HIV Envelope Binding by Macrophage-Expressed Gp340 Promotes HIV-1 Infection.

Abstract

The scavenger receptor cysteine-rich protein gp340 functions as part of the host innate immune defense system at mucosal surfaces. In the genital tract, its expression by cervical and vaginal epithelial cells promotes HIV trans-infection and may play a role in sexual transmission. Gp340 is an alternatively spliced product of the deleted in malignant brain tumors 1 (DMBT1) gene. In addition to its innate immune system activity, DMBT1 demonstrates instability in multiple types of cancer and plays a role in epithelial cell differentiation. We demonstrate that monocyte derived macrophages express gp340 and HIV-1 infection is decreased when envelope cannot bind it. Inhibition of infection occurred at the level of fusion of M-, T-, and dual-tropic envelopes. Additional HIV-1 envelope binding molecules, such as dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN), mannose binding lectin, and heparan sulfate, enhance the efficiency of infection of the cells that express them by increasing the local concentration of infectious virus. Our data suggest that gp340, which is expressed by macrophages in vivo, may function to enhance infection in much the same manner. Its expression on tissue macrophages and epithelial cells suggests important new opportunities for HIV-1 pathogenesis investigation and therapy.

Introduction

The identification of the receptors responsible for HIV fusion and infection was followed by the identification of cell surface receptors that, while not required for fusion or infection, promote the infection of expressing cells or enhance the transfer of virus to other cells (trans-infection). These latter molecules do not alter HIV envelope protein (Env) structure, but appear to function, in part, by concentrating and stabilizing HIV. Like CD4, CCR5, and CXCR4, the main coreceptors for HIV infection, many of these “enhancer” molecules are members of the immune system and function in innate and adaptive immunity (reviewed in (1)). The best example of this family of HIV receptors is dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN), which functions in the immune system by binding naïve T cells expressing ICAM-3 to aid in the sampling of MHC-peptide complexes and in the accumulation of pathogens by antigen presenting cells (APC) (2-4). Monocyte derived dendritic cell (DC) expressed DC-SIGN binds HIV envelope through high-mannose carbohydrates (5, 6) and increases the efficiency of infection (3, 7). Other immune molecules that bind HIV-1 Env have been described and include the mannose receptor (MR), which is expressed by DC and macrophages and binds pathogens to aid in antigen presentation (8, 9), syndecans (10, 11), heparan sulfate (12-14), galactosyl ceramide (GalCer) (15, 16), and potentially others (17).

Several components of human saliva have been shown to inhibit HIV infection in vitro (18, 19). One of these, SAG, was identified as an alternatively spliced derivative of the DMBT1 gene, a presumed tumor suppressor (20, 21) and modulator of epithelial cell differentiation (22). A membrane bound version of this molecule, gp340, has been identified on macrophages in vivo (23) and on genital tract epithelial cells (24). Gp340 contains multiple scavenger receptor cysteine rich (SRCR) domains, and acts as an opsonin receptor for pathogens including multiple types of bacteria and surfactant protein A (25) and D (26). SAG/gp340 contributes to innate immunity by agglutinating bacteria and promoting adherence to oral surfaces, thus regulating the composition of the pellicle flora (20, 27-29). Bacterial agglutination may aid in the clearance and immune presentation of pathogens (30), particularly if SAG/gp340 shares the ability of lung derived soluble gp340 to induce chemokinesis in local macrophages (25). Gp340 expressed by genital tract epithelial cells binds HIV and promotes infection of target cells (24). In this report, we demonstrate that macrophage cell surface expressed gp340 promotes infection by HIV. The identification of gp340 as a cell associated promoter of HIV infection adds to an increasing list of immune molecules whose functions have been usurped by HIV to promote infection.

Materials and Methods

Cells and viruses

PBMC were collected from the blood of seronegative donors through an Institutional Review Board approved protocol. Monocyte derived macrophages (MDM) were prepared as previously described (31) in DMEM (Mediatech, Herndon, VA) supplemented with 10% FBS (HyClone, Logan, Utah) and 2mM glutamine (Invitrogen, Carlsbad, CA) (complete medium). M-CSF (2 ng/ml), GM-CSF (10 ng/ml) (R&D Systems, Minneapolis, MN), or no cytokines were added during MDM generation in preliminary experiments. Similar results were obtained with each type of MDM preparation in flow cytometric analysis of gp340 expression, and M-CSF was used for all experiments reported in this study. 293T, U937, A301, and SupT1 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in complete medium. HIV-1 strains Ba-L, JR-FL, UGO24, N7, and 89.6 were obtained from the Center for AIDS Research, University of Pennsylvania (Philadelphia, PA). The pNL4-3 backbone HIV plasmid with the luciferase gene in place of nef and lacking Env, and plasmids encoding JR-FL, Ba-L, ADA, UGO24 and 89.6 Env were kindly supplied by Robert W. Doms (University of Pennsylvania). Co-transfection of plasmids encoding the indicated Env and the backbone HIV-1 plasmid into 293T cells was used to prepare Env pseudotyped luciferase reporter viruses as previously described except that FuGene 6 Transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) was used for the transfections (32). Recombinant vaccinia virus vP11T7_gene1 (expression vector for T7 RNA polymerase), vSIMB_E:L (SP6 RNA polymerase under control of a synthetic vaccinia virus early:late promoter), and reporter plasmid containing the luciferase gene under control of the SP6 promoter were the kind gift of Stuart N. Isaacs (University of Pennsylvania) (32).

Antibodies and peptides

Anti-human gp340 antibodies 116 and BR-55 both murine mAb that recognize the Lewis-Y antigen, 143 mAb, GT199 mAb, DAPA (murine polyclonal), and 1527 (rabbit polyclonal) were used (24, 33). Anti-human gp340 antibody H12 (mouse monoclonal) was the kind gift of J. Mollenhauer (34). Anti-human gp340 antibodies m213-06, m213-01 (mouse monoclonals) and R6499 (rabbit polyclonal) were the kind gift of U. Holmskov (23). Anti-CD4 mAb leu3a was obtained from Becton-Dickenson Biosciences (Lexington, KY). FITC conjugated anti-mouse IgG and anti-rabbit IgG and peroxidase labeled goat anti-rabbit IgG were purchased from Sigma Chemical Co. (St. Louis, MO). Peptides 6284, CTRPNYNKRKRIHIG, and scrambled 6284, RCIHNRTIKGPYNKR, were used (24).

FACS analysis

MDM were detached from plates with PBS + 5 mM EDTA and stained with the indicated primary antibodies in staining buffer (PBS, 1% FBS, 4 mM CaCl₂, 0.02% NaN₃) for 30 min on ice. Cells were washed with staining buffer and then stained for 30 min with FITC conjugated anti-mouse or anti-rabbit IgG secondary antibody. Cells were analyzed on a FACScan (Becton-Dickenson) flow cytometer and analyzed with CELLQuest software (Becton-Dickenson).

Surface Plasmon Resonance analysis

Biacore analysis was performed on a Biacore 3000™ instrument (Biacore, Inc., Piscataway, NJ) using a CM5 sensor chip. The data were evaluated using BIAevaluation 3.0 software (Biacore, Inc.). The chip surface was activated by injecting 35 μl of a 1:1 mixture of 0.05M N-hydroxysuccinimide and 0.2M N-ethyl-N′- (dimethylaminoprophyl) carbodiimide at 5 μl/min. Purified gp340 (5 μg/ml in 10mM NaOAc, pH=4) was immobilized to a density of approximately 2000 response units (RU) and blocked with 35 μl of 1 M ethanolamine, pH=8.5. OVA was immobilized on a second chip and used as a background control. Running buffer was PBS pH=7.4 + 0.005% Tween p-20, 0.1% soluble carboxymethyl dextran, and 5 mM CaCl₂. To evaluate inhibition of binding of Env to gp340, anti-gp340 antibody was injected over surface bound gp340 at the indicated concentrations at a flow rate of 25 μl/min. After a 2 min injection of antibody, the surface was washed for 2 min in running buffer prior to injection of 50 nM Ba-L gp120 (Robert Doms) at a flow rate of 25 μl/min and measurements were taken every second.

Gp340 binding ELISA

ELISA plates were coated with gp340 purified from saliva (33) at 1 μg/ml and then blocked with BSA. Antibodies were added at 10 μg/ml for 30 min followed by the addition of HIV gp120 (IIIB) (obtained from Robert Doms) (1 μg/ml). After 30 min, plates were washed and bound Env was detected with a rabbit anti-IIIB antiserum (Robert Doms) and peroxidase labeled anti-rabbit IgG.

Infection assays

MDM were pre-treated with antibodies for 1 hour at 37°C and then incubated with the indicated viruses overnight in the continued presence of antibody (10 μg/ml). Cells were then washed 3 times to remove input virus and cultured in fresh media containing the same antibody. Cells were fed every 3 to 4 days with fresh media containing antibody. At indicated time points, supernatants were collected for analysis of p24 Gag protein content using the Coulter p24 antigen assay research kit (Beckman-Coulter, Inc., Fullerton, CA). For pseudovirus infections, the virus was not removed until the 3rd day when the cells were washed, lysed in luciferase lysis buffer, and assayed for luciferase activity using the Promega luciferase kit (Promega Corporation, Madison, WI). For cell line infections, U937, A301, or SupT1 cells were transfected with pDest12.2-DMBT1 (35) or control pDest12.2 plasmid using Fugene 6 Transfection Reagent followed 24 h later by incubation with or without peptides (10 μg/ml) and infection with IIIB virus. Peptides were replaced with fresh medium on day 3. 7 days later, p24 Gag content was measured by ELISA.

Fusion assays

Fusion assays were performed as previously described except that FuGene 6 was used for transfections (32).

Statistical analysis

Luciferase and p24 Gag protein quantification was performed on samples run in duplicate or triplicate for each condition and each sample was analyzed in duplicate. Standard error of the mean was calculated with Microsoft Excel software. The student's t-test was used with 2-tails and equal variance.

Results

Analysis of HIV-1 Env-gp340 interaction

HIV Env binding to gp340 has previously been demonstrated by indirect methods including the ability to strip Env from T-tropic strains of HIV-1 (36, 37) and by direct methods including surface plasmon resonance analyses (33). Multiple antibodies have been developed that recognize gp340. To determine whether these antibodies were capable of inhibiting the binding of HIV gp-120 to gp340, a direct binding ELISA with gp340 coated plates and Env binding measured with an Env-specific Ab was used. Abs 143, 116, BR-55, DAPA, and H12 inhibited Env binding to gp340 while GT199 and m213-06 did not (Figure 1A). To confirm these data, biosensor chip bound gp340 was first treated with increasing concentrations of anti-gp340 mAb 143 followed by HIV Ba-L Env. Gp-120 binding was substantially blocked by mAb 143 in a concentration dependent manner (Figure 1B). Similar data were obtained with mAb 116, which recognizes the Lewis Y epitope (33). Thus, HIV-1 envelope binds immobilized gp340, and multiple anti-gp340 antibodies can inhibit this binding.

Figure 1.

Anti-gp340 antibodies inhibit Env binding to gp340. A) Gp340 purified from saliva was bound to ELISA plates. The indicated anti-gp340 Abs were added at 10 μg/ml for 30 min followed by addition of HIV IIIB Env with antibodies for an additional 30 min. mIg is purified non-immune murine Ig, mIgG1 is a mAb that does not bind gp340. Plates were washed and Env binding detected with specific antisera. Data is given as the optical density of the ELISA. No Env indicates background staining in the absence of added Env or antibody. Medium indicates no gp340-specific antibody added. Triplicate analyses with SEM are shown. P-values less than 0.001 were observed in comparing Abs 143, 116, BR-55, DAPA, and H12 with their appropriate control. B) Gp340 was coupled to carboxymethyl dextran attached to a glass supported gold surface on the sensor chip of a Biacore 3000® by direct amine coupling. Anti-gp340 antibody 143 was injected over gp340 bound sensor chips at the indicated concentrations and then washed until initial fall off of binding (2 min). 50 nM recombinant Ba-L gp120 was then injected and binding was measured at 1-second intervals. Binding values of gp120 free buffer injected over gp340 plus antibody-loaded chips were subtracted from data shown. Shown is a representative experiment of three (A) or two (B) performed.

Gp340 expression has been observed on alveolar macrophages and perivascular macrophages by immunohistochemical analysis of fixed tissue (22, 26). We used flow cytometric analysis with specific antibodies to verify gp340 expression on MDM. Anti-gp340 antibodies H12, 116, and DAPA recognized surface gp340 on MDM (Figure 2). Similar results were obtained with gp340 specific antibodies 143, m213-06, GT199, and R6499. We previously demonstrated that cell surface gp340 on cell lines binds HIV Env and promotes trans-infection, which can be inhibited by a peptide derived from the base of the V3 loop, 6284 (CTRPNYNKRKRIHIG) (24). Similar studies on MDM expressing gp340 did not reveal a significant reduction in Env binding with the addition of peptide 6284. This is likely due to the observation that macrophages express multiple HIV binding molecules including CD4, MR (38), syndecan (39), and DC-SIGN (40) in addition to gp340.

Figure 2.

Gp340 is expressed on MDM. Monocytes were isolated from PBMC and cultured for 7 days in the presence of M-CSF to allow differentiation into macrophages. Cells were detached and stained with the indicated primary antibody followed by FITC conjugated anti-mouse IgG. Negative control received an IgG1 isotype control antibody followed by FITC-anti-mouse IgG. Similar staining was observed for mouse Ig, the appropriate control for DAPA. Flow cytometric histograms shown are gated on large (forward-side scatter) cells, which were typically 90-97% of the live cells in the culture. Data are representative of greater than three repetitions for each antibody.

Macrophages express low levels of CD4, CCR5, and CXCR4 compared to CD4⁺ T cells (41) and are easily infected by M-tropic strains but poorly infected by T-tropic strains of HIV-1 (31, 42). To determine the role of gp340 in HIV-1 macrophage infection, MDM were pre-treated with pre-immune Ig, isotype control mAb, or anti-gp340 antibody prior to infection with the primary CCR5 using HIV-1 isolate N7 (10 ng/well). BR-55 (binds Lewis-Y) mAb inhibited infection of MDM (Figure 3A). Specific or control antibody was added every 3 days when fresh medium was added throughout the infection. As preliminary experiments demonstrated long-lived inhibition of infection when antibody was added throughout infection, subsequent experiments were terminated on day 7-10 post infection. Gp340-specific antibody inhibited in a dose dependent manner but never inhibited more than 90% at the highest concentrations (Figure 3B). The polyclonal anti-gp340 antibody DAPA strongly, but incompletely, inhibited infection of MDM with the dual tropic 89.6 virus (Figure 3C). MDM, pre and post treated with an HIV blocking anti-CD4 mAb (Leu-3a) had no infection while DAPA antibody strongly but incompletely inhibited infection with UGO24 virus, a primary T cell tropic virus that replicates in MDM (Figure 3D). Thus, the addition of an antibody that inhibits HIV Env binding to gp340 reduced infection of macrophages. The addition of mAb GT199, that does not inhibit Env binding to gp340 (Figure 1A) but binds gp340, did not inhibit HIV infection. Our data demonstrate that certain antibodies to gp340 that block Env binding, inhibit the infection of MDM by M-, dual-, and T-tropic viruses (Figure 3). Similar levels of inhibition were observed with the V3 loop derived peptide 6284 that has been demonstrated to inhibit Env binding to gp340 and trans-infection (24). These data suggest that gp340 on the surface of MDM is used by HIV to promote infection.

Figure 3.

Gp340-specific antibodies added before infection and present throughout reduce HIV infection of MDM. MDM were pre-treated with the indicated Ab (10 μg/ml) for 1 hour and then infected with HIV-1 (10 ng p24 Gag protein/well). After an overnight incubation, cells were washed 3 times and cultured in media with antibody added every 3 to 4 days. Supernatants were collected at the indicated time points and assayed for p24 Gag protein content by ELISA. HIV-1 viruses and gp340-specific antibodies used: A) N7 primary isolate and BR-55 mAb; B) Ba-L strain and BR-55 mAb, assayed on day 7; C) 89.6 strain and DAPA polyclonal Ab; and D) UGO24 strain and DAPA Ab, assayed on day 7. Error bars are SEM. Data are representative of 3-8 experiments. P-values were less than 0.001 in comparing gp340 Ab to control in A, C, and D.

While the use of specific antibodies and Env derived peptide inhibitors of Env binding to gp340 are consistent with the hypothesis that gp340 on macrophages promotes infection, we used a third method to definitively prove this. Cell lines capable of supporting HIV infection, including ones of macrophage lineage, were transiently transfected with a gp340 expression plasmid that results in surface gp340 expression (24). Infection of U937, A301, and SupT1 cells was enhanced when cells expressed gp340, compared to control vector transfected cells, and this enhancement in infection was overcome with the addition of the inhibitory V3 peptide 6284 (Figure 4). The lower levels of enhancement 2.5 to 4-fold of infection by gp340 compared to the near 90% inhibition of infection observed by its blocking likely reflects incomplete expression observed with transient transfection.

Figure 4.

Transient delivery of gp340 enhances infection in cell lines that support HIV infection. U937, A301, and SupT1 cells were transfected with pDest12.2 (Control) or pDest12.2-Dmbt1 (gp340) plasmids followed 24 h later with the addition of peptide 6284 or a scrambled version and then infected with HIV IIIB (10 ng/well). 7 days later, p24 Gag protein content was measured. Error bars are SEM and data is representative of three experiments. P-values were less than 0.002 in comparing 6284 peptide to scrambled peptide for all cell lines.

To determine where in the HIV-1 viral life cycle gp340 promotes infection, single replicative cycle pseudovirus infections were used. Pseudoviruses were constructed by cotransfection of a backbone plasmid lacking the Env gene and containing a reporter gene, firefly luciferase, and a second plasmid encoding HIV Env. The encapsulated genomic RNA does not encode Env, so the pseudovirus cannot produce infective progeny. Luciferase activity is dependent on the amount of virus used to infect the cells (Figure 5A). Both 116 mAb and DAPA polyclonal Ab strongly but incompletely inhibited pseudovirus infection and this inhibition was not overcome by increasing input virus, suggesting that at the amounts of virus used, the gp340 effect was not saturable (Figure 5B). Data are shown as the percent inhibition comparing the gp340-specific Ab to its control (isotype mAb or preimmune Ig). Raw values from infection in the absence of antibody are shown in Figure 5A.

Figure 5.

Anti-gp340 antibodies block infectivity of single replicative cycle virus. Cultured macrophages were pre-treated with no Ab (A), 116 mAb (10 μg/ml), DAPA Ab (10 μg/ml) and their controls (10 μg/ml) (B) and then infected with Ba-L envelope pseudotyped luciferase reporter virus at the indicated p24 concentration/well. After 3 days, cell lysates were analyzed for luciferase activity. Data in (B) is presented as the percent inhibition of infection comparing isotype control mAb to 116 and preimmune Ig to DAPA. Infections were done in duplicate and analyzed in duplicate and error bars are the SEM for the quadruplicate measurements. Data are representative of three experiments.

Viral replication proceeds through multiple defined steps. Cell surface infectivity enhancing molecules that promote HIV infection of cells do so through multiple mechanisms that affect specific steps in viral replication. This includes increasing local concentrations of infectious virus (DC-SIGN, DC-SIGNR) (3, 43, 44), delivering activation signals to target cells through chemokine receptors (45) and promoting fusion through interactions with Env (heparin sulfate) (46). Fusion assays were performed to determine whether gp340 binding to Env enhanced infection at this level. We attempted to use the gp340 expression plasmid as a source of cell surface gp340 in standard fusion assays employing quail cells (QT1) (47), but these cells did not express cell surface gp340 after transfection. We then adapted the fusion assay to use macrophages expressing endogenous gp340, CD4, and coreceptor. 293T cells expressing JR-FL or 89.6 Env and containing a plasmid encoding luciferase under an SP6 promoter were co-cultured with primary MDM containing SP6 RNA polymerase delivered by vaccinia virus. Luciferase activity was directly related to the amount of fusion that occurred between the Env-expressing 293T cells and the MDM expressing gp340, CD4, and CCR5 or CXCR4. Pre-incubation of MDM with anti-gp340 antibodies DAPA and 116 or V3 peptide 6284 inhibited fusion with JR-FL (Figure 6A) and 89.6 (Figure 6B) Env expressing fusion partners. Fusion to 293T cells expressing the HXB2 Env was also inhibited by anti-gp340 Abs, but the level of fusion was typically very low.

Figure 6.

Inhibition of Env binding to gp340 diminishes fusion of HIV-1 Env expressing 293T cells to MDM. MDM were infected with recombinant vaccinia virus expressing the SP6 RNA polymerase. 293T cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and transfected with T7-driven Ba-L Env (A) or 89-6 Env (B) plasmids and an SP6-promoter driven luciferase reporter plasmid. MDM were pretreated with the indicated antibody (10 μg/ml) or peptide (10 μg/ml) for 1 hour and then cocultured with 293T cells. Cell lysates were assayed for luciferase production after 7 hours. Error bars are SEM for triplicate cultures. Data is representative of at least 3 repetitions. P-values were less than 0.002 in comparing gp340 Abs or 6284 peptide to their controls.

Discussion

Gp340 functions in the innate immune system and acts as an epithelial cell differentiation antigen. Deletions in its gene are associated with certain types of cancer. Gp340 expression by genital tract epithelial cells promotes transfer of virus to target cells (24). We now demonstrate that macrophage expression of gp340 enhances HIV infection through its ability to bind HIV Env and promote fusion. This likely occurs by increasing local concentrations of virus at the cell surface similar to DC-SIGN (43). The blocking of Env binding to cell surface expressed gp340 by specific antibodies or an inhibitory peptide derived from gp120 reduces infection of macrophages by up to 90%. To clearly distinguish that gp340 is responsible for the enhancement, it was delivered to cell lines capable of supporting HIV replication by transient transfection. A 2.5 to 4-fold increase in infection was observed. The use of transient transfection, while only delivering gp340 to a fraction of the cells, avoids the selection of clones that may differ in their ability to support HIV replication due to other factors. Finally, we identify that gp340 enhances infection by increasing fusion.

The observation that gp340 is a cell surface expressed HIV-1 binding protein on macrophage lineage cells in vitro (Figure 2) and in vivo (26, 35, 48) suggests a wide range of possible effects this molecule could have during HIV infection. Originally identified as an antimicrobial molecule in saliva and cloned as a brain tumor suppressor, it is present on the surface of some astrocytes and microglia (23) and, thus, could play a role in HIV pathology of the brain. The molecule serves multiple functions depending on the cell type expressing it. These functions include an observed alteration of the gene in malignant transformation (26, 34), a role in epithelial cell differentiation (22), the ability to activate alveolar macrophages (25), as a link between the innate and adaptive immune system (22, 23), and as an anti-bacterial and anti-viral molecule in the oral cavity (36). We conclude that gp340 promotes infection of macrophages based on three types of data. First, antibodies that disrupt gp340-Env binding inhibit infection, while antibodies that bind gp340 but do not inhibit Env binding, do not block infection. Second, a peptide derived from HIV Env that blocks Env binding to gp340, but not its scrambled control, blocks infection. Third, delivery of gp340 to cell lines that support HIV infection increases their infectability. The epitope specificity of most of the gp340 specific blocking antibodies has not yet been determined and the observation that multiple antibodies inhibit Env-gp340 interaction suggests that they recognize epitopes associated with Env binding, or that certain antibody bindings disturb a global structure required for Env binding. The former is supported by data demonstrating that multiple SRCR domains in gp340 independently bind Streptococcus mutans (49), due to the presence of a conserved region present in each of 13 SRCR regions, which are all highly conserved. The likely mechanism of gp340 action is concentration of virus at the site of fusion. Preliminary studies using Biacore analysis suggest that Env binding to gp340 does not increase the affinity of Env binding to CD4, but we cannot exclude that gp340 alters exposure of other regions of Env required for fusion and entry. It is unknown whether gp340 signals cells in any of its expressed forms, although it has been reported to induce chemokinesis in alveolar macrophages (25), suggesting some ability to signal. Further studies to delineate the full function of this molecule and the results of its binding to HIV Env are needed.

We have demonstrated that multiple human vaginal and cervical epithelial cell lines expressed gp340 and use it to promoted HIV infection of target cells in an in vitro model of genital tract transmission (24). The presence of an HIV binding protein that promotes infection on genital tract mucosa presents a potential new mechanism for mucosal transmission. The rhesus monkey homologue of gp340 is expressed on endometrial tissue and is increased by progesterone treatment (50). It is also expressed by rhesus genital tract tissue (DW, unpublished observations) offering a model system to test its role in transmission. The rabbit epithelial polarity reversal gene hensin is recognized by anti-human gp340 antibodies and was shown to be a differentially spliced DMBT1 homologue (51). Thus, if gp340 is present on genital tract epithelial cells and acts as a polarity reversal promoter in these cells, it might provide a mechanism for HIV-1 to traverse intact epithelia during sexual transmission, i.e. transcytosis.

Our data demonstrate that gp340 binds HIV-1 Env with high affinity and inhibition of this binding diminishes infection of macrophages, demonstrating that cell surface expressed gp340 enhances infection. The mechanism of enhancement of infectivity is an enhancement of viral fusion likely through increasing the local concentration of HIV at the surface of the cell. We cannot discount an ability of gp340 to alter HIV Env such that fusion is more easily accomplished, although we observed that its binding to Env does not increase Env's binding to CD4. Understanding the multifaceted interaction between HIV-1 and host cells is of enormous importance in the understanding of HIV infection of target cells, HIV-induced pathology, and in the design of new treatment strategies to disrupt such interactions and prevent infection. The identification of gp340 as another member of the immune system whose role has been usurped by HIV presents a new direction of study in the effort to understand HIV immunopathogenesis.

Abbreviations used in this paper

DMBT1

deleted in malignant brain tumors 1

DC-SIGN

dendritic cell specific ICAM-3 grabbing non-integrin

Env

envelope protein

DC

dendritic cells

SRCR

scavenger receptor cysteine rich

MDM

monocyte derived macrophages