Cell-free human immunodeficiency virus type 1 transcytosis through primary genital epithelial cells

Abstract

Although the transport of human immunodeficiency virus type 1 (HIV-1) through the epithelium is critical for HIV-1 colonization, the mechanisms controlling this process remain obscure. In the present study, we investigated the transcellular migration of HIV-1 as a cell-free virus through primary genital epithelial cells (PGECs). The absence of CD4 on PGECs implicates an unusual entry pathway for HIV-1. We found that syndecans are abundantly expressed on PGECs and promote the initial attachment and subsequent entry of HIV-1 through PGECs. Although CXCR4 and CCR5 do not contribute to HIV-1 attachment, they enhance viral entry and transcytosis through PGECs. Importantly, HIV-1 exploits both syndecans and chemokine receptors to ensure successful cell-free transport through the genital epithelium. HIV-1-syndecan interactions rely on specific residues in the V3 of gp120 and specific sulfations within syndecans. We found no obvious correlation between coreceptor usage and the capacity of the virus to transcytose. Since viruses isolated after sexual transmission are mainly R5 viruses, this suggests that the properties conferring virus replication after transmission are distinct from those conferring cell-free virus transcytosis through the genital epithelium. Although we found that cell-free HIV-1 crosses PGECs as infectious particles, the efficiency of transcytosis is extremely poor (less than 0.02% of the initial inoculum). This demonstrates that the genital epithelium serves as a major barrier against HIV-1. Although one cannot exclude the possibility that limited passage of cell-free HIV-1 transcytosis through an intact genital epithelium occurs in vivo, it is likely that the establishment of infection via cell-free HIV-1 transmigration is a rare event.

FIG. 1.

Expression of HIV-1 receptors on human primary adult genital epithelial cells. (A) PGECs (10⁵ cells) were incubated with isotype controls or anti-CD4 SIM.4, anti-DC-SIGN DC28, anti-CXCR4 12G5, anti-CCR5 2D7, anti-HSPG 10E4, anti-CSPG CS-56, anti-GalCer MAB342, anti-CD147 HIM6, anti-cytokeratin-7 CK7, anti-cytokeratin-7 CK8, anti-cytokeratin-18 CK18, anti-cytokeratin-19 CK19, anti-CD4 RP4-T4, anti-syndecan-1 ID4, anti-syndecan-2 10H4, anti-syndecan-3 1C7, and anti-syndecan-4 8G3 antibodies (1 μg) in 500 μl of PBS containing 0.25% human serum. Values are geometric means expressed in fluorescence units (log scale) and are representative of the results obtained with four independent donors. PGECs were treated with heparinase, chondroitinase ABC, and phospholipase C or were left untreated. (B) Immunostained cryosections of human ectocervical and vaginal epithelial tissue, revealing the expression of HSPGs (10E4 monoclonal antibody [MAb]), syndecan-1 (BB4 MAb), and syndecan-2 (10H4 MAb) and the absence of syndecan-3 (1C7) and syndecan-4 (8G3).

FIG. 2.

Establishment of a cell-free HIV-1 transcytosis assay. (A) Attachment: PGECs were exposed to 10 ng of p24 of HIV-1 (JR-CSF) at 4°C or 37°C. After different intervals of time, cells were washed, detached and lysed. Amounts of attached virus were determined by p24 ELISA of cell lysates. (B) Internalization: HIV-1 was added to PGECs as described above, and at different intervals of time, cells were washed, trypsinized, washed again, and immediately lysed. Amounts of internalized HIV-1 were quantified by p24 ELISA of cell lysates. (C) Transcytosis: HIV-1 was added to the apical surface of PGECs, and amounts of transcytosed viruses were quantified by p24 ELISA of the lower chamber corresponding to the basal surface. (D) Infectivity of transcytosed viruses: medium from the basal chamber was collected at different intervals of time, filtered, standardized for p24 content, and added to TZM-bl indicator cells. Infection was measured 48 h postinfection by determining levels of beta-galactosidase activity. Results are representative of the results of four independent experiments using PGECs derived from each of the four donors. Error bars represent standard deviations.

FIG. 3.

Cell-free R5 and X4 viruses cross PGECs in a Gp120-dependent manner. (A) Attachment: PGECs were exposed to 10 ng of p24 of HIV-1 for 1 h at 37°C. After different intervals of time, cells were washed, detached, and lysed. Amounts of attached virus were determined by p24 ELISA of cell lysates. (B) Internalization: HIV-1 was added to PGECs for 4 h at 37°C, and cells were washed, trypsinized, washed again, and immediately lysed. Amounts of internalized HIV-1 were quantified by p24 ELISA of cell lysates. (C) Transcytosis: HIV-1 was added to the apical surface of PGECs for 8 h at 37°C, and amounts of transcytosed viruses were quantified by p24 ELISA of the lower chamber corresponding to the basal surface. Results are representative of four independent experiments using PGECs derived from four donors.

FIG. 4.

Chemokine receptors facilitate cell-free HIV-1 transcytosis through PGECs. (A) Attachment: PGECs preincubated with a panel of inhibitors (anti-CD4 MAb, anti-GalCer, anti-CCR5 MAb, anti-CXCR4 MAb, AMD3100, and TAK779) were exposed to 10 ng of p24 of pNL-JR-FL (R5) or pNL-92UG024.2 (X4) for 1 h at 37°C. Cells were then washed, detached, and lysed. Amounts of attached virus were determined by p24 ELISA of cell lysates. (B) Internalization: viruses were added to PGECs for 4 h at 37°C. Cells were washed, trypsinized, washed again, and immediately lysed. Amounts of internalized HIV-1 were quantified by p24 ELISA of cell lysates. (C) Transcytosis: HIV-1 was added to the apical surface of PGECs for 8 h at 37°C, and amounts of transcytosed viruses were quantified by p24 ELISA of the lower chamber corresponding to the basal surface. Results are representative of four independent experiments using PGECs derived from four donors.

FIG. 5.

HSPGs promote both HIV-1 attachment to and internalization into PGECs. (A) Attachment: PGECs, pretreated with heparinase or chondroitinase or left untreated, were exposed to 10 ng of p24 of pNL derivatives for 1 h at 37°C. Cells were then washed, detached, and lysed. Amounts of attached virus were determined by p24 ELISA of cell lysates. (B) Internalization: viruses were added to PGECs for 4 h at 37°C. Cells were washed, trypsinized, washed again, and immediately lysed. Amounts of internalized HIV-1 were quantified by p24 ELISA of cell lysates. (C) Transcytosis: HIV-1 was added to the apical surface of PGECs for 8 h at 37°C, and amounts of transcytosed viruses were quantified by p24 ELISA of the lower chamber corresponding to the basal surface. Results are representative of four independent experiments using PGECs derived from four donors.

FIG. 6.

Requirements for Gp120-HSPGs interactions in HIV-1 transcytosis through PGECs. (A) HIV-1 was added to the apical surface of PGECs for 8 h at 37°C in the presence of increasing concentrations of soluble HSPGs, and amounts of transcytosed viruses were quantified by p24 ELISA of the lower chamber corresponding to the basal surface. (B) Attachment: PGECs were exposed to 10 ng of p24 of wild-type, gp160-deficient, V3-deficient, R298A mutant, and R326A mutant viruses for 1 h at 37°C. Cells were then washed, detached, and lysed. Amounts of attached virus were determined by p24 ELISA of cell lysates. (C) Transcytosis: HIV-1 was added to the apical surface of PGECs for 8 h at 37°C in the presence of increasing concentrations of soluble HSPGs, and amounts of transcytosed viruses were quantified by p24 ELISA of the lower chamber corresponding to the basal surface. Results are representative of four independent experiments using PGECs derived from four donors.