Macaques remain the principal model system for analyzing HIV transmission. During vaginal exposure to SIV in macaques, the first cells that are infected are Langerhans cells, submucosal CD4+
T cells, and DCs (18
). A cellular barrier segregates the vaginal, cervical, and uterine lumens from these targets of infection, so the mechanisms for HIV-1 traversing this barrier and the host components contributing to this process remain an important area of study in HIV transmission. In primary genital tract epithelial cells and the endometrial carcinoma line HEC1A, direct transcytosis of virus through the body of the cells has been shown to be partially dependent on HSPG moieties expressed on the apical membrane. More specifically, the syndecan family has been implicated in mediating some of the transcytosis observed in these cell lines (7
). Here, we demonstrate that gp340 also directly contributes to the transcytosis observed in HEC1A cells and primary human endocervical tissue, which is distinct from that mediated by syndecans. Initially, the secreted DMBT1 splice variant SAG was identified as a component of human saliva important in inhibiting oral transmission of HIV-1 (33
). However, the cell-associated variant gp340 was shown to bind and facilitate trans
infection of HIV-1 when expressed on luminal lining cells of the genital tract (47
) and promote cis
infection of macrophages (10
). We also demonstrate that HEC1A cells express gp340 and that expression mediates the ability of these cells to bind, transmit, and transcytose HIV-1. Further, we show that human endocervical tissue can mediate transcytosis of HIV-1 in a manner dependent on the interaction of gp340 with the viral envelope.
Most host cellular factors that interact with HIV Env and do not mediate fusion do so through glycosylation-based interactions with the complex sugars that coat the exterior of the viral envelope protein. The 24 N-glycosylation moieties on the Env protein are highly mutable, and interactions with them are, for the most part, relatively nonspecific. This makes them poor targets for therapeutics and vaccines. The association between HIV Env and gp340, however, is a protein-protein interaction (53
). Based on peptide binding studies and subsequent computer modeling of the interaction of Env with gp340, the interaction sites correspond to the base of the Env V3 loop and the SRCR domains of gp340 (54
). The amino acid sequence of the V3 loop base is highly conserved. Further, this site contains Arg298, which is critical for interaction with syndecans (13
), suggesting that the site may support interactions with multiple host factors important for transcytosis.
gp340 in human alveolar macrophages can be found trafficking between the extracellular membrane and intracellular endosomes after ligand binding (32
). Vaginal and cervical cells that express gp340 are capable of binding, harboring, and then transferring virus to susceptible targets of infection (7
) up to 4 days post viral pulsing (47
). Further, treatment of these and other gp340-expressing cell lines with trypsin following viral pulsing does not ablate their abilities to maintain and transmit virus. Interestingly, the rabbit orthologue of gp340 functions as a modulator of polarity in epithelial cells (48
). While a polarity reversal function for human gp340 has yet to be demonstrated, it remains a distinct possibility, given our finding that gp340 mediates transcytosis.
Studies of HIV transmission in the human population demonstrate that transmission is an uncommon event, with a probability of infection per exposure between 0.005 and 0.3% (17
). Despite measurable levels of transcytosed virus, our results support the finding that the epithelial cell barrier represents a significant impediment to viral transmission. Even with a viral dose that exceeds that found in human semen by severalfold, ~0.6% of the virus successfully traverses a single cell layer by transcytosis, but our preliminary results suggest that this small amount of transcytosed virus remains capable of infecting target cells. This was reduced and infection was inhibited by blocking the binding of Env to both gp340 and HSPG moieties. This suggests that blockage of cellular receptors that mediate transcytosis could serve as an effective means of reducing transmission.
Limitations remain in the use of in vitro systems including human genital tract tissue as a model of HIV-1 transmission. Given the inability to demonstrate comparable in vivo functionality for one of the more highly studied HIV host factors, DC-specific intercellular adhesion molecule-grabbing integrin, an in vivo correlation in a physiologically relevant model is necessary to further support the role of gp340 in HIV transmission. In order to understand the function of gp340 in in vivo transcytosis, a macaque model of the interaction of gp340 with virus must be developed. Only recently has the chromosomal locus of the gp340 orthologue in macaques been identified. Interestingly, in macaques, gp340 is linked to a progesterone response element (1
). The macaque protein has not been characterized, nor has its tissue distribution been identified, but our preliminary studies demonstrate that the SRCR region of macaque gp340 can bind HIV Env and macaque vaginal and cervical tissue-expressed gp340 mRNA (data not shown). Comparison of the basic cell biology of macaque gp340 and testing of its role in vaginal transmission remain important next steps in understanding human HIV transmission during heterosexual intercourse.
This research presents gp340 as a facilitator of transcytosis in HEC1A cells and normal human endocervical tissue. Through inhibition studies directed at blocking the interaction of gp340 with HIV Env, the contribution of gp340 to viral transcytosis was identified and determined to account for about half. With the help of combinational treatments, the roles of gp340 and HSPG moieties in transcytosis appear distinct based on the ability to inhibit selectively and additively. Blockage of both receptors that mediate transcytosis in HEC1A cells or human tissue nearly blocked their ability to transcytosis virus. This suggests that there might still exist other factors that contribute to transcytosis in these cells or that existing inhibitors are not 100% effective. However, discovery of gp340 as a host cell factor that promotes HIV infection through two mechanisms, trans
infection, with promotion of viral infectivity (47
), and transcytosis, continues the identification of the earliest events in HIV transmission and provides an additional target for the development of preventive therapeutics.