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

Inhibition of MVB and vacuole formation reduced HIV-1 sequestration and virus spread to lymphocytes.

<p>(A) Polarized tonsil cells were transfected with control siRNA or siRNAs against Hrs and rabankyrin-5. After 72 h, expression of Hrs and rabankyrin-5 was examined by Western blot. The mean density of protein bands is shown under the blot. β-Actin was detected to confirm equal loading. Immunoblots were performed at least twice and a representative figure is shown. (B, C) Tonsil epithelial cells transfected with control siRNA or siRNAs specific to Hrs or rabankyrin-5 at 72 h after transfection were exposed to HIV-1_92UG029 or HIV-1_SF170. After 3 days, one set of siRNA-transfected cells were examined for intracellular HIV-1 (upper panels). The next set of siRNA-transfected cells were cocultured with activated CD4+ T lymphocytes (lower panels). Four hours later, lymphocytes were collected and grown for 4 days for HIV-1_92UG029 and 10 days for HIV-1_SF170. HIV-1 infection was examined by ELISA p24. Data represent one of three independent experiments and are shown as mean ± SEM of triplicate values. ***<i>P</i> < 0.0001 and ****<i>P</i> < 0.00001, compared with the control siRNAs.</p

Spread of sequestered HIV-1 from cervical and foreskin epithelial cells to lymphocytes.

<p>(A) Cell surface expression of ICAM-1 was examined in AP and BL membranes of polarized CK#4 and FK#2 cells by domain-specific biotinylation assay. The mean density of ICAM-1 protein bands is shown under the blot. β-Actin was detected to confirm equal loading. Experiments were performed at least twice and a representative figure is shown. (B, C) Polarized cervical CK#4 (B) and foreskin FK#2 (C) cells containing intracellular HIV-1_SF33 and activated PBMC were preincubated with antibodies against ICAM-1 and LFA-1 or with their isotype controls, respectively. Then, epithelial cells and PBMC were cocultured for 4 h. PBMC were collected, grown for 4 days, and examined by ELISA p24. (D, E) Polarized cervical and foreskin epithelial cells sequestering X4-tropic HIV-1_92UG029 and R5-tropic HIV-1_SF170 viruses were cocultured with CD4+ T lymphocytes in the presence of absence of ICAM-1 and LFA-1 antibodies or their isotype controls. Lymphocytes were collected and grown for 4 days (HIV-1_92UG029) or 7 days (HIV-1_SF170), and infection was examined by ELISA p24. (B-E) Data are shown as mean ± SEM of three independent experiments, each in triplicate (n = 3). **<i>P</i> < 0.001, ***<i>P</i> < 0.0001, ****<i>P</i> < 0.00001, compared with the control isotype antibodies.</p

Analysis of transcytosed and intracellular virions in polarized tonsil, cervical and foreskin epithelial cells.

<p>HIV-1_SF33 (20 ng/ml; p24) was added to the AP surface of polarized tonsil (A), cervical (B), and foreskin (C) epithelial cells, and 4 h later uninternalized virions were removed with trypsin. At days 1, 3, 6 and 9 after inoculation of virus, the culture medium from lower chambers and trypsinized cells were collected for detection of transcytosed virions (lower panels) and intracellular virus (upper panels). Transcytosed and intracellular virions were examined by ELISA p24. # Not detected. Data are shown as mean ± SEM (bars) of triplicate values.</p

Induction of HIV-1 release from sequestration in epithelial cells.

<p>(A) Polarized tonsil epithelial cells containing HIV-1_SF33 for 6 days were treated with ionomycin (10 μM), cytochalasin D (12 μg/ml), or a combination of ionomycin and cytochalasin D for 30 min. Intracellular virus and virus released from AP and BL medium were examined by ELISA p24 (upper panel) and TER was measured in control and treated cells (lower panel). Mean values ± SEM of three independent experiments (in triplicate or quadruplicate) are shown (n = 3). *P < 0.01 and **P < 0.002, BL virus release of ionomycin+cytochalasin D-treated cells compared with BL virus release of cells treated independently with ionomycin or cytochalasin D. (B, C) Polarized tonsil epithelial cells containing sequestered R5-tropic HIV-1_SF170 (B) and X4-tropic HIV-1_92UG029 (C) at day 6 were treated with ionomycin or a combination of ionomycin and cytochalasin D for 30 min. TER of polarized cells was measured in control and treated cells. Combined AP and BL medium and trypsinized cells were examined for released and intracellular virus, respectively. Mean values ± SEM of three independent experiments (in triplicate) are shown (n = 3). ***<i>P</i> < 0.0001, virus release from cells treated with ionomycin+cytochalasin D compared with ionomycin alone. (D) Polarized tonsil epithelial cells containing sequestered HIV-1_SF33 at day 6 were treated with ionomycin for 30 min or untreated. Activated CD4+ T lymphocytes were infected with culture medium collected from untreated and ionomycin-treated cells. After 10 days, CD4+ T lymphocytes were examined for HIV-1 infection by using ELISA p24. Results were reproduced in three independent experiments and are shown as mean ± SEM (n = 3). # Not detected.</p

Release and spread of sequestered HIV-1 from epithelial cells to PBMC through interaction of ICAM-1 of tonsil epithelial cells and LFA-1 of lymphocytes.

<p>(A) Activated PBMC were added to AP or BL membranes of polarized tonsil epithelial cells containing HIV-1_SF33 for 6 days, and after 4 h cocultured cells were fixed and immunostained for CD45 (green) and cytokeratin (red). Nuclei are counterstained with TO-PRO (blue). (B) Activated PBMC were added to AP and BL membranes of polarized tonsil cells containing HIV-1_SF33, HIV-1_SF170, or HIV-1_92UG029 for 6 days. After 4 h incubation, PBMC were collected from AP and BL chambers separately and used for detection of CD45 by Western blotting. (C) ICAM-1 was detected in AP and BL membranes of polarized tonsil cells with or without intracellular HIV-1_SF33 by domain-specific biotinylation assay. (D) LFA-1 was detected in activated and nonactivated PBMC by Western blot. (B, C and D) The mean density of CD45, ICAM-1 and LFA-1 protein bands is shown under the blot. The presence of β-actin ensured equal loading. Immunoblots were performed at least twice and a representative figure is shown. (E, F) Polarized tonsil cells containing intracellular HIV-1_SF33 and activated PBMC were preincubated with anti-ICAM-1 and LFA-1 antibodies, respectively, or with isotype controls. Epithelial cells and lymphocytes were cocultivated for 4 h from the AP or BL membranes of polarized cells. Epithelial cells that were not cocultivated with PBMC served as a control. TER was measured (E, lower panel). Culture medium was examined for released virus (E, upper panel). PBMC were grown for 7 days and examined by ELISA p24 (F). Data represent one of two independent experiments and are shown as mean ± SEM of triplicate values. ***<i>P</i> < 0.0001, ****<i>P</i> < 0.00004, compared with the control isotype antibodies. # Not detected. (G) Polarized tonsil epithelial cells containing X4-tropic HIV-1_92UG029 or R5-tropic HIV-1_SF170 were cocultured with CD4+ T lymphocytes from AP or BL membranes of epithelial cells in the presence or absence of antibodies against ICAM-1 and LFA-1. After 4 h lymphocytes were collected and grown for 4 days (X4-tropic HIV-1_92UG029) or 7 days (R5-tropic HIV-1_SF170). Lymphocytes were then examined for HIV-1 infection by ELISA p24. Data are shown as mean ± SEM of three independent experiments (in triplicate) (n = 3). **P<0.001, and ***P<0.0007, **** P<0.00005.</p

Disruption of cortical actin cytoskeleton is critical for HIV-1 release.

<p>(A) Polarized tonsil epithelial cells containing intracellular HIV-1_SF33 for 6 days were incubated with activated PBMC for 4 h from AP or BL surfaces of polarized cells. Then PBMC were removed and epithelial cells were fixed. In parallel experiments, polarized cells were treated with TNF-α and/or IFN-γ for 24 h, and ionomycin and cytochalasin D for 30 min. Cells without PBMC coculture and not treated served as a control. Cells were then fixed and costained for fluorescence-labeled phalloidin (green) and for HIV-1 p24 (red). Similar results were obtained in 5 independent experiments. (B) Experiments were performed by cocultivation of tonsil cells containing HIV-1_SF33 with PBMC, or treated with cytokines, ionomycin and cytochalasin D as described above (A). Then cells were lysed and subjected to ultracentrifugation. Supernatant (S) containing G-actin and pellet (P) containing F-actin were detected by Western blot. A representative Western blot was selected from two independent experiments. The mean density of the protein bands is shown under the blot.</p

Cocultivation of activated PBMC and CD4+ T lymphocytes with epithelial cells containing HIV-1 induces virus release.

<p>(A) Activated and nonactivated PBMC were added to the AP surface of polarized tonsil epithelial cells (2:1), and after 1, 2, 3, 4 and 5 h the TER was examined. As a control, one set of polarized tonsil cells were not cocultivated. Another set of cells were treated with 10 mM EDTA for 30 min. (B) Activated CD4+ T lymphocytes were added to the AP surface of tonsil epithelial cells, and after 4 h the TER was examined. (A, B) Mean values ± SEM of three independent experiments (in triplicate) are shown (n = 3). ****<i>P</i> < 0.00001, TER of polarized cells cocultivated with activated PBMC or CD4+ T lymphocytes compared with TER of control cells. (C) Activated and nonactivated PBMC with or without ionomycin were added to the BL surface of tonsil cells containing intracellular HIV-1_SF33 at day 6. After 4 h of cocultivation AP and BL medium was collected separately, centrifuged, and supernatants were examined for released virus using ELISA p24. Polarized epithelial cells and culture medium without cocultivation with PBMC were used as a control. (D) Activated PBMC were added to the AP or BL surface of tonsil cells containing HIV-1. Four hours later, AP and BL medium was examined for HIV-1 p24. (E) Polarized tonsil epithelial cells were grown in Transwell inserts with 3-μm or 0.42-μm pore size and exposed to HIV-1_SF33 from AP membranes. Epithelial cells with sequestered virus at day 6 were cocultured with activated PBMC from the AP surface of polarized cells grown in inserts with 3.0-μm pore size and from BL membranes in inserts with 3-μm or 0.42-μm pore size. After 4 h, the TER of polarized cells was measured, and the culture medium from AP and BL membranes were combined for ELISA p24. (F). Matching tonsil keratinocytes and CD4+ T lymphocytes were isolated from the same tonsil of 2 independent donors. HIV-1_SF33 was added to the AP surface of polarized tonsil epithelial cells, and at day 6 intracellular virus was examined by ELISA p24 (F, upper panel). One set of cells containing virus were used for cocultivation of autologous CD4+T lymphocytes. After 4 h, lymphocytes were collected, grown for 12 days and examined by ELISA p24 (F, lower panel). Data in panels C-F represent one of two or three independent experiments and are shown as mean ± SEM of triplicate values. (D) *<i>P</i> < 0.01, ****<i>P</i> < 0.00007, BL release of virus compared with AP release.</p

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

<div><p>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.</p></div