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Published as doi: 10.1096/fj.07-9443com.
 (The FASEB Journal. 2008;22:1055-1064.)
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(The FASEB Journal. 2008;22:1055-1064.)
© 2008 FASEB

An intercellular adhesion molecule-3 (ICAM-3) -grabbing nonintegrin (DC-SIGN) efficiently blocks HIV viral budding

Qiuwei Wang and Shen Pang1

University of California, Los Angeles, School of Dentistry, Los Angeles, California, USA

1Correspondence: UCLA School of Dentistry, 10833 Le Conte Ave., Los Angeles, CA 90095, USA. E-mail: spang{at}ucla.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Efficient inhibition of the HIV infection life cycle at the stages of viral infection, reverse transcription, and post-translational processing has been extensively studied. However, efficient inhibition of HIV assembly and budding has not been reported. Here, we report that dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3) -grabbing nonintegrin (DC-SIGN) and its related protein, DC-SIGNR, effectively block HIV budding from infected cells. Cotransfection of DC-SIGN or DC-SIGNR with HIV demonstrated 95–99.5% inhibition of viral production from host cells. DC-SIGN or DC-SIGNR can also effectively inhibit 90–95% of HIV generation from infected cells. DC-SIGN efficiently reduces the amount of gp120 present on the cell plasma membrane, and completely strips off gp120 from the virions produced by the host cells, suggesting that blockage of HIV budding is due to internalization of gp120 by DC-SIGN.—Wang, Q., Pang, S. An intercellular adhesion molecule-3 (ICAM-3) -grabbing nonintegrin (DC-SIGN) efficiently blocks HIV viral budding.


Key Words: gp120 • viral production


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
METHODS TO INTERRUPT THE HIV life cycle at the stages of viral entry, reverse transcription, integration, and post-translational processing have been well documented. Anti-HIV drugs designed to attack viral entry (e.g., T-20, enfuvirtide, Fuzeon), reverse transcription (AZT), and post-translational protein modification (protease inhibitors such as indinavir, ritonavir, and nelfinavir) have been used clinically. However, blockage at these stages has limitations, in that once the virus has passed through these stages, treatment fails. Therefore, approaches are necessary to target other stages in the viral life cycle, including viral assembly and budding.

Our approach was to find a protein to interrupt viral assembly and/or budding. In this study, we focused on a protein that is expressed in dendritic cells (DCs). The normal function of DCs is to present pathogen-derived antigens to T-lymphocytes. DCs scavenge pathogens before migrating to the lymph nodes, where they present processed antigens to resting T-cells and initiate an adaptive immune response (1 2) . It is proposed that HIV can be internalized into DCs by either DC engulfment or receptor-mediated viral fusion, because many DCs express low levels of CD4 and CCR5 or CXCR4 coreceptors (3 4 5 6 7) and several lectins (8) . The presence of a C-type (calcium-dependent) lectin, dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3) -grabbing nonintegrin (DC-SIGN), in some subsets of DCs may also help the virus to attach to and fuse into DCs (9) . However, there are studies demonstrating that although DCs can be infected, viral generation from DCs is much lower than from infected CD4-positive T-lymphocytes (10 11 12 13 14 15 16 17) . HIV production from infected DCs in vivo is often 10- to 100-fold lower (17) than from CD4-positive T-lymphocytes, suggesting that there is a specific mechanism of DCs that can efficiently block viral release from the infected cells.

DC-SIGN, which is specifically expressed on certain DC subsets located in mucosal tissues, has been proposed to play an essential role in transmission of HIV from DCs to T-cells (9 , 18 , 19) . DC-SIGN binds the HIV glycoprotein, gp120, with a high affinity, and transmits the virus to T-cells (9 , 18 , 19) . In addition to playing roles in viral transmission, this molecule may also play other roles. On the basis of the low production of HIV from DCs (10 11 12 13 14 15 16 17) , we hypothesized that DC-SIGN might block viral generation in DCs. To investigate the role of DC-SIGN in viral production, gene transfection was used to express this protein in 293T and HeLa-CD4 cells, into which X4 or R5 HIV clones were introduced by either infection or transfection. To accurately quantify viral infection in this study, we used HIV clones that express the enhanced green fluorescent protein (EGFP) (20 21 22) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and maintenance
SV40-transformed human embryonic kidney (HEK) 293T cells, CEM CD4-positive T-lymphocytes, and CD4-modified HeLa-CD4 cells have been described previously (18 19 20) . 293-CCR5/CD4 is a human embryonic kidney (HEK 293) cell line transformed to express CCR5 and CD4 (23) . 293-DC-SIGN is a DC-SIGN gene-expressing 293 cell line obtained from Dr. Benhur Lee (UCLA). All cell lines were maintained in RPMI medium plus 10% fetal bovine serum (FBS).

Plasmids
Both DC-SIGN and the DC-SIGN-related sequence (DC-SIGNR) (24) were obtained from the National Institutes of Health AIDS Reagent Program. Plasmids containing HIV genomes have been described previously (20 21 22) . All plasmids were purified by cesium chloride gradient ultracentrifugation.

EGFP-expressing HIV-1 clones
HIVNL4–3-EGFP-Env(+) is derived from HIVNL4–3, with part of its nef gene replaced by EGFP. Two EGFP-expressing R5 clones, HIVJRCSF-EGFP (clade B, R5) and HIV94UG114-EGFP (clade D, R5), were constructed, using an approach similar to that for constructing HIVNL4–3-EGFP-Env(+) (22) .

Cotransfection of 293T cells with DC-SIGN or DC-SIGNR and HIV plasmid clones
293T cells were plated into 12-well plates at 105/well 24 h prior to transfection. An appropriate amount of DC-SIGN, DC-SIGNR, or control plasmid (0.03 to 3.0 µg) was mixed with 3.0 µg of plasmid containing the HIVNL4–3-EGFP, HIV94UG114-EGFP, or HIVJRCSF viral genomes. Calcium precipitation was used to cotransfect plasmids into cells. At 16 h post-transfection, cell cultures were washed, and 1 ml of new medium was added to each well.

Viral infection
We plated 2 x 104 cells into 24-well plates 24 h prior to infection. To infect HeLa-CD4 or 293-CCR5/CD4 cells, 30 µl of medium from virus-producing cell cultures was added to the cultures to a 0.5-ml total volume. The infected cell cultures were washed 16 h postinfection, and growth medium (RPMI+10% FBS) was replaced. Two wells were devoted to each infection.

Quantification of viral infection
At 2 days postinfection, the infected cells became EGFP-positive. By counting EGFP-positive cells, viral infectivity could be very precisely calculated. Since infected cells might grow more slowly than uninfected cells, EGFP-positive colonies containing one or several cells were counted as one infection unit. One infection unit should be derived from one originally infected cell; therefore, a colony was considered as one infection unit whether it contained only 1 or more than 10 cells. The number of infection units counted from one well was divided by 4 x 104 (at 24 h, the cells in 24-well plates may double their numbers from 2x104 to 4x104) to obtain the percentage of infected cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DC-SIGN and DC-SIGNR inhibit viral production in transfected cells
DC-SIGN or a gene that is highly homologous to it, known as DC-SIGNR, was cotransfected into 293T cells with a plasmid that contained an EGFP-expressing HIV genome. Three EGFP-expressing HIV clones, HIVNL4–3-EGFP-Env(+), HIVJRCSF-EGFP, and HIV94UG114-EGFP, were used. Insertion of EGFP into the viral genome does not alter viral tropism, as indicated by our previous studies (23) . In such cotransfections, the plasmid that carries a viral genome produces both viral RNA genomes and viral proteins, and the plasmid carrying the DC-SIGN gene produces the DC-SIGN protein. Plasmids carrying the influenza hemagglutinin (HA) membrane protein, the {theta}-defensin pseudogene that has a termination codon in its open reading frame, or no gene, used as a control. Transfection efficiency was determined by measuring percentages and intensities of green fluorescent light density in transfected cultures. DC-SIGN did not demonstrate significant inhibition by EGFP expression (Fig. 1 A), suggesting that after transfection, HIV gene expression was not blocked by DC-SIGN. At 2 days post-transfection, 30 µl of medium from each of the cotransfected 293T cell cultures was collected and used to infect either HeLa-CD4 (for X4 viral clones such as HIVNL4–3-EGFP-Env(+)) or 293-CCR5/CD4 cells (for R5 clones HIVJRCSF-EGFP or HIV94UG114-EGFP). The medium from DC-SIGN- or DC-SIGNR-cotransfected 293T cells demonstrated much lower infectivity than the controls. By counting EGFP-positive cells, it was found that DC-SIGN and DC-SIGNR-inhibited virus generation by 99–99.5% (Fig. 1B ). Similar experiments were also performed with cotransfected HeLa-CD4 cells, and the results were very similar to those seen with cotransfected 293T cells (Fig. 1C ).


Figure 1
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  		Figure 1. Blockage of HIV generation by DC-SIGN(R) in cotransfected cells. A) Transfection efficiency of 293T cells when infected by plasmid pNL4–3-EGFP-Env(+) or with plasmids containing DC-SIGN(R) or control plasmids, including the HA gene, the human {theta}-defensin pseudogene, which has a termination codon in its open reading frame, and the pCDNA3.1 plasmid with no gene insert (control). B) Infectivity of viral preparations collected from cotransfected 293T cultures. At 24 h post-transfection, 30 µl of medium collected from cotransfected cell cultures (A) was used to infect HeLa-CD4 cells. Two days postinfection, EGFP-positive colonies in HeLa-CD4 cell cultures were counted to evaluate viral infectivity. C) Infectivity of viral preparations collected from cotransfected HeLa-CD4 cultures. Methods were identical to those used to cotransfect 293T cells. The harvested HIV94UG114-EGFP samples from various treatments were assessed by infecting 293-CCR5/CD4 cells. D) Dosage studies of DC-SIGN. DC-SIGN (0.03 to 3.0 µg) with either HIV94UG114-EGFP or HIVNL4–3-EGFP-Env(+) was used to cotransfect 293T cells. Inhibitory effects were quantified by counting EGFP-positive cells, as described in A. E) p24 assay of DC-SIGN-cotransfected 293T cells. 293T cells were cotransfected with various doses of DC-SIGN and different HIV clones. Wild-type HIV clones NL4–3 and JRCSF were also assayed. Error bars indicate SD.

DC-SIGN has no significant effect on viral protein expression (Fig. 1A ). Therefore, DC-SIGN was postulated to be effective in blocking stages of the HIV life cycle that occur later than viral protein translation; i.e., viral assembly, or budding.

To determine the efficiency of DC-SIGN in blocking viral generation, dosage studies were carried out. The results demonstrated that inhibition by DC-SIGN was consistent with the doses of DC-SIGN plasmid used for cotransfection (Fig. 1D ). When 0.3 µg of DC-SIGN plasmid was used to cotransfect 293T cells with 3 µg of HIV plasmid, over 92% inhibition was observed, suggesting that DC-SIGN is a potent inhibitor. Because the DC-SIGN expression levels may not be linearly related to the amounts of DC-SIGN plasmid used for transfection, the cells that were transfected by 0.3 µg of DC-SIGN plasmid may not express exactly 1/10th of the amount of DC-SIGN protein as the cells transfected by 3 µg of DC-SIGN plasmid. Because of the limited resources in each cell, it is unlikely that every transfected DC-SIGN DNA molecule can be transcribed and then translated. Therefore, when 0.3 µg of DC-SIGN plasmid was used, we obtained 92% inhibition instead of 10% inhibition as compared with the cell cultures transcribed with 3 µg of DC-SIGN plasmid.

We assayed p24 Gag protein from isolated viral medium samples. DC-SIGN- (or DC-SIGNR-) cotransfected 293T cells demonstrated ~95% inhibition of p24 (Fig. 1E ), indicating that DC-SIGN can significantly reduce viral generation. DC-SIGN inhibition of wild-type HIV clones such as HIVNL4–3 and HIVJRCSF, which were not modified with the EGFP gene, demonstrated very similar results, as seen with p24 assays (Fig. 1E ), indicating that the EGFP gene is not involved in such inhibition.

Inhibition of HIV production by DC-SIGN in infected cells
In HIV-infected cells, HIV is integrated into the host genome. To determine whether DC-SIGN can also effectively inhibit viral production in cells that have been infected by HIV, 293T cells were infected with high doses of HIV pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). It was observed by fluorescent microscopy that over 95% of cells were infected, as demonstrated by the presence of EGFP-positive cells. The infected cells were seeded into 24-well cell culture plates 24 h prior to transfection with plasmids containing DC-SIGN or DC-SIGNR (hereafter DC-SIGN(R) will be designated DC-SIGN and DC-SIGNR collectively). Control plasmids carrying either the influenza HA gene or no gene were also used to transfect the HIV-infected cells. At 24 h post-transfection, virus-containing medium from each well of the transfected cell cultures was used to infect HeLa-CD4 or 293-CCR5/CD4 cells. The results demonstrated that DC-SIGN(R) was able to efficiently inhibit HIV production (Figs. 2 A, B). The results also further confirmed that DC-SIGN(R)-mediated inhibition occurs at late stages of the HIV life cycle, viral assembly and/or budding, because when DC-SIGN was used to transfect cells, HIV had completed the stages of entry, reverse transcription, integration, and viral gene transcription.


Figure 2
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  		Figure 2. Blockage of HIV generation by DC-SIGN in HIV-infected cells and HIV infection of DC-SIGN stably expressing cells. A) 293T cells infected by HIVNL4–3-EGFP-Env(+) or HIV94UG114-EGFP were transfected by DC-SIGN(R). At 24 h post-transfection, 100 µl of medium from each well was used to infect HeLa-CD4 or 293-CCR5/CD4 cells. B) Inhibition of HIV generation was assessed by p24 assays. C) Effect of DC-SIGN in DC-SIGN stably expressing 293 cells. HIVNL4–3-EGFP-Env(+) pseudotyped with VSV-G was used to infect either 293 or 293-DC-SIGN cells. At 3 h postinfection, infected cell cultures were washed three times to reduce background levels of HIV. Two days postinfection, 30 µl of medium collected from each well was used to infect HeLa-CD4 cells. Error bars indicate SD. D) Infection of Raji, Raji/DC-SIGN, NC-37, and NC-37/DC-SIGN by VSV-G-pseudotyped HIVNL4–3-EGFP-Env(+). Equal amounts (106 cells) of Raji/DC-SIGN, NC-37/DC-SIGN, and their parental DC-SIGN-negative cell lines, Raji and NC-37, were infected with VSV-G-pseudotyped HIVNL4–3-EGFP-Env(+). When an amount of virus with a p24 count of 200 ng was used to infect these cells, ~10% of cells was infected. Three hours postinfection, the cells were washed three times. E) Inhibition of HIV generation from Raji and NC-37 cells by DC-SIGN. Two days postinfection, virus generated from these four cell cultures, Raji, Raji/DC-SIGN, NC-37, and NC-37/DC-SIGN, was collected, and one-fifth was used for infection of HeLa-CD4 cells.

DC-SIGN is stably expressed in subsets of DCs (25) . It was of interest to determine whether expression of this protein is the key to low production of HIV in DC-SIGN-positive dendritic cells. The DC-SIGN stably expressing cell line, 293-DC-SIGN (a gift from Dr. B. Lee, University of California, Los Angeles), was used to investigate whether DC-SIGN can block HIV production in cells that stably express DC-SIGN. Because the 293-DC-SIGN cell line does not express CD4, HIV, which only has gp120 on its surface, cannot efficiently infect this cell line. Instead, a VSV-G-pseudotyped HIV clone, HIVNL4–3-EGFP, was used to infect the 293-DC-SIGN cell line and its control, 293 cells. HIVNL4–3-EGFP was prepared by cotransfecting this HIV plasmid clone into 293T cells with a plasmid that expresses VSV-G. These virus preparations showed high infectivity for both the 293 and 293-DC-SIGN cell lines (over 90% cells were EGFP-positive). Two days postinfection, the medium collected from the infected cell cultures was used to infect HeLa-CD4 cells. The results demonstrated that expression of DC-SIGN in the 293 cell line efficiently blocked viral production (Fig. 2C ). Therefore, stably expressed DC-SIGN can efficiently inhibit HIV production, similar to transfected DC-SIGN.

We also studied the effect of DC-SIGN on blocking of HIV production, using cell lines Raji, Raji/DC-SIGN, NC-37, and NC-37/DC-SIGN (25 26 27 28) . Previous studies demonstrated that Raji/DC-SIGN is very similar to B-THP-1/DC-SIGN, and both Raji/DC-SIGN and B-THP-1/DC-SIGN were extensively used for study of DC-SIGN-mediated HIV infection (9 , 25 26 27 28) . We used the VSV-G-pseudotyped HIV clone, HIVNL4–3-EGFP, to infect the Raji/DC-SIGN and NC-37/DC-SIGN cell lines and their DC-SIGN-negative parental cell lines. Infection levels of these two pairs of cell lines were very similar (Fig. 2D ). Two days postinfection, the virus generated from infected cell cultures was collected and used to infect HeLa-CD4 cells. The viral preparations from Raji/DC-SIGN and NC-37/DC-SIGN cell cultures demonstrated significantly lower infectivity than their parental cell lines, Raji and NC-37 (Fig. 2E ). Because some cells in Raji/DC-SIGN and NC-37/DC-SIGN cell lines express very low levels or no DC-SIGN (25) , and virus generated from these cells was not blocked, these two DC-SIGN-transduced cell lines did not show 99–99.5% inhibition of HIV production. However, inhibition by DC-SIGN in these two cell lines was still significant.

Both the extracellular and transmembrane domains are essential for HIV blockage
There are three distinct portions of the DC-SIGN lection, the cytoplasmic, the transmembrane, and the extracellular domains. The extracellular domain contains a carbohydrate recognition domain (CRD), which is essential for DC-SIGN to bind to glycoproteins, including gp120. The cytoplasmic domain is required for DC-SIGN to internalize HIV viral particles (19) . Therefore, for DC-SIGN to perform its role in HIV transmission, both the extracellular and cytoplasmic domains are critical. To determine whether these two domains are required for DC-SIGN-mediated blockage of HIV generation, we subcloned the DC-SIGN domains (Fig. 3 A). A DC-SIGN subclone that spans the transmembrane (TM) and extracellular (ExCel) domains, TM-ExCel, containing 364 aa residues (344 of ExCel and 20 of the TM domains), demonstrated the greatest inhibition of HIV production (~99%) compared to other individual DC-SIGN subclones (Fig. 3B ). However, the ExCel domain alone did not significantly inhibit HIV generation, suggesting that the TM domain is essential for DC-SIGN to exert its inhibition of viral generation. Correspondingly, the TM-ExCel DC-SIGN subclone also demonstrated significant inhibition of HIV virion generation (80%), as seen in p24 assays (Fig. 3C ). Although the TM-ExCel DC-SIGN subclone alone was not as efficient as full-length DC-SIGN (Fig. 3B ), it is undisputable that the TM-ExCel DC-SIGN subclone is sufficient to block HIV budding. This result suggests that DC-SIGN blockage of viral production uses a different mechanism than DC-SIGN to bind and internalize HIV; it uses a mechanism that does not require involvement of the cytoplasmic domain of DC-SIGN.


Figure 3
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  		Figure 3. DC-SIGN domains required for viral inhibition. A) Diagram of DC-SIGN domains and DC-SIGN subclones that contain the DC-SIGN domains in a plasmid vector. B) Infectivity assay of DC-SIGN subclones. C) p24 assay of DC-SIGN subclones blocking HIV generation (TM, transmembrane; Cyto, cytoplasmic; ExCel, extracellular). The Cyto-TM plasmid contains the cytoplasmic and transmembrane domains (60 aa residues), and TM-ExCel contains both transmembrane and extracellular domains (364 aa residues).

Our results demonstrate that retention of the TM domain is required for DC-SIGN to inhibit HIV generation, suggesting that inhibition occurs at the stage of viral budding, the only step that is involved with the host cell membrane during viral generation.

Reduction of gp120 from the cell membrane by DC-SIGN
As previous studies have demonstrated, DC-SIGN has high affinity for interacting with cell-free HIV gp120 envelope protein (18) . It is important to clarify whether the interaction between gp120 and DC-SIGN is also fundamental in DC-SIGN blockage of HIV budding. We constructed an EGFP-tagged DC-SIGN subclone by fusing the EGFP protein to the N terminus of the TM-ExCel DC-SIGN sequence. EGFP-tagged TM-ExCel DC-SIGN demonstrated inhibition almost identical to that of TM-ExCel DC-SIGN, with no EGFP in HIV production (Fig. 4 A), suggesting that fusion of EGFP does not change the structure of the functional domains of DC-SIGN when blocking HIV budding. Transfection of EGFP-tagged TM-ExCel DC-SIGN demonstrated that DC-SIGN localizes to the cell membrane (Fig. 4B , top left). To study the interaction between HIV gp120 and DC-SIGN, we cotransfected 293T cells with EGFP-tagged TM-ExCel DC-SIGN and a plasmid that contains the gp120 gene driven by the CMV promoter. As control, pCDNA3.1, a plasmid that contains the CMV promoter but not the gp120 gene, was used. Our results demonstrated that gp120 significantly decreases the amount of DC-SIGN present on the cell membrane (compare Fig. 4B , left, bottom, and top photos, and see histogram on the right), suggesting that DC-SIGN can interact with gp120 molecules.


Figure 4
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  		Figure 4. The interaction between DC-SIGN and gp120. A) EGFP-tagged TM-ExCel DC-SIGN demonstrated an effect similar to TM-ExCel DC-SIGN in blocking HIV generation. B) EGFP-tagged TM-ExCel DC-SIGN located on the cell membrane (top) and gp120 could decrease the amount of DC-SIGN on the cell membrane (bottom). gp120 decreases the amount of GFP-tagged TM-ExCel DC-SIGN on the cell membrane (right; results were from three experiments; error bar indicates SD). The numbers of EGFP-positive cells and the intensity of green fluorescence emitted from these cells were assessed. C) DC-SIGN efficiently reduced the abundance of EGFP-tagged gp120 located on the cell membrane. D) Colocalization of Ds-Red-tagged gp120 and EGFP-tagged TM-ExCel DC-SIGN. E) Deletion of repetitive sequences significantly decreased DC-SIGN-mediated blockage of HIV production.

The interaction of gp120 with DC-SIGN was also studied by fusing DC-SIGN to internalized EGFP-tagged gp120. The EGFP-tagged gp120 molecules localize to the cell membrane even without gp41 (Fig. 4C , left panels), since the signal sequence (amino acid residues 1–32) contains a TM domain. It is likely that when gp41 is absent, gp120 molecules can remain in the cell membrane before removal of the signal sequence. Our results demonstrated that DC-SIGN can very efficiently decrease the amount of gp120 on the cell membrane (Fig. 4C ). These results further confirmed our speculation that DC-SIGN interacts with gp120. An interaction such as this may ultimately internalize gp120. To further study the interaction between gp120 and DC-SIGN, we used DsRed protein (purchased from BD Clontech) to tag gp120. DsRed-tagged gp120 was used to cotransfect 293T cells with EGFP-tagged TmExCel-DC-SIGN. In some transfected cells, colocalization of DsRed-tagged gp120 and EGFP-tagged TmExCel-DC-SIGN was observed (Fig. 4D ), suggesting that these two molecules can interact with each other. DC-SIGN blockage of HIV budding can also be partially compensated for by high levels of expression of gp120 (Fig. 4A ), further supporting our expectation that the interaction between DC-SIGN and gp120 is the key to blocking DC-SIGN-mediated HIV budding.

We also studied the potential function of the repetitive sequences of DC-SIGN. Previous studies demonstrated that removal of the repetitive sequence prevents formation of DC-SIGN tetramers (18) . The DC-SIGN monomers have much lower binding affinity to gp120 than the tetramers (18) . Therefore, it was expected that the removal of the seven (23 bp each) and half (15 bp) repeats from DC-SIGN would significantly decrease the ability of DC-SIGN to block HIV production. The results clearly indicated that the removal of these 23-bp repeats very significantly decreased the blockage effect of DC-SIGN (Fig. 4E ), supporting the hypothesis that the binding between DC-SIGN and gp120 is the key to DC-SIGN-mediated HIV blockage.

HIV gp120 is stripped from the viral envelope by DC-SIGN
DC-SIGN inhibition has been demonstrated by both p24 assays and infection of HeLa-CD4 and/or 293-CCR5/CD4 cells. When p24 assays were used, the data indicated that DC-SIGN inhibited ~95% of HIV generation (Fig. 1E ). It is very likely that there are still some viral particles generated from DC-SIGN-positive cells; however, most of these viral particles are not infectious, so that virus from DC-SIGN-transfected cells demonstrated less than 1% infectivity compared with the virus from controls. One explanation is that the viral particles generated from DC-SIGN-expressing cells do not have gp120 on their surfaces. To investigate such a possibility, we collected viral particles from 293T cultures cotransfected by DC-SIGN and pNL4–3-EGFP-Env(+), and the control culture, 293T transfected by only pNL4–3-EGFP-Env(+).We used Western blot analysis to quantify gp120 in virions. We found that gp120 is completely absent in virions from the 293T culture cotransfected by both DC-SIGN and the pNL4–3-EGFP-Env(+) plasmid (Fig. 5 A), indicating that DC-SIGN very efficiently strips gp120 from the viral envelope. However, gp41 was not completely removed by DC-SIGN (Fig. 5A ). Therefore, DC-SIGN has two means of inhibiting HIV generation: 1) blocking viral budding so that the number of viral particles generated from infected cells is decreased by 70–97%, and 2) stripping gp120 from the viral envelope so that the remaining 3–30% of viral particles generated from the infected cells are not infectious.


Figure 5
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  		Figure 5. Western blot analysis to detect gp120, gp41, and DC-SIGN. A) Western blots to assess the amount of gp120 and gp41 present on the viral envelope. Viral preparations from 293T cells transfected by pNL4–3-EGFP-Env(+) alone or by pNL4–3-EGFP-Env(+) with pCDNA3-DC-SIGN were concentrated. Equal amounts of HIV (200 ng p24 protein count) of these two viral preparations were subjected to Western blot analysis, using monoclonal antibodies obtained from the NIH AIDS Reagent Program. B) Western blot analysis to quantify the amount of DC-SIGN present in viral preparations. The same amount of viral preparations (200 ng p24 count), as shown in A, was loaded for Western blot analysis. Three DC-SIGN(R) monoclonal antibodies from the NIH AIDS Reagent Program (cat. 5442 and 6886) were combined for detecting DC-SIGN(R). C) Detection of DC-SIGN(R) expression in lymphocytes, glial cells, and epithelial cell lines. Proteins isolated from the CD4-positive cell line, CEM (2.0 µg), and CD4-negative cell lines (30 µg) were subjected to Western blot analysis. The cell lines are LNCaP, prostate epithelial cells; HOT, oral, epithelial; Ca SKi, cervical, epithelial; A172, glial; 293T, embryonic kidney epithelial cell lines; and CEM, a T-lymphocyte cell line. D) A model to summarize the effect of DC-SIGN on blocking HIV production. In pathway A, DC-SIGN internalizes the complex of gp120 and gp41. The precursor of gp120 and gp41, gp160, is produced on the endoplasmic reticulum (ER). After folding, protease will remove the signal peptide and separate gp120 and gp41. The gp41-gp120 complex will transfer to the plasma membrane, and there, the binding of DC-SIGN to gp120 internalizes the complex and degrades it. In pathway B, DC-SIGN internalizes gp120 but not gp41. Some gp160 may be cleaved before folding. If so, gp41 will not associate with gp120. DC-SIGN is able to internalize gp120 but not gp41. Some virions containing these gp41 molecules bud out, so this glycoprotein can be detected in viral preparations from DC-SIGN-expressing cell cultures, as shown in A.

We also quantified the presence of DC-SIGN on the viral envelope. As a membrane protein, DC-SIGN may integrate into the viral envelope. Viral preparations from DC-SIGN and HIV-cotransfected 293T cells were concentrated for Western blot analysis. Our results demonstrated that this protein was not taken up by the virus during viral budding (Fig. 5B ). It is likely that during viral budding, the viral prebudding complex avoids the membrane areas that contain DC-SIGN molecules. If so, in DC-SIGN-expressing cells, viral budding areas would be significantly smaller than the infected cells that do not express DC-SIGN.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results propose a role for DCs in human immunity. As a type of antigen-presenting cell, the surface lectins of DCs are believed to interact with pathogens before engulfing them. Our results suggest that DCs can engulf certain pathogens, including HIV, and permit certain types of pathogens to continue their life cycles so that more pathogen-derived peptides can be generated (29) . Certain lectins such as DC-SIGN can block these pathogens at the stages of assembly and budding, so that only very limited numbers of viral particles can be generated from DCs. Using such a mechanism, DCs are able to efficiently present pathogen-derived peptides on their surfaces without sacrificing themselves to the process of pathogen replication.

It was also noted that the levels of DC-SIGN in DCs may not be as high as in transfected 293T cells or in DC-SIGN lentiviral vector-transduced CEM or Raji cells. Therefore, although it is very likely that DC-SIGN can block HIV production in DCs, the real effect of such blockage in DC-SIGN-positive DCs may still need to be quantified. Previous studies demonstrated that although DCs can be infected, much fewer virions can be generated from DCs than from infected CD4-positive T-lymphocytes (10 11 12 13 14 15 16 17) , suggesting that there are certain proteins that can efficiently block viral release from the infected cells. Whether DC-SIGN is the only critical protein in such inhibition merits further exploration, since there are other lectins expressed in DCs and some of them can also bind to gp120 (30) .

Although the expression levels of DC-SIGN in DC-SIGN-positive DCs may not be high, because the copy numbers of HIV in infected DCs may also not be high, it is still possible that low expression of DC-SIGN can efficiently block HIV budding in infected cells. If so, decreases in DC-SIGN expression when DCs become mature may be a mechanism of DC-mediated transinfection.

Previous studies demonstrated that DC-SIGN plays an essential role in transmission of HIV from DCs to T-cells (9 , 18 , 19) . These results, combined with ours, suggest that DC-SIGN plays dual roles in HIV infection. DC-SIGN-mediated viral transmission requires a clathrin involved in endocytosis, with the cytoplasmic domain of DC-SIGN being essential (19) . Since DC-SIGN-mediated inhibition of HIV budding uses a different mechanism, the cytoplasmic domain of DC-SIGN is not required (Fig. 3) . The different requirements of these two mechanisms may be important in development of new therapies for HIV-infected patients. We may use C-terminal-truncated DC-SIGN to transduce cells. Truncation of the cytoplasmic domain still retains blockage of DC-SIGN, which abrogates the capacity of DC-SIGN in viral transmission.

The gp160 precursor for the two envelope glycoproteins, gp120 and gp41, is synthesized on ribosomes associated with the endoplasmic reticulum (ER), and forms a trimeric structure (31) . This is transported from the ER to the Golgi complex, where the mannose-rich oligosaccharide side chains are added and the precursor is cleaved to gp120 and gp41. These two viral proteins are noncovalently associated following cleavage of the precursor, and move to the plasma membrane. For this reason, together with our results, we propose a model for this process (Fig. 5D ), in which most gp160 is processed through pathway A (Fig. 5D ). The gp120 and gp41 complex is targeted by DC-SIGN, resulting in no infectious viral particles being generated. In pathway B (Fig. 5D ), some gp160 may not be correctly folded and cleaved. Membrane-bound gp120 can be degraded by DC-SIGN, but the gp120-free gp41 molecules would not be affected. The virus generated from the DC-SIGN-expressing cells contain gp41 but not gp120, as shown in Fig. 5A . It is expected that only a small fraction of gp160 enters pathway B, so that the amount of virus generated from DC-SIGN-expressing cells is significantly less than from cells that do not express DC-SIGN.

In humans, DC-SIGN has been reported to be expressed in vivo by DCs, macrophages, activated B-cells, the dermis of the skin, the placenta, the intestinal and genital mucosa, and lymphoid tissues (8) . DC-SIGNR is expressed in several types of endothelial cells (32) . We have studied several epithelial cell lines and found low expression levels of DC-SIGN or DC-SIGNR (Fig. 5C ). In addition to binding HIV gp120, DC-SIGN(R) was also able to bind the hepatitis C virus (HCV) E2 glycoprotein (33) and CMV envelope glycoprotein B (34) . It will be of interest to determine whether DC-SIGN can also block production of HCV and CMV in host cells.

The strong inhibition of viral production by DC-SIGN suggests the possibility of using this protein for treatment of HIV-infected patients. Expression of this protein in various CD4-positive cells should inhibit viral production from infected cells. Because it can also enhance the immune response (29) , DC-SIGN is expected to be useful for in vivo studies for developing an HIV vaccine. We also noted that DC-SIGN(R) is expressed in the CD4-positive T cell line, CEM, and certain CD4-negative cell lines (Fig. 5C ). However, most of these proteins may lack part of the DC-SIGN(R) sequence. The full-length DC-SIGN is at least 45,774 Da (if not glycosylated), and DC-SIGNR is at least 45,350. The band of ~40 kDa may either lack the TM or a part of the CRD (35) . A minor band of ~45 kDa in CEM cell preparations has been detected (Fig. 5C ). It is not clear whether this band represents a functional isoform of DC-SIGN, since the soluble DC-SIGN form that lacks the TM domain is only 0.6 kDa smaller than that containing the full-length TM domain (35) . Characterizing the expression and splicing of DC-SIGN in CD4-positive T-lymphocytes and macrophages will be necessary to find an approach to use endogenous DC-SIGN to block HIV production in patients.


   ACKNOWLEDGMENTS
 
Work reported here was partially supported by U.S. National Institutes of Health grant AI047722. DC-SIGN, DC-SIGNR, and mABs for DC-SIGN were obtained through the AIDS Research and Reference Reagent Program. DC-SIGN and DC-SIGNR were from Drs. S. Pöhlmann, F. Baribaud, F. Kirchhoff, and R. W. Doms (30 , 32 , 36 ), and mABs were from Drs. F. Baribaud, S. Pöhlmann, J. A. Hoxie, and R. W. Doms (30 , 32 , 36 , 37 ). We thank Dr. Benhur Lee of the Department of Microbiology, Immunology, and Molecular Genetics at the University of Los Angeles (Los Angeles, CA, USA) for providing 293-DC-SIGN cells, and Wendy Aft for editing the manuscript.

Received for publication July 31, 2007. Accepted for publication October 4, 2007.


   REFERENCES
TOP
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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