1 infection was quantitatively evaluated, and the percentage of cells positive for gB was determined. Error bars indicate SEM. <b>C.</b> Cells were incubated with antibodies against nectin-1 for 1 h and then infected with HSV-1. Cells were fixed after 24 h, HSV-1 infection was confirmed by detection of goat anti-HSV-1 immune serum, and the number of infected cells was counted. ab, cells incubated with antibodies. c, control cells without antibodies. Error bars indicate SEM. *P<0.01, **P<0.001, all compared with the control group.</p