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The Journal of Immunology, 2007, 179, 3126 -3132
Copyright © 2007 by The American Association of Immunologists, Inc.
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gp340 Expressed on Human Genital Epithelia Binds HIV-1 Envelope Protein and Facilitates Viral Transmission1

Earl Stoddard*, Georgetta Cannon*, Houping Ni*, Katalin Karikó{dagger}, John Capodici*, Daniel Malamud2,{ddagger} and Drew Weissman3,*

* Department of Medicine and {dagger} Department of Neurosurgery, Division of Infectious Diseases, School of Medicine and {ddagger} Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During sexual transmission of HIV in women, the first cells likely to be infected are submucosal CD4+ T cells and dendritic cells of the lower genital tract. HIV is segregated from these target cells by an epithelial cell layer that can be bypassed even when healthy and intact. To understand how HIV penetrates this barrier, we identified a host protein, gp340, that is expressed on genital epithelium and binds the HIV envelope via a specific protein-protein interaction. This binding allows otherwise subinfectious amounts of HIV to efficiently infect target cells and allows this infection to occur over a longer period of time after binding. Our findings suggest a mechanism of viral entry during heterosexual transmission where HIV is bound to intact genital epithelia, which then promotes the initial events of infection. Understanding this step in the initiation of infection will allow for the development of tools and methods for blocking HIV transmission.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The spread of HIV continues, with nearly 40 million infected individuals and >5 million newly infected every year. Worldwide, the principle means of infection remains heterosexual transmission. In many areas of the world, particularly sub-Saharan Africa, HIV infection has become more prevalent in women than men (unaids.org). This trend, in conjunction with inequitable sexual rights for women in many parts of Asia and Africa, makes it important to identify the mechanisms of male-to-female spread of HIV and to implement new methods for preventing sexual transmission. Normally, the mucosal barrier acts as a significant blockade for pathogens seeking to gain entry into the human body. HIV enters either through breaks in this barrier or through some active mechanism that allows it to transverse the mucosal barrier. Identification of mediators of transmission is the subject of this study.

Macaque studies indicate that the first targets of SIV infection during nontraumatic vaginal application are Langerhans cells (LC),4 subepithelial dendritic cells (DC) and CD4+ T cells of the lower genital tract (1, 2, 3). The lower genital tract barrier is composed of a columnar monolayer in the endocervix and stratified squamous epithelium in the vagina and ectocervix along with their associated mucous and glycoprotein secretions. The necessary events in transmission preceding infection of LCs, subepithelial DCs, and CD4+ T cells remain unclear. In vitro models of female genital tissue have been designed to explore transmission, but their in vivo relevance is controversial (4, 5). Disruption of these epithelial barriers by ulcerating lesions or other local factors (reviewed in Ref. 6), allowing direct access to underlying cells capable of propagating virus, is a proposed mechanism for penetrating the outer mucosa (2, 7). However, infection in the presence of a seemingly undamaged epithelium also occurs (8). Previous experiments have suggested that primary vaginal epithelial cells can sequester and transmit HIV to activated PBMCs in culture, even though the epithelial cells do not express measurable levels of virus receptors (9). This indicates that other factors within this subset of cells may interact directly with HIV and facilitate virus transfer to target cells.

A number of host factors not directly involved with fusion have been shown to interact with HIV and influence the infection. For example, DC-specific intercellular adhesion molecule-grabbing integrin (DC-SIGN), which is expressed mainly on macrophages and subsets of DCs, binds and sequesters HIV and subsequently presents the virus to target CD4+ T cells for infection (10, 11, 12, 13, 14). The syndecan family of receptors has been shown to bind HIV-1 through interactions between its heparin sulfate moieties and HIV-1 gp120 and to mediate transinfection of HIV (15). Heparin sulfate has also been indicated to play a role in the transcytosis of HIV-1 across primary genital tract epithelium in vitro, but similar studies in cell lines were inconclusive (16, 17). Mannose receptors (MR) on primary monocyte-derived macrophages can bind HIV and facilitate transmission to T cells in coculture, as well (18). Salivary agglutinin (SAG) is another host protein that binds HIV-1 envelope protein (Env), but in a different manner. SAG, first identified as a component of human saliva with anti-HIV-1 activity, was subsequently shown to form a protein-protein interaction with the HIV-1 gp120 protein (19, 20, 21). These studies do not eliminate the potential for glycan(s) aiding binding or proper epitope confirmation but do show that a specific V3 loop peptide is sufficient to bind gp340 (22). SAG is a splice variant encoded by the DMBT1 gene that also encodes other variants including cell surface-associated gp340 that acts as an immune scavenger receptor (23, 24, 25) and epithelial cell differentiation Ag (25). gp340 plays a role in innate immune surveillance of bacteria in the lung and oral cavities and in the host defense against influenza A virus (26, 27, 28). A correlation has also been drawn between mutations in the DMBT1 gene and occurrence of certain epithelial-derived malignant tumor (23, 29). In this study, we show that gp340 expressed on primary female genital epithelial cells binds HIV-1 and enhances transmission by allowing otherwise subinfectious amounts of HIV to efficiently infect target cells and allowing this infection to occur over a longer period of time after binding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Virus, cell lines, Abs, and inhibitors

HIV-1 BL2, Ba-L, 89.6, and IIIB viruses were obtained from the Center for AIDS Research at the University of Pennsylvania (Philadelphia, PA). 293, HEC1A, and FaDu cells (American Type Culture Collection; ATCC) were grown in DMEM supplemented with 10% FBS and 2 mM L-glutamine (Invitrogen Life Technologies). AGS cells (ATCC) were grown in F12 supplemented with 10% FBS and 2 mM L-glutamine. VK2E6E7, Endo1E6E7, and Ect1E6E7 cell lines were obtained from R. Fichorova (Fearing Research Laboratory, Boston, MA) and grown in KFM medium (Invitrogen Life Technologies) as described (30). gp340-specific mAbs 303 mAb (31) and 116, mouse polyclonal Ab DAPA, and rabbit polyclonal Ab 1527 were used. HIV-1 V3 loop-specific peptides 6283, VQINCTRPNYNKRKR, and 6284; CTRPNYNKRKRIHIG; and control peptide 6220, KEATTTLFCASDAKA were obtained from the AIDS Reference and Reagent Program. Scrambled peptide 6284, RCIHNRTIKGPYNKR, was synthesized. pDEST-DMBT1 was obtained from Jan Mollenhauer (Deutsches Krebsforschungs-zentrum, Heidelberg, Germany) (32, 33).

Stable clone construction and selection

293 cells were transfected with pDEST-DMBT1 in Fugene 6 (Roche Diagnostics) per the manufacturer’s instructions and selected with 500 µg/ml G418 (Invitrogen Life Technologies). Potential clones were measured for extra- and intracellular expression of gp340 by flow cytometry as described below.

Cellular staining for expression of gp340

Cells were grown in six-well plates and removed with PBS without Ca2+ or Mg2+ with 5 mM EDTA. Optimal staining was observed when 4 mM Ca2+ was used throughout the staining. Polyclonal Ab 1527 in PBS, 2% FCS, 4 mM Ca2+ was added to disrupted cells for 20 min on ice. For intracellular protein analysis, saponin (Sigma-Aldrich) (0.25%) was added to all staining steps. PE-conjugated goat F(ab')2 anti-rabbit IgG (CalTag) was used as a secondary Ab. Cells were analyzed on a FACScan flow cytometer using Cell Quest Pro software (BD Biosciences).

PBMC purification and stimulation

PBMCs of healthy volunteers were isolated from leukapheresis packs obtained under an institutional review board-approved protocol. Cells were subjected to Ficoll-Hypaque density gradient centrifugation. Purified PBMCs were stimulated in RPMI 1640 supplemented with 10% FBS and 2 mM L-glutamine (complete medium) plus 4 µg/ml PHA (Sigma-Aldrich) and 20 U/ml IL-2 (AIDS Reference and Reagent Program) at 37°C for 48 h, washed, and cultured in IL-2 (20 U/ml).

Viral binding assay

293, HEC1A, AGS, VK2E6E7, Endo1E6E7, and Ecto1E6E7 were seeded in 96-well plates and grown to 80–95% confluency. Cells were treated or not with heparinase III (5 U/ml), and then inhibitors were incubated with cells for 1 h at 37°C. Following pretreatments, 5 ng of HIV were added to each well in the continued presence of inhibitors and allowed to incubate for 2 h at 37°C. Cells were washed multiple times after viral pulsing. Between washes, plates were centrifuged at 400 x g for 7 min. Cells were lysed in cell lysis buffer (Promega), and samples were measured for HIV-1 p24 content by ELISA.

Transinfection assay

Parental or gp340-expressing 293 cells or VK2E6E7, Endo1E6E7, Ect1E6E7, FaDu, or AGS cells seeded in 96-well plates to 80–95% confluency, except when prolonged culture in the absence of targets was performed where 50% confluency was used, were incubated with various IU inputs of HIV in 100 µl final volume for 2 h at 37°C and then extensively washed. Cells were exposed to trypsin (0.05%), EDTA for 10 min at room temperature where indicated (34). Additional inhibitors, peptides or Abs, were added to cells 30 min before pulsing with virus and replaced after cells were washed. Plates were centrifuged at 400 x g for 7 min between each wash. After viral incubation and washing, PHA- and IL-2-stimulated PBMCs (PHA-blasts) were cocultured with the pulsed cells for 1 day and then moved to new wells for 6 days at 37°C with IL-2. Infection was monitored by measuring supernatant-associated p24 Gag protein content on day 7. The 50% tissue culture-infective dose TCID50 was calculated using the Reed-Münch formula (35).

Tissue isolation, staining, and analysis

Fresh vaginal and cervical tissues were obtained during total abdominal hysterectomy from the Cooperative Human Tissue Network (University of Pennsylvania). Tissue was flash frozen, sectioned, and stained with 303 mAb (31) and goat anti-mouse Ig-peroxidase and then counterstained with hematoxylin.

Statistics

Means, SDs, and p values for paired samples were calculated using Microsoft Excel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cell surface expression of gp340 enhances HIV binding

Human papilloma virus E6 and E7 gene-transformed vaginal, endocervical, and ectocervical primary epithelial cells (VK2E6E7, Endo1E6E7, and Ect1E6E7, respectively) were obtained from Dr. Raina Fichorova (Fearing Research Laboratory, Boston, MA) (30). Established cell lines, HEC1A (endometrial) and AGS (stomach), were obtained from the ATCC. Each of the cell lines expressed detectable levels of gp340 on the cell surface by flow cytometric analysis (Fig. 1A). These gp340-expressing cell lines were analyzed for their ability to bind HIV-1. As certain of these types of cells were demonstrated to bind HIV via heparin sulfate moieties (16, 17), they were tested with and without pretreatment with heparinase III. Heparin sulfate knockdown by heparinase III treatment was confirmed by FACS analysis of each cell line using the anti-HSPG IgM clone 10E4 (data not shown). Cells that express gp340 on their cell surface demonstrated a high capacity to bind HIV-1 (Fig. 1B). This binding was inhibited by peptide 6284. In contrast, binding was not inhibited by pretreatment with scrambled peptide 6284 + heparinase III or heparinase III alone. HIV binding was similarly inhibited by peptide 6284 in the absence of heparinase III pretreatment (data not shown).


Figure 1
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  		FIGURE 1. Cell lines derived from human cervical, vaginal, and intestinal epithelium express gp340. A, Endo1E6E7, VK2E6E7, Ect1E6E7, HEC1A, and AGS cells were stained with gp340-specific mAb 116 followed by goat anti-mouse IgG-PE secondary Ab. Cells were analyzed on a FACScan flow cytometer. Figure 1, Isotype control staining; {blacksquare}, mAb 116 staining. B, 293, VK2E6E7, Endo1E6E7, Ecto1E6E7, AGS, and HEC1A cells were pretreated with medium alone, peptide 6284 (10 µg/ml) and heparinase III (5U/ml), scrambled peptide 6284 (10 µg/ml) and heparinase III, or heparinase III alone. Cells were then pulsed with BL2 virus (5 ng/well). After washing, the cells were lysed and measured for p24 content by ELISA. Experiments were performed in four well replicates. Bars, SD.

 
gp340 enhances HIV transinfection

Secreted gp340 found at high levels in the oral cavity has been demonstrated to inhibit the infectivity of HIV-1 likely through its ability to bind the envelope protein (19, 21). gp340 also exists in a cell surface-associated form, although mRNA containing exon 55, which encodes the putative transmembrane domain, has not yet been detected. Cell lines expressing gp340 on their extracellular membranes were established by transfecting a plasmid encoding the presumed full length secreted form of gp340 (Refs. 32 and 33 and Fig. 2A). Control untransfected 293 cells had very low levels of endogenous gp340 as did many human-derived epithelial and myeloid cell lines (Fig. 2A and data not shown). gp340-expressing cell lines were measured for their ability to transmit cell-free HIV to human PHA-blasts. Transfected cells were incubated with or without HIV-1-specific peptide 6284, derived from the base of the V3 loop that has been shown to block Env binding to gp340 (22) or control peptides. Decreasing amounts of HIV (primary M-tropic strain BL2) were added and incubated with the cells in the continued presence of peptides. Infectious units of added virus were calculated on PHA-blasts using the Reed-Münch technique (35). After extensive washing, the cells were cocultured for 7 days with PHA-blasts. Infection was monitored by p24 Gag protein content in the culture supernatant in each well and scored for the presence or absence of infection. The assay used six replicates of cells for each viral dilution and condition and calculated the number of wells that became infected at each amount of added virus. Thus, a finding that 50% of the wells became infected when 0.11 IU were added and 100% of the wells became infected when 0.33 IU of HIV were added for gp340 expressing 293 cells determines that gp340 increased the TCID50 of the added virus ~6-fold compared with that of the nonexogenously expressing 293 cells (Fig. 2B). When gp340-expressing cells were preincubated with peptide 6284 corresponding to HIV-1 Env, the enhancement was abolished (Fig. 2B). Incubation with numerous control peptides that shared the charge and amino acid makeup of 6284 had no effect on enhancement of infection by gp340 (data not shown). To demonstrate statistical significance, comparisons of the average p24 in the 6 wells for each condition at each viral input were performed. At both 0.33 and 0.11 IU, the difference between clone 1D4 and untransfected 293 cells (mean p24 of 179.2 pg/ml vs 63.8 pg/ml at 0.33 IU and 74.2 pg/ml vs7 pg/ml at 0.11 IU) was significant (p = 0.008 and 0.03, respectively). Similarly, the difference between transmission by clone 1D4 in the presence or absence of peptide 6284 (mean p24 of 179.2 pg/ml vs 80.2 pg/ml at 0.33 IU and 74.2 pg/ml vs 7 pg/ml at 0.11 IU) was significant (p = 0.03 for both). Treatment of PHA-blasts with peptide inhibitors, 6283 and 6284, before and during viral challenge showed no ability to inhibit infection (data not shown). Other transfected 293 clones expressing gp340 could transmit HIV to target cells to a degree that correlated strongly with their overall expression levels (data not shown). None of the transfected 293 clones or untransfected 293 cells could replicate virus in the absence of coculture. Trypsinization with a protocol that completely removed surface bound virus 2 h after viral pulsing reduced (~40%) but did not abolish transinfection to cocultured target cells (Fig. 2C).


Figure 2
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  		FIGURE 2. 293 cells stably expressing cell surface gp340 enhance the transfer of HIV to target cells. A, 293 cells and clone 1D4, stably expressing gp340, were stained for surface expression of gp340 using rabbit polyclonal Ab 1527. Isotype control stained cells shown are 293 cells, but the 1D4 clone gave the same pattern. B, Clone 1D4 and 293 cells were pulsed with increasing IU amounts of HIV BL2 virus with or without a V3-derived peptide 6284 (10 µg/ml) that blocks Env-gp340 binding, washed, and exposed to PHA-blasts for 7 days. C, Clone 1D4 and 293 cells were pulsed with increasing IU of HIV IIIB virus with or without pretreatment with trypsin-EDTA. IUs were calculated using PHA-blasts as target cells in a TCID50 assay. The experiment in B was performed in replicates of six wells per viral addition, and data are expressed as the percentage of the wells that became infected. C, Means and SD for replicates of three.

 
AGS cells can transmit virus to target cells

To examine native expressed gp340, the gastrointestinal carcinoma cell line AGS, which expresses gp340 (23), was analyzed for its ability to transmit HIV in vitro. AGS cells enhanced transmission of HIV to target CD4+ T cells. Cells were infected in 50% of the wells when 0.08 IU of virus were pulsed per well. This represented a 6-fold enhancement in the viral infectivity by gp340. Peptides 6283 and 6284, derived from the proposed gp340 binding site in Env, abolished the enhancement in infectivity. Control peptide 6220, derived from another part of Env, or peptides that shared the charge and amino acid composition of 6283 and 6284, had no impact on the enhancement in infectivity (Fig. 3 and data not shown). The difference between peptide 6284 and control peptide was significant at viral inputs of 2, 0.4, and 0.08 IU (p = 0.03, 0.01, and 0.02). For peptide 6283 compared to peptide 6220, statistical significance was found at viral inputs of 0.4 and 0.08 IUs (p = 0.05 and 0.04). The magnitude of the inhibition by the active peptides correlated closely with the affinity with which these peptides interacted with gp340 (D. Weissman, unpublished observation).


Figure 3
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  		FIGURE 3. Transmission of HIV by AGS cells expressing endogenous gp340 is inhibited by peptide corresponding to the V3 domain of HIV. AGS cells were preincubated with peptides 6283 or 6284 and control peptide 6220 (10 µg/ml) for 15 min and then pulsed with decreasing amounts of HIV strain BL2. After viral exposure, the cells were washed four times to remove unbound virus and cocultured with T cell blasts in the absence of peptides for 24 h. The T cell blasts were then moved to new wells, and p24 Gag Ag content was measured 7 days later. IUs were calculated using PHA-blasts as target cells in a TCID50 assay. The experiment was performed in replicates of six per virus concentration. Data are expressed as the percentage of the six wells in which the cells became infected for each virus concentration.

 
Vaginal and cervical epithelia express gp340

Physiologically normal genital tract tissue was obtained from subjects undergoing total abdominal hysterectomy. Tissue sections were immunohistochemically stained for gp340 with a specific mAb. In the vaginal epithelium, the stratified squamous epithelia showed significant and diffuse expression of gp340 (Fig. 4A). Expression in cervical tissue was seen on the outer luminal surface of the columnar epithelial cells but most strongly along the basilar region (Fig. 4, B and C). Expression patterns between three independent donors were comparable.


Figure 4
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  		FIGURE 4. Human genital tract tissues express gp340. Physiologically normal vaginal (A) and cervical (B and C) tissues were stained with mouse anti-gp340 mAb 303. Cell nuclei were counterstained with hematoxylin. Bars, 25 µm.

 
Primary vaginal and cervical cell lines transmit HIV to PHA-blasts

VK2E6E7, Endo1E6E7, and Ecto1E6E7 were tested for their ability to transmit cell-free virus to target PHA-blasts. FaDu, a pharyngeal epithelial cell line that does not express gp340, was used as control. To determine the contribution of gp340 in the context of other HIV-binding macromolecules that are could be present on these cells, gp340-specific Abs and HIV-specific peptides that block the interaction of gp340 with Env were used.

Cell lines were pulsed with decreasing amounts of HIV Ba-L, washed, and had target cells immediately added or added after 2 or 4 days of culture. Adding 0.08 IU of HIV per well to Ect1E6E7 cells led to infection in 50% of wells, and virus incubated with Ect1E6E7 for 4 days remained infectious (Fig. 5A). When FaDu cells were pulsed with 250 IU of HIV, <10% of the added virus could be recovered following immediate coculture, and infectivity was lost when virus was incubated with the cells for 2 or 4 days before coculture (Fig. 5B). Similar results were obtained using the VK2E6E7 and Endo1E6E7 cell lines or when the primary isolate CCR5-using BL2 or laboratory isolates 89.6 and IIIB viral strains of HIV-1 were used (data not shown). None of the genital tract-related cell lines replicated virus in the absence of coculture with target cells (data not shown). To confirm the contribution of gp340 to the observed increase in viral transmission associated with the genital tract epithelial cell lines, cells were incubated with gp340 specific Abs (116 and DAPA). The ability of VK2E6E7 cells to transmit HIV to PHA-blasts was markedly decreased by preincubation with these gp340-specific Abs that were previously demonstrated to inhibit Env binding to gp340 (Ref. 19 and Fig. 6). Similar levels of decrease in transmission were observed when Ect1E6E7 and Endo1E6E7 cells lines were used (data not shown). The observation that both multiple gp340-specific Abs that inhibit Env binding and HIV V3-derived peptides that inhibit gp340-Env interaction reduced genital tract cell line mediated transinfection demonstrates that gp340 mediated this effect. It has been recently demonstrated that a specific arginine in the V3 region mediates binding to syndecan (36). Heparinase III treatment, despite showing a large decrease in surface expression of syndecan-1 by FACS, had no significant impact on the ability of the cells to transmit virus, demonstrating that syndecans did not mediate transinfection in our system (data not shown).


Figure 5
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  		FIGURE 5. Genital tract epithelial cell lines transinfect HIV-1. Ectocervical epithelial cells (Ect1E6E7; A) and a pharyngeal epithelial cell line control FaDu (B) were pulsed with decreasing IU amounts of HIV Ba-L and then washed four times. Either immediately or 2 or 4 days later, PHA-blasts were added. PHA-blasts were removed from Ect1E6E7 and FaDu cells after 18 h and cultured. Seven days post-coculture, supernatant-associated p24 Gag protein content was measured by ELISA. IUs added were calculated using PHA-blasts as target cells in a TCID50 assay. Data are expressed as the percentage of wells containing cells demonstrating infection for each amount of virus added.

 

Figure 6
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  		FIGURE 6. Transmission of HIV-1 by vaginal epithelial cells to target T cells in culture is mediated by gp340. Vaginal epithelial cells (VK2E6E7) were incubated with isotype control or gp340-specific Abs 116 or DAPA and then pulsed with HIV-1 BL2 virus. After extensive washing, PHA-blasts were added for 24 h and then moved to new plates without VK2E6E7 cells. After 7 days, p24 Gag protein content in the supernatant was measured. Samples were run in triplicate and SEM is given.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
During heterosexual transmission, the initial cells likely infected by HIV are LCs, submucosal DCs, and CD4+ T cells of the vagina and cervix based on macaque studies (1, 2, 3, 5, 9). The mechanism(s) by which HIV-1 transverses the epithelial barriers that separate the virus from these target cells are not well defined. We identify gp340 as a host protein that is diffusely expressed in the female genital tract and enhances early transmission events by genital tract cells through stabilizing infectious virus. A secreted form of gp340, SAG, was originally characterized as an anti-HIV-1 component of human saliva that binds Env with high affinity and inhibits HIV infection (19, 20). In this report, we demonstrate that gp340 is expressed on genital tract epithelial cells in vivo and that cell lines derived from such tissues express gp340. gp340 on these cell lines allows otherwise subinfectious amounts of HIV to efficiently infect target cells and allows this infection to occur over a longer period of time after binding. The underlying mechanism of the increase in infectivity will require further studies. The increased binding and viral transmission mediated by gp340 can be inhibited with either V3 loop-specific peptides that block gp340-gp120 interaction or gp340-specific Abs. Thus, gp340 expressed by primary genital tract epithelial cells reduces the amount of HIV required to infect target cells and preserves infectivity for multiple days following exposure to the virus suggesting that HIV uses this binding to enhance genital tract transmission.

Heterosexual transmission of HIV-1 is an inefficient process with population studies suggesting that the risk of infection per exposure is 0.01 to 0.15% (37). The presence of ulcerative and nonulcerative sexually transmitted diseases (STD) increases this risk per exposure. Mucosa-penetrating lesions with surrounding inflammation and target cells in ulcerative STDs are a mechanism for increased transmission, whereas the presence of increased targets of infection in epithelial and subepithelial tissues has been proposed as a mechanism for enhanced infection in nonulcerative STDs (9, 38, 39). How the virus crosses healthy genital tract tissue is unknown. Passive diffusion through tiny perforations in the epithelial layer has been theorized (9). The results of this study with the observation that genital tract epithelial cells express an HIV Env-binding protein that increases viral infectivity would enhance infection in the presence of large, tiny, or no epithelial perforations. Multiple mechanisms for gp340-mediated enhancement in vivo could be postulated. gp340 could concentrate virus at the mucosal surface and facilitate greater virion exposure to underlying targets of infection in the setting of any perforations. Further, gp340 expression is up-regulated in the presence of cell damage and thus would be present in higher concentrations at sites of ulceration or sites of perforation. In the absence of perforations, allowing otherwise subinfectious amounts of HIV to efficiently infect target cells and allowing this infection to occur over a longer period of time would enhance infection of DC that constantly probe epithelial cell layers.

A number of different HIV-binding macromolecules have been identified (reviewed in Ref. 38). In the context of vaginal and cervical epithelia, heparin sulfate molecules on the cell surface have been shown to facilitate some binding of HIV-1 (19). However, this glycosylation-based interaction was not the only means by which HIV-1 bound to these cells as evidenced by the inability to completely abate binding in the presence of specific inhibitors. In fact, treatment of the genital tract epithelial cell lines used in this study with heparinase did not alter their ability to bind or transinfect HIV-1. Other well-studied HIV-1 binding molecules such as CCR5, CXCR4, CD4, MR, and DC-SIGN are not expressed on genital epithelia, although their potential importance to infection in many compartments of the body is clearly documented (10, 15, 18, 40). Thus, genital tract epithelial cells can mediate transinfection of HIV-1 through specific protein-protein interactions between cell-associated gp340 and HIV-1 Env. The potential mechanisms used to increase viral infectivity and half-life demonstrated by these studies including trafficking to subcellular compartments as has been observed for DC-SIGN (7) are the subject of future investigation. Cell surface- and endosome-associated gp340 was detected in alveolar macrophages, suggesting that gp340 is capable of trafficking to intracellular compartments (23). The ability of gp340 to internalize virus and whether this plays a role in transinfection must be tested.

The protein-protein interaction between gp340 and HIV-1 Env is uncommon and interesting among identified host HIV-1 Env-binding molecules. Most identified non-CD4 or coreceptor interactions with Env, including DC-SIGN and MR, are mediated through glycosyl groups that coat viral proteins (12, 13, 41). The Env protein of HIV-1 is covered with 24 N-glycosylation moieties the sugars of which serve to shield it from immune recognition (42). Binding via glycosyl moieties is relatively nonspecific; thus, directed inhibitors of the interaction have reduced potential. gp340 contains 14 scavenger receptor cysteine-rich (SRCR) domains, which are a highly conserve motif in the innate immune system (43). Computer modeling using crystal structures from a homologous SRCR domain and peptide inhibition studies demonstrated that the binding site for gp340 appears to be near the base of the V3 loop of Env (20) and within the SRCR domain of gp340. Prior studies with SAG demonstrated that the addition of soluble CD4 enhanced binding to Env suggesting binding is to a site that is partially shielded (20), supporting the V3 studies. The role of the V3 loop in selecting coreceptor usage is well documented, but it is the combined binding of both the V3 loop and the bridging sheet domains of Env that mediate coreceptor binding leading to fusion and infection. Envs lacking V3 loops have been demonstrated to infect cells as well, suggesting that its presence is not absolutely required for infection (44, 45). Our results suggest that gp340 binding to Env does not interfere with viral fusion and that the presence of gp340 on a cell with CD4 and coreceptor, in fact, promotes fusion (D. Weissman and G. Cannon, unpublished observations). Whether this binding stabilizes the reactive intermediate of Env after CD4 binding is unknown. If this were to occur, it would allow subinfectious amounts of HIV to efficiently infect target cells and allow this infection to occur over a longer period of time after binding.

gp340 secreted from salivary glands is known as SAG (19, 20). Oral mucosal cells do not express cell surface gp340, but the oral cavity has high levels (1–10 µg/ml) of soluble gp340. SAG inhibits HIV infection by binding to Env. This difference in promotion vs inhibition of infection by cell-associated vs soluble gp340 is a property that is similarly observed for CD4, another HIV Env-binding molecule with protein-protein interaction. Genital tract secretions have very low levels of soluble gp340 (unmeasurable in 80% of samples; D. Malamud, unpublished observations). The disparity between high levels of soluble gp340 and inhibition of HIV infection in the oral cavity in contrast to the genital tract, where there are high levels of cell-associated enhancing gp340 and little HIV-inhibiting soluble gp340, may account for the differences in transmission between these two locations.

Immunohistochemical staining of surgically excised tissue with gp340-specific mAbs demonstrated expression on both vaginal and cervical epithelial cells. For vaginal sections, the expression was seen diffusely on the stratified squamous layer. This would be a relevant expression site for HIV-1 infection because the genital mucosa and epithelial cell layer act as the principle barrier separating HIV-1 from its target DCs and T cells. Cervical staining for gp340 was strongest along the basal membrane of the columnar epithelial cells but was also present along the apical region of these cells. The polar expression is noteworthy because of described functions of another mammalian homolog of gp340. The rabbit gene Hensin was shown to be important in maintaining and switching polarity in epithelial-derived cells (46, 47, 48). It is unclear whether gp340 has a similar function, but it is intriguing to consider the possible impact of a polarity-switching protein that binds HIV-1 in the genital epithelial barrier. This process, known as trans-cytosis, has been described for cells derived from the intestinal and genital tracts (16, 17). We demonstrate that cells derived from these tracts express gp340 and that this expression of gp340 results in their ability to transinfect. If human gp340 shares the polarity reversal function expressed by rabbit gp340 (Hensin), such a protein would present an interesting means of infection in the absence of breaks in the female mucosal barrier that expose HIV directly to its targets of infection.

An important next series of studies will need to confirm whether gp340 plays a role in the in vivo mucosal transmission of HIV. Such studies are extremely important given the recent suggestions for a lack of a physiological role for the most studied of the HIV accessory protein, DC-SIGN. Macaque models present the most viable means of measuring gp340 significance. However, the macaque gp340 gene has only recently been identified and not yet cloned (49). Cloning and testing the ability of macaque gp340 to bind and transmit HIV-1, HIV-2 or SIV will be a necessary step preceding the use of macaques. In addition, inhibitors that function in the genital tract environment of the interaction of gp340 with HIV (or SIV) Env in vivo will need to be developed.

Our results identify a protein, gp340, expressed on vaginal and cervical epithelium that binds HIV Env with high affinity through a specific protein-protein interaction. Cells that express gp340 are able to mediate transinfection of HIV and increase both the infectious titer and half-life of the virus. This enhancement of infectivity is dependent on gp340 interaction with HIV Env. Taken together, our observations provide an interesting potential mechanism by which otherwise subinfectious amounts of HIV-1 actively bind gp340 and are presented to the underlying target cells in the genital mucosa during genital tract contact with HIV. More studies will be necessary to understand the role that gp340 might play in vivo, but the existence of such a complex interaction in such a relevant compartment points to an important role in early HIV-1 infection in females. Therapeutics directed at blocking the interaction between Env and gp340 could become a potent and specific microbicide.


   Acknowledgments
 
The pDEST-DMBT1 plasmid was provided by Jan Mollenhauer. Tissue samples were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute. Other investigators may have received specimens from the same subjects. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health; HIV-1 MN Env (15-mer) Peptide Complete Set from the Division of AIDS, National Institute of Allergy and Infectious Diseases; and IL-2 from Hoffmann-La Roche.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI50484, DE14825, and AI060505. The Cooperative Human Tissue Network is funded by the National Cancer Institute. Back

2 Current address: Department of Basic Sciences, College of Dentistry, New York University, 345 East 24th Street, Room 904 S, New York, NY 10010. Back

3 Address correspondence and reprint requests to Dr. Drew Weissman, University of Pennsylvania, 522B Johnson Pavilion, Philadelphia, PA 19104. E-mail address: dreww{at}mail.med.upenn.edu Back

4 Abbreviations used in this paper: LC, Langerhans cell; DC, dendritic cell; Env, envelope protein; DC-SIGN, DC-specific intercellular adhesion molecule-grabbing integrin; MR, mannose receptor; SAG, salivary agglutinin; PHA-blasts, PHA- and IL-2-stimulated PBMCs; TCID50, 50% tissue culture-infective dose; STD, sexually transmitted disease; SRCR, scavenger receptor cysteine-rich. Back

Received for publication August 17, 2006. Accepted for publication June 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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