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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.
Women account for more than half of the newly human immunodeficiency virus (HIV)-infected adults worldwide, and most women acquire HIV type 1 (HIV-1) through heterosexual exposure (23, 44, 46, 49, 50). Heterosexual transmission accounts for 80% of the infections of the 40 million people now infected with HIV-1 (2, 61). For successful heterosexual transmission, HIV-1 first has to cross the mucosal barrier of the genital tract to infect CD4+ T cells. Although this initial transport of HIV-1 through the epithelium is absolutely critical for HIV-1 colonization, it has been poorly studied and, most importantly, the mechanisms controlling this process remain obscure. Although some studies suggest that HIV-1 may enter the body through lesions (16), others suggest that lesions are not required for infection and that HIV-1 is capable of crossing the epithelial barrier by an undefined mechanism (35, 36, 37, 55). HIV-1 infection is not particularly easy to acquire sexually; male-to-female transmission incidence has been estimated to be 0.002% to 0.02% for each sexual act (4, 25, 40), suggesting that the genital epithelium serves as a major barrier against HIV-1. Semen contains cell-free or cell-associated virus (12, 13, 19, 42, 45, 58), and each source of HIV-1 infectivity may require a different mechanism to establish infection. The relative transmissibility of cell-free versus cell-associated virus is still unclear. The ectocervix, endocervix, and vagina of the female genital tract are all considered potential portals for HIV-1. Endocervix cells are apparently resistant to infection by both cell-free and cell-associated virus (15, 26). Since genital epithelial cells lack CD4, cell-free HIV-1 has to utilize unconventional mechanisms to cross primary genital epithelial cell layers. Several receptors have been reported to facilitate HIV-1 entry into CD4-negative cells. Specifically, galactosyl ceramide (GalCer) (27, 28, 64), adhesion molecules such as ICAM-1 and LFA-1 (20, 21, 47), C-type lectins such as DC-SIGN, DC-SIGNR, langerin, and the mannose receptor (7, 24, 43, 57), and proteoglycans such as chondroitin sulfate (CSPGs) and heparan sulfate proteoglycans (HSPGs) (3, 9, 10, 38, 63) all have been shown to promote HIV-1 attachment and/or entry into cells that lack CD4. To date, there has been no demonstration that these receptors are capable of mediating fusion between viral and cellular membranes. Thus, these receptors represent prime candidates for cell-free HIV-1 passage through genital epithelial cells.
Proteoglycans are proteins that bear covalently-linked long unbranched anionic-sulfated glycosaminoglycans (i.e., chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate) (8). Glycosaminoglycans are sulfated polysaccharides consisting of disaccharide repeats (40 to 100 repeats) of uronic acid (or galactose) and hexosamines. The precise nature of the disaccharide repeats governs the functions of the proteoglycan. HSPGs promote HIV-1 attachment to host cells. Specifically, soluble heparin or heparan sulfate (38, 39, 41, 51, 52) inhibits HIV-1 attachment. Furthermore, the removal of cell surface heparan sulfate chains of HSPGs by heparinase diminishes both HIV-1 attachment to, and infectivity of, CD4+ HeLa cells and macrophages (38, 51). A specific class of HSPGs—the syndecans—when expressed together with CD4 and chemokine receptors (in cis) promotes HIV-1 attachment to target cells (51). Although syndecans do not alleviate the requirement for CD4 and chemokine receptors for viral entry (51), these in-cis attachment receptors may amplify HIV-1 infection by promoting viral adsorption to the surface of permissive cells. Syndecans may also function as in-trans receptors for HIV-1. Specifically, syndecans expressed on the surface of endothelial cells capture HIV-1 (9). The exposed viral glycoprotein gp120 serves as the main ligand for the heparan sulfate chains of syndecans (9, 14). Syndecans such as DC-SIGN preserve and enhance the in-trans infectivity of a broad range of primate lentiviruses, including primary viruses produced from physiological peripheral blood mononuclear cells (9). Given their proven efficacy as attachment receptors, syndecans may play a central role in the initial contact between HIV-1 and the host prior to the establishment of the infection. We thus investigated the respective contribution of attachment (HSPGs, C-type lectin receptors, and GalCer) and entry receptors (CD4, CXCR4, and CCR5) for the apical-basal transport of cell-free HIV-1 through primary genital epithelial cells, which represent the initial portal for HIV-1 colonization.
TZM-bl cells (generously contributed by John C. Kappes, Xiaoyun Wu, and Tranzyme Inc.) were obtained through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. TZM-bl cells express CD4, CXCR, and CCR5, which render them susceptible to infection, and contain an integrated Escherichia coli lacZ gene driven by the HIV long terminal repeat (59). Upon infection, Tat production from the integrated provirus leads to activation of the lacZ reporter, resulting in synthesis of beta-galactosidase in these cells. Infected cells are identified by enzymatic activity measurement 48 h postinfection, allowing quantitation after a single round of infection as described previously (59). Primary genital epithelial cells (PGECs) were provided by B. Kahn of the Department of Obstetrics and Gynecology at Scripps Clinic. By rotation of cotton swabs against the vaginal walls, several million cells were collected per individual. Cells were immediately placed in sterile phosphate-buffered saline (PBS), held at 4°C, and transported to the laboratory. After centrifugation (300 × g for 5 min), the cell pellet was digested in 1 mg/ml of collagenase-dispase (Roche Molecular Biochemicals) containing 1 mg/ml of DNase (Sigma) and 0.15 mg/ml of Na-p-tosyl-l-lysine chloromethyl ketone (Sigma) for 1 h at 37°C. The digest was spun down (1,000 × g for 20 min) and resuspended in 250 mg/ml of PBS-bovine serum albumin (PBS-BSA). After additional centrifugation, the pellet was resuspended in 5 mg/ml of PBS-BSA and loaded onto a 50% Percoll gradient. PGECs were then isolated from contaminating cells by fluorescence-activated cell sorting (FACS) as previously described (18) and propagated into collagen type I-coated T-25 flasks in Dulbecco's modified Eagle medium F12 medium containing 10% fetal calf serum and epithelial cell growth supplement (100 μg/ml) (Sigma). As a second source of PGECs, cells were obtained from Whittaker (customized request). PGECs were passaged fewer than three times prior to use in order to maintain their original features.
All cloned viruses were produced from electroporated Jurkat-CCR5 (generous gift from M. Emerman) and normalized by p24 enzyme-linked immunosorbent assay (ELISA) (Perkin Elmer Life Sciences). We generated a panel of pNL4.3-derived infectious molecular clones in which the envelope gene has been replaced with envelope genes carried by a selection of isolates, including wild-type pNL4.3 (generously contributed by Malcolm Martin through the NIH AIDS Research and Reference Reagent Program) (1), the gp120-deficient virus pNL4.3-E- (generously contributed by Nathaniel Landau through the NIH AIDS Research and Reference Reagent Program) (11, 29), and pNL-ADA, pNL-JRFL, pNL-YU2, pNL91US005.11, pNL-RW020.5 and pNL-92BR020.4 as R5 viruses and pNL-HXB, pNL-92UG021.6, and pNL-92UG024.2 as X4 viruses (a generous gift from P. Bieniasz) (66).
One million cells were incubated with antibodies (1 μg) in 500 μl of PBS containing 0.25% human serum. Anti-CD4 SIM.4 immunoglobulin G1 (IgG1) (generously contributed by James Hildreth), anti-DC-SIGN DC28 IgG2a (generously contributed by F. Baribaud, S. Pohlmann, J.A. Hoxie, and R.W. Doms), anti-CXCR4 12G5 IgG2a (generously contributed by James Hoxie), and anti-CCR5 2D7 IgG1 (generously contributed by Millennium Pharmaceuticals, Inc. and BD Biosciences PharMingen) were obtained through the NIH AIDS Research and Reference Reagent Program. Anti-HSPG 10E4 IgM was obtained from Seikagaku, anti-chondroitin sulfate CS-56 IgM was obtained from Sigma, anti-GalCer MAB342 IgG2b, anti-cytokeratin-7 (CK7) OV-TL12130 IgG1, anti-cytokeratin-8 (CK) C51 IgG1, anti-cytokeratin-18 (CK18) RCK108 IgG1, and anti-cytokeratin-19 (CK19) RCK106 IgG1 were obtained from Chemicon, anti-CD147 HIM6 IgG1 was obtained from BD Biosciences PharMingen, anti-syndecan-1 (CD138) ID4 IgG1 was obtained from Biogenesis, and anti-syndecan-2 10H4 IgG1, anti-syndecan-3 1C7 IgG1, and anti-syndecan-4 8G3 IgG1 were provided by G. David. Note that the antibody staining was performed on adherent PGECs prior to Cell Stripper (CellGro, Mediatech, Inc.) detachment. Cell surface removal of heparan and chondroitin sulfates by using heparinase (heparitinases I and III; Seikagaku [30 and 6 mIU/ml]) or chondroitinase ABC (Sigma) (10 U) and glycosylphosphatidylinositol-linked protein removal by phospholipase C (Sigma) (25 U) were performed as described previously (51).
Paraffin sections of paraformaldehyde-fixed material (4% in 0.1 M phosphate buffer) were blocked with PBS containing 1% BSA and 10% goat serum and incubated for 1 h with primary antibody. Primary antibody binding was detected using biotin-conjugated goat anti-mouse (Dako) (1/300) in PBS-BSA-goat serum, a Vectastain Elite kit (Vector), and diaminobenzidine tetrahydrochloride (Sigma) as the substrate as described previously (9).
TZM-bl cells (100,000 cells/ml) were exposed to an increasing volume of medium present in the basal chamber (which contains transcytosed viruses) for 2 h. Cells were washed to remove unbound virus, and viral infection was measured 48 h postinfection by determination of beta-galactosidase activity.
PGECs were seeded onto the upper face of collagen I- and fibronectin-coated 12-mm-diameter, 3-μm-pore-size polycarbonate membrane transwells at a density of 105 cells/well and were cultured until formation of tight junctions was achieved. The inserts were fed every 2 days. The monolayer on the filter effectively divides the well into an apical compartment and a basolateral compartment. The integrity of each cell monolayer was measured using an endothelial volt-ohm meter (Millipore). To ensure the integrity of the PGEC barrier, we monitored the elevated transendothelial electrical resistance of each cell monolayer and measured the paracellular passage of the extracellular marker inulin by determination of the permeability coefficient as evaluated by a diffusion assay using 14C-carboxylated inulin (Sigma) (molecular weight, 5,000) in the upper chamber as described previously (10). After verifying the integrity of the monolayer on the transwell filters, PGECs were exposed to HIV-1 (added to the upper chamber) and attachment, internalization, and release of HIV-1 into the basal chamber were monitored as indicated in the Results section. Note that the integrity of the monolayer of PGECs was preserved even 8 h post-virus exposure (data not shown). The CCR5 antagonist TAK779 was generously contributed by the Division of Acquired Immunodeficiency Syndrome, National Institute of Allergy and Infectious Diseases, NIH, and distributed with the permission of Takeda Chemical Industries Ltd. (5), whereas the CXCR4 antagonist AMD3100 was generously contributed by the Division of Acquired Immunodeficiency Syndrome, National Institute of Allergy and Infectious Diseases, NIH, and distributed with the permission of AnorMED (17, 52).
To date, two major classes of cell surface HSPGs, the syndecans and the glypicans, have been identified. The four members of the syndecan family (syndecan-1 to syndecan-4) are transmembrane proteins consisting of a short cytoplasmic domain and an extended extracellular domain, which bears three heparan sulfate chains (far from the plasma membrane) and one or two chondroitin sulfate chains (close to the plasma membrane) (8). In contrast to syndecans, glypicans (glypican-1 to glypican-6) are GPI-linked proteins consisting of a globular extracellular domain that bears two to three heparan sulfate chains and no chondroitin sulfate chains (8). To determine whether HSPGs affect HIV-1 passage through the primary genital epithelial cells, we first analyzed the expression of syndecans and glypicans on these cells both in vitro and in vivo.
We found that PGECs express both heparan and chondroitin sulfates (Fig. (Fig.1A).1A). Heparinase treatment decreased HS levels, whereas chondroitinase and phospholipase treatments did not, suggesting that syndecans rather than glypicans or CSPGs dominate on the surface of PGECs. High levels of syndecan-1 and syndecan-2 likely contribute to the high HSPG levels (Fig. (Fig.1A).1A). Primary vaginal epithelial cells express undetectable levels in CD4 and DC-SIGN and low levels in CXCR4, CCR5, and GalCer. We expressed these FACS data as a bar graph instead of as histograms simply for space considerations. Given that PGECs highly express CK19 but poorly express CK8 and CK18 (Fig. (Fig.1A),1A), this suggests that our isolated PGECs are mainly vaginal and ectocervical cells rather than endocervical cells as described previously (18). These data are representative of the results obtained with four independent donors. Consistent with these in vitro immunostaining data, we found that both cervical and vaginal epithelia express high levels of HSPGs (10E4), likely contributing to the high levels of syndecan-1 (BB4) and syndecan-2 (10H4) (Fig. (Fig.1B).1B). Corroborating our FACS data, syndecan-3 and syndecan-4 are absent or weakly expressed on the genital epithelium (Fig. (Fig.1B).1B). Syndecan-1 and syndecan-2 staining perfectly delineates the surface of the apical side of the genital epithelium. This demonstrates that syndecans are expressed on the surface of an important route of entry for HIV-1 and may thus have profound impact on HIV-1 transmission by acting as attachment receptors.
After analyzing the cell surface expression of candidate attachment and entry HIV-1 receptors on PGECs, we developed assays to discriminate between initial viral attachment, viral internalization, and viral release from PGECs. PGECs were seeded onto the upper face of collagen I- and fibronectin-coated 12-mm-diameter, 3-μm-pore-size polycarbonate membrane transwells at a density of 105 cells/well and cultured until formation of tight junctions was achieved. The inserts were fed every 2 days. The monolayer on the filter effectively divides the well into an apical compartment and a basolateral compartment. The integrity of each cell monolayer was measured using an epithelial volt-ohm meter. The trans-epithelial electrical resistance of each cell monolayer must exceed 600 Ω/cm2. To ensure the integrity of the epithelial cell barrier, we also determined the paracellular passage of the extracellular marker inulin with the permeability coefficient as measured by a diffusion assay using 14C-carboxylated inulin in the upper chamber as described previously (10). We observed that the integrity of PGEC monolayers on transwells was achieved between day 5 and day 7 after plating. The development of an in vitro transmigration assay is critical, because it allows the examination of the movement of HIV-1 from the apical surface to the basal surface of the epithelium in an “isolated” system independent of external factors, which may interfere with the assay. Indeed, the in vivo analysis of HIV-1 transmigration is complicated by the possibility of the “Trojan horse” transport of the virus via macrophages and dendritic cells, as well as by the presence of external body fluids (pH, antibodies).
HIV-1 (primary R5 JR-CSF virus) was added to the apical surface of the PGEC monolayer. First, after different time intervals, PGECs were washed to remove unbound virus, detached (Cell Stripper), and lysed. Amounts of attached virus were determined by p24 ELISA of PGEC lysates. Experiments were conducted both at 4°C and 37°C (Fig. (Fig.2A).2A). We found that HIV-1 rapidly attached to PGECs, with a plateau occurring after 1 h (Fig. (Fig.2A).2A). During the first hour, we did not observe significant differences in the efficiency of attachment between 4°C and 37°C, suggesting that the initial attachment of HIV-1 to PGECs is temperature independent. Second, we examined how long it takes HIV-1 to enter into PGECs. HIV-1 was added to PGECs, and, at different time intervals, cells were washed, trypsinized, washed again, and lysed. Amounts of internalized HIV-1 were quantified by p24 ELISA of PGEC lysate. Less than 0.1% of attached particles penetrated PGECs at 4°C; this suggests that HIV-1 internalization into PGECs is strictly temperature dependent. Only 9% to 11% of attached viruses entered PGECs (Fig. (Fig.2B).2B). Viral internalization into PGECs is a slower process than attachment, since it took 2 h for HIV-1 to enter PGECs at 37°C (Fig. (Fig.2B).2B). Third, we examined how long it takes HIV-1 to transmigrate through the PGEC monolayer. To mimic physiological transmigration conditions, HIV-1 was added to the apical surface of PGECs and amounts of transcytosed viruses were quantified by measuring capsid levels in the lower chamber corresponding to the basal surface. We found that 4 h was sufficient for HIV-1 to transmigrate through the PGEC monolayer and observed a peak after 8 h (Fig. (Fig.2C).2C). We did not observe an increase in p24 levels in the basal chamber (Fig. (Fig.2D),2D), suggesting that PGECs do not support HIV-1 replication, as previously reported (63). To determine whether viruses that have crossed PGECs represent infectious particles, the medium from virus-exposed PGECs was collected at different time intervals, filtered, and added to indicator TZM-bl cells. Prior to 4 h, no p24 was detected in the basal chamber. Importantly, basal chamber medium collected between 4 and 8 h represented a source of infection for TZM-bl cells (Fig. (Fig.2D),2D), indicating that the viruses that had crossed PGECs represented infectious particles. We chose TZM-bl cells as targets because, in contrast to peripheral blood mononuclear cells, which usually contain more CCR5+ than CXCR4+ CD4+ T cells, TZM-bl cells express similar levels of CXCR4 and CCR5. Importantly, medium from the basal chamber of PGECs collected after 16 h had almost lost its potential to infect TZM-bl cells (Fig. (Fig.2D),2D), suggesting that when particles which have crossed PGECs do not rapidly encounter target cells, they rapidly lose their infectivity, probably due to particle degradation or gp120 shedding. Since less than 0.01% of viruses (from the original inoculum) successfully transcytosed the monolayer, this suggests that PGECs form a nearly impermeable barrier against cell-free HIV-1. These results also demonstrate that a subset of cell-free viruses rapidly attaches to the apical surface of PGECs, enters into PGECs, and is released from the basal surface of PGECs as infectious particles.
We then examined the influence of the viral glycoprotein on cell-free HIV-1 transcytosis through PGECs. We used pNL4.3-derived infectious molecular clones in which the envelope gene was replaced with envelope genes derived from a panel of isolates, including pNL-ADA, pNL-JRFL, pNL-YU2, pNL91US005.11, pNL-RW020.5, and pNL-92BR020.4 as R5 viruses and pNL4.3, pNL-92UG021.6, and pNL-92UG024.2 as X4 viruses (66). All viruses were grown in Jurkat-CCR5 cells, standardized for p24, and tested for transmigration through PGECs as described above. We found that all viruses efficiently attached to and entered the monolayer whereas a virus deleted for gp160 (pNL4.3-E-) did not (Fig. (Fig.3,3, top and middle panels). This suggests that gp120 is the main ligand for HIV-1 attachment to and entry into the genital epithelium. R5 viruses are generally more preferentially released from the epithelial cell monolayer than X4 viruses, suggesting a preferential transport for R5 viruses through the primary genital cells (Fig. (Fig.3,3, bottom panel). This is in accordance with the results of two previous studies, which showed that primary gastrointestinal epithelial cells (34) and immortalized cervical epithelial cells (63) selectively transfer R5 viruses. However, some R5 viruses (e.g., pNL-RW020.5) do not efficiently cross PGECs whereas some X4 viruses (e.g., pNL-92UG024.2) do cross PGECs efficiently. Thus, coreceptor usage does not represent an absolute precondition for cell-free virus transit through PGECs. Thus, the predominance of R5 viruses during the early steps of sexual transmission apparently cannot be explained by the possibility that cell-free R5 viruses cross the genital epithelial cells more efficiently than X4 viruses. Given that the gp160-deficient virus fails to cross the epithelial barrier (no capsid is released into the basal chamber), this demonstrates that the integrity of the tight junctions was maintained and that, most importantly, the epithelial monolayer does not allow inappropriate paracellular transport. All transcytosed viruses efficiently infect indicator TZM-bl cells (data not shown), suggesting that cell-free viruses that do cross the genital epithelium represent infectious particles that may potentially initiate HIV-1 colonization.
After showing that gp120, but not coreceptor usage, governs HIV-1 transmigration through PGECs, we asked whether CCR5 or CXCR4 play a role in this process. To address this issue, CCR5 or CXCR4 was blocked with either neutralizing monoclonal antibodies (2D7 against CCR5 and 12G5 against CXCR4) or small antagonists (TAK779 against CCR5 and AMD3100 against CXCR4). PGEC monolayers were preincubated with 10 μg/ml of neutralizing antibodies or with 100 nM of TAK779 (5) or 1.2 μM AMD3100 (17, 52) for 1 h at 37°C prior to addition of R5 (pNL-JR-FL) or X4 (pNL-92UG0214.2) viruses. Note that anti-CD4, -CXCR4, and -CCR5 agents, when used at the concentrations indicated above, blocked 90% to 100% of the infectivity of all viruses used with TZM indicator cells in this study (data not shown). CXCR4 and CCR5 inhibitors did not block viral attachment to PGECs (Fig. (Fig.4),4), suggesting that HIV-1 uses receptors other than CXCR4 and CCR5 to initially attach to the surface of PGECs. However, CCR5 and CXCR4 inhibitors diminished HIV-1 entry into and transmigration through PGECs, suggesting that chemokine receptors facilitate HIV-1 transcytosis. Both anti-CD4 and anti-GalCer antibodies failed to prevent HIV-1 attachment, entry, and transcytosis (Fig. (Fig.4),4), suggesting that HIV-1 crosses the genital epithelium in a CD4- and GalCer-independent manner. Together, these data indicate that cell-free HIV-1 requires chemokine receptors but not CD4 and GalCer to efficiently transcytose through PGECs.
Given that CXCR4 and CCR5 facilitate HIV-1 entry, but not attachment, our data suggest that HIV-1 requires additional receptors to attach onto PGECs. We thus examined whether syndecan-1 and syndecan-2, richly expressed on PGECs (Fig. (Fig.1),1), participate in HIV-1 attachment to the genital epithelial monolayer. To address this issue, PGECs were pretreated or not pretreated with heparinase or chondroitinase to remove cell surface heparan or chondroitin sulfate moieties, respectively. We verified by FACS that heparan and chondroitin sulfates were removed, using the 10E4 antibody for heparan sulfates and the CS-56 antibody for chondroitin sulfates (Fig. (Fig.1A)1A) as described above. We observed that the enzymatic treatment conditions (enzyme concentration, incubation time, temperature) as well as the source (or the lot number) of these enzymes were of critical importance in significantly reducing the levels of heparan and chondroitin sulfates on the surface of PGECs. Therefore, for each enzymatic treatment, we verified by FACS that these enzymes removed heparan or chondroitin sulfate moieties (at least 80% removal) prior to addition of virus as shown above (Fig. (Fig.1A).1A). Importantly, heparinase treatment of PGECs reduces attachment, internalization, and transcytosis of a majority of the HIV-1 strains tested (Fig. (Fig.5).5). Given that attachment was only partially reduced compared to internalization, which was remarkably inhibited, this suggests that HSPGs (syndecan-1 and syndecan-2) not only participate in the initial attachment but also facilitate the subsequent viral internalization. Similar inhibitory results were obtained using the anti-heparan sulfate 10E4 antibody (10 μg/ml) (data not shown). Chondroitinase does not significantly influence HIV-1 transcytosis (Fig. (Fig.5).5). Given that chondroitin sulfates, like heparan sulfates, are long linear anionic chains, this suggests that HIV-1 attaches to the genital epithelium via HSPGs in a specific manner. Interestingly, pNL-RW020.5 transcytosis was only slightly reduced after heparan sulfate removal (Fig. (Fig.5),5), suggesting that the dependence on syndecan-1 and syndecan-2 for PGEC crossing may differ between HIV-1 strains. Importantly, heparinase and chondroitinase treatments did not affect the expression of CXCR4, CCR5, GalCer, or other cell surface antigen such as CD147 (Fig. (Fig.1A),1A), suggesting that these enzymes specifically removed heparan and chondroitin sulfate moieties without altering other cell surface molecules. By demonstrating that syndecans promote viral transcytosis through PGECs, our data suggest that syndecans may have an impact on HIV-1 transmission.
We then examined whether HIV-1 exploits specific sulfations within the heparan sulfate of syndecans. Specifically, we examined whether 2-O, 3-O, or 6-O sulfations or N-sulfation is required for efficient HIV-1 binding to genital epithelial syndecans. To address this issue, we examined the capacity of chemically modified heparan sulfate derivatives to block HIV-1 transcytosis. Specifically, we analyzed the inhibitory effects of heparin, N-desulfated heparin, over-sulfated heparin, 2-O-desulfated and 3-O-desulfated heparin, and 6-O-desulfated heparin. The disaccharide composition was determined by complete depolymerization of the chains by hydrazinolysis. Nitrous acid cleavage and borotritide reduction followed by reverse-phase ion-pairing chromatography using a Hi-Chrom 50DS C18 column were conducted as described previously (6). Soluble heparin derivatives were tested for their capacities to block HIV-1 transcytosis through PGECs. To exclude the participation of CCR5, PGECs were pretreated with the anti-CCR5 2D7 antibody. Oversulfated heparin was more potent than native heparin, suggesting that the degree of sulfation influences HIV-1-syndecan interaction (Fig. (Fig.6).6). 2-O-desulfated, 3-O-desulfated, and N-desulfated heparin exhibited an intermediate inhibitory effect, whereas completely 6-O-desulfated heparin was least effective. This suggests that the 6-O-sulfate group (GlcNAc 6OSO3) plays a critical role in HIV-1 binding to genital epithelial syndecans. It is critical to emphasize that the 6-O-desulfated heparin is still highly sulfated due to intact 2-O and 3-O sulfations. This suggests that the interaction between HIV-1 and genital epithelial syndecans is not simply the result of random interactions between basic residues in gp120 and negative charges in syndecans but is the result of specific interactions between gp120 and a well-defined sulfation process, 6-O sulfation.
We found that gp120 and syndecans are required for effective HIV-1 transcytosis (Fig. (Fig.2).2). We then asked which domains of gp120 are responsible for syndecan contact. We previously reported that a basic residue located at the base of the V3 region (arginine 298) is required for HIV-1 binding to syndecans artificially expressed in a B-cell line (14). We thus examined whether this residue is also important for HIV-1 binding to syndecans expressed on PGECs as well as for transcytosis. To address this issue, we replaced arginine 298 by an alanine within the proviral clone encoding HXB2 ConsB virus, which contains the consensus B sequence of the V3 of R5 HIV-1 isolates (14), creating the R298A mutant virus. As a control, we substituted the other highly conserved arginine located at the C-terminal base of the V3 (arginine 326), creating the R326A mutant virus (14). To exclude the participation of CCR5, PGECs were pretreated with the anti-CCR5 2D7 antibody. In contrast to wild-type and R326A viruses, the R298A virus, like the gp160- and V3-deficient viruses, attaches to and crosses the genital monolayer poorly (Fig. (Fig.6).6). This suggests that a highly conserved arginine (Arg298) at the base of the V3 region is critical for the initial contact between the virus and the genital epithelium. Importantly, wild-type, R298A, and R326A viruses bind DC-SIGN efficiently and incorporate wild-type amounts of gp120 (14). These findings indicate that a basic residue located at position 298 of the V3 of gp120 is required for the attachment of cell-free HIV-1 to the surface of the genital epithelium via the syndecans and for subsequent transcytosis.
During sexual transmission, HIV-1 has to cross the genital epithelium, which serves as a barrier against pathogens. To cross the genital epithelium, HIV-1 may exploit several routes. First, HIV-1 may directly transcytose through the genital epithelium as cell-free virus. Second, HIV-1 may take advantage of genital lesions that can permit either cell-free or cell-associated virus to cross the epithelial barrier. Third, HIV-1 may cross the genital epithelium associated with cells (e.g., spermatozoa), a process that is likely facilitated by the expression of adhesion molecules on genital epithelial cells. In the present study, we investigated the transcellular migration of HIV-1 as a cell-free virus through an intact PGEC monolayer. The absence of CD4 on PGECs implicates an unusual entry pathway for HIV-1. Since we found that HSPGs serve as major attachment receptors for HIV-1 (9, 10, 51), we asked whether they contribute to the capacity of HIV-1 to transcytose through PGECs. First, we found that both in vitro and in vivo human PGECs express high levels of HSPGs likely contributed by syndecan-1 and syndecan-2. This is in accordance with our previous observations that adherent cells express high HSPG levels whereas suspension cells lack HSPGs (9, 10, 51). We did not detect CD4 or DC-SIGN, suggesting that these receptors are normally absent on the surface of PGECs. In contrast, PGECs express low but significant levels of CXCR4, CCR5, and GalCer. The cell surface expression pattern of PGECs “passaged” a small number of times may differ from that of PGECs “passaged” a larger number of times. For example, we found that after 10 passages, PGECs derived from genital scraping from one donor (among four donors) lost their CXCR4 expression but increased their GalCer expression (data not shown). This underscores the importance of the number of passages of PGECs for transmigration studies. However, we observed constant patterns of HSPGs and syndecans on PGECs passaged a smaller or larger number of times (data not shown).
After demonstrating that HSPGs are abundantly expressed on PGECs, we examined their role during HIV-1 transcytosis. We developed a transmigration assay using Transwell filters, which allowed us to distinguish between viral attachment, entry, and transmigration. We found that HIV-1 rapidly attaches to the surface of PGECs in a temperature-independent manner. Using neutralizing antibodies, we found that CD4, GalCer, CXCR4, and CCR5 do not participate in the initial attachment of HIV-1 to PGECs. The finding that the affinity of HIV-1 for CCR5 and CXCR4 in the absence of CD4 is low (62) may explain why HIV-1 does not use CXCR4 and CCR5 to bind PGECs. However, the removal of HSPGs, but not of CSPGs, diminishes the capacity of HIV-1 to attach to PGECs, suggesting that HIV-1 exploits HSPGs to mediate its initial adsorption onto PGECs. We showed that the presence of gp120 is required for the attachment of HIV-1 to PGECs. Indeed, a gp120-deficient virus fails to attach to and transcytose through PGECs. Moreover, we found that the presence of a basic residue located at the base of the V3 loop (Arg298) is necessary for HIV-1 attachment to and transcytosis through PGECs. Given that Arg298 is critical for HIV-1 binding to syndecans (14), our finding further supports the notion that HIV-1 exploits syndecans (syndecan-1 and syndecan-2) to facilitate its adsorption onto the genital epithelium. We previously demonstrated that a cell-free virus already lost its infectivity after a single day whereas cell-associated virus kept its infectivity even after a week (9); thus, a rapid adsorption onto the genital epithelium via syndecans may greatly enhance the endurance of the virus during the early steps of sexual transmission. Indeed, one can envision that a cell-free virus is more susceptible than a cell-associated virus to degradation (pH, proteases) or neutralization (defensins) in the genital environment.
Blocking CXCR4 or CCR5, but not GalCer, reduces HIV-1 entry and transcytosis through PGECs, suggesting that CXCR4 and CCR5 play a significant role in cell-free HIV-1 transmission. This is in accordance with the results of a recent study, which demonstrated that high doses of a RANTES analog that neutralizes CCR5 prevent HIV (simian immunodeficiency virus/HIV-1) vaginal transmission in rhesus macaques (31). Thus, although CXCR4 and CCR5 do not mediate the initial attachment of the virus onto PGECs, they facilitate HIV-1 entry into PGECs. Interestingly, syndecans dually facilitate HIV-1 attachment to, and entry into, PGECs. Our finding that syndecans promote HIV-1 attachment to PGECs is in accordance with previous studies, including ours, which showed that HSPGs (e.g., syndecans) enhance HIV-1 adsorption onto cells that express CD4 such as macrophages (51), CD4+ HeLa cells (38), and CD4+ T-cell lines (30, 39, 41, 48, 66) and cells that lack CD4 such as primary human vein endothelial cells (9), human genital epithelial cells (63), or HeLa cells (38). This is also in accordance with previous studies, which showed that the removal of HSPGs reduces HIV-1 internalization into brain microvascular endothelial human cells that compose the blood-brain barrier (3, 10). Interestingly, the low (0.01%) efficiency of HIV-1 entry into PGECs greatly contrasts with the higher (0.1%) efficiency of HIV-1 entry into primary brain microvascular brain endothelial cells that compose the blood-brain barrier (10). Our data are also in accordance with the findings of Wu et al., who showed that HSPGs are involved in HIV-1 uptake by immortalized ectocervical cells (63).
We obtained several lines of evidence indicating that HIV-1-syndecan interactions rely on specific residues in the V3 of gp120 (i.e., arginine 298) and specific sulfations within the heparan sulfate chains of syndecans (i.e., 6-O sulfation). Since gp120 serves as the main ligand for syndecans (9, 14), it is likely that they facilitate HIV-1 adsorption onto PGECs via gp120-syndecan interactions. However, it is more difficult to understand how they facilitate the subsequent transcytosis steps, namely, internalization and transport through PGECs. One can envision that syndecans facilitate HIV-1 transmigration into PGECs either by rescuing HIV-1 from abortive endocytosis or by directing HIV-1 particles into “safe” transcytosis routes. Interestingly, both syndecans and chemokine receptors enhance HIV-1 transcytosis through PGECs. Previous studies showed that syndecans are associated with lipid rafts (22, 33, 54, 56, 65) and form complexes with CXCR4 and CCR5 on the cell surface (54). This suggests the possibility that syndecans and chemokine receptors are in close proximity on the surface of PGECs and that they cooperate for successful cell-free virus transcytosis. The affinity of gp120 to CXCR4 and CCR5 is low in the absence of CD4 (62). However, blocking CD4 does not prevent HIV-1 transcytosis through PGECs via CCR5 or CXCR4. Either the low affinity of HIV-1 for CXCR4 and CCR5 is nevertheless sufficient for HIV-1 transcytosis via the chemokine receptors, or other PGEC surface receptors (i.e., syndecans), by interacting with gp120, enhance HIV-1 binding to CCR5 and CXCR4.
Although we showed that gp120 is necessary for HIV-1 transmigration through PGECs, we did not observe any obvious correlation between coreceptor usage and the capacity of the virus to transcytose. Since the viruses isolated early after sexual transmission are mainly R5 viruses (32, 60, 67), our data suggest that the properties conferring virus replication early after transmission are distinct from those conferring cell-free virus transcytosis through the genital epithelium. Thus, it is likely that factors other than CCR5 usage on PGECs dictate R5 virus predominance during the early steps of HIV-1 colonization.
Although we showed in the present study that HIV-1 possesses the capacity to cross PGEC monolayers as infectious particles, the efficiency of cell-free HIV-1 transmigration is extremely poor (i.e., less than 0.02% of the inoculum). This supports the notion that the epithelium serves as a major barrier against pathogens. This is in accordance with the results of a previous study which showed using an explant culture model that cell-free HIV-1 poorly crosses intact stratified epithelium (26). Given that our data indicate that cell-free HIV-1 inefficiently crosses a monolayer of PGECs, this suggests that the block in HIV-1 transcytosis through the genital epithelium occurs precociously, probably during the crossing of the first layer of genital epithelial cells. Our data are also in accordance with the recently reported low male-to-female sexual transmission incidence (53). Although one cannot exclude the possibility that a low level 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. One can envision that cell-free HIV-1 mainly enters the body through genital lesions (16). Moreover, one cannot exclude the possibility that HIV-1 transmigrates through the genital epithelium mainly, or even exclusively, as a cell-associated virus (12, 13, 19, 42, 45, 58). Further work is required to compare the respective contributions of cell-free and cell-associated transmigration through the genital epithelium to HIV-1 invasion.
In this study, we examined cell-free HIV-1 transcytosis through artificial PGEC monolayers. We found that HSPGs, especially syndecan-1 and syndecan-2, 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. Gp120 is absolutely necessary for transcytosis, but no correlation between coreceptor usage and PGECs transmigration was identified. In conclusion, HIV-1 requires the presence of both syndecans and chemokine receptors to ensure successful cell-free transport through the genital epithelium. Given that HIV-1-syndecan interactions are based on electrostatic contacts between basic residues in gp120 and sulfate groups, our study suggests that HIV-1 may exploit these interactions to pass safely through the genital epithelium.
We thank J. Kuhns for secretarial assistance. We thank M. Emerman for providing us with the Jurkat-CCR5 cells, P. Bieniasz for the pNL-envelope series, and J. Esko for heparin analogs.
This is publication no. 18273 from the Department of Immunology, The Scripps Research Institute, La Jolla, CA.
We acknowledge financial support from the U.S. Public Health Service (grant no. AI054196) to P.A.G.
Published ahead of print on 18 October 2006.