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
), 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
). 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
HeLa cells (38
), and CD4+
T-cell lines (30
) 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
). 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
), 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
) 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
), 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
). 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.