Measles virus targets DC-SIGN to enhance dendritic cell infection

Abstract

Dendritic cells (DCs) are involved in the pathogenesis of measles virus (MV) infection by inducing immune suppression and possibly spreading the virus from the respiratory tract to lymphatic tissues. It is becoming evident that DC function can be modulated by the involvement of different receptors in pathogen interaction. Therefore, we have investigated the relative contributions of different MV-specific receptors on DCs to MV uptake into and infection of these cells. DCs express the MV receptors CD46 and CD150, and we demonstrate that the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a novel receptor for laboratory-adapted and wild-type MV strains. The ligands for DC-SIGN are both MV glycoproteins F and H. In contrast to CD46 and CD150, DC-SIGN does not support MV entry, since DC-SIGN does not confer susceptibility when stably expressed in CHO cells. However, DC-SIGN is important for the infection of immature DCs with MV, since both attachment and infection of immature DCs with MV are blocked in the presence of DC-SIGN inhibitors. Our data demonstrate that DC-SIGN is crucial as an attachment receptor to enhance CD46/CD150-mediated infection of DCs in cis. Moreover, MV might not only target DC-SIGN to infect DCs but may also use DC-SIGN for viral transmission and immune suppression.

FIG. 1.

Recombinant DC-SIGN interacts with MV strains ED and WTF. DC-SIGN-Fc interactions with MV ED and WTF were determined by a DC-SIGN-Fc binding ELISA. Viruses were coated with an anti-F antibody. DC-SIGN-Fc binding to different concentrations of MV WTF (A) or ED (B) or their mock controls was measured by ELISA. The specificity of the DC-SIGN-Fc-MV interaction was determined by measuring binding in the presence of mannan and EGTA. An Fc chimera with an identical Fc domain (ICAM-3-Fc) was used as an Fc control. Data for one representative experiment of three are shown. Errors bars represent standard deviations of triplicates.

FIG. 2.

DC-SIGN binds the MV glycoproteins F and H. (A) DC-SIGN-Fc capture ELISA was used to determine the interaction of DC-SIGN-Fc with glycoproteins in MV (ED) lysates. MV proteins were immunoprecipitated with DC-SIGN-Fc and detected with MV-specific antibodies for F (A5047) or H (L77). Error bars represent standard deviations of duplicates. Data for one representative experiment of three are shown. (B) Recombinant F and H interact with DC-SIGN. F and H were expressed in the Meljuso cell line and then used to coat beads. The interactions of these F- and H-coated beads with CHO-DC-SIGN transfectants were measured by a fluorescent bead binding assay. The specificity of the DC-SIGN interaction was determined by measuring binding in the presence of mannan and EGTA. Error bars represent standard deviations of duplicates. Data for one representative experiment of three are shown.

FIG. 3.

Cellular DC-SIGN is a receptor for MV. (A) K562 cells express the MV receptor CD46, whereas Raji cells express the MV receptors CD46 and CD150. Open histograms represent the isotype controls, and filled histograms represent specific antibody staining. The mean fluorescence intensity of specific staining is depicted. Expression is shown in log scale and ranges from 10⁰ to 10⁴. (B) Specific binding of MV ED-coated beads to the receptor CD46 or CD150 was determined by measuring binding in the presence of blocking antibodies for CD46 (13/42) or CD150 (5C6). Standard deviations for the fluorescent bead adhesion assay were <5%. Data for one representative experiment of three are shown. (C) K562-DC-SIGN transfectants express high levels of DC-SIGN. Open histograms represent the isotype controls, and filled histograms represent specific antibody staining. The mean fluorescence intensity of specific staining is depicted. Expression is shown in log scale and ranges from 10⁰ to 10⁴. (D) MV ED and WTF interactions with K562-DC-SIGN transfectants were determined using the fluorescent bead adhesion assay. Specificity was determined by measuring binding in the presence of blocking antibodies against DC-SIGN (AZN-D1 and AZN-D2), mannan, EGTA, or CD46 (13/42). Standard deviations for the fluorescent bead adhesion assay were <5%. Data for one representative experiment of three are shown.

FIG. 4.

DC-SIGN is not an entry receptor for MV. (A) CHO transfectants express CD46, CD150, or DC-SIGN. Open histograms represent the isotype controls, and filled histograms indicate the specific antibody staining. The mean expression levels are depicted. Expression is shown in log scale and ranges from 10⁰ to 10⁴. (B and C) CHO transfectants were infected with MV ED, WTF (MOI, 0.5), or a mock control. MV H expression was measured at 48 h postinfection to determine the level of infection. Open histograms represent the isotype controls. Percentages represent the positive cells compared to the isotype controls (B). Representative phase-contrast photos were taken to show syncytium formation of the different transfectants. Arrows indicate examples of observed syncytia (C).

FIG. 5.

DC-SIGN is a major attachment receptor for MV on immature DCs. (A) Immature DCs express CD46 and CD150 and high levels of DC-SIGN. Open histograms represent isotype controls, and filled histograms indicate specific antibody staining. The mean fluorescence intensity of specific staining is depicted. Expression is shown in log scale and ranges from 10⁰ to 10⁴. (B and C) Binding of MV ED and WTF to DCs was determined using the fluorescent bead adhesion assay. Specificity was determined by measuring binding in the presence of mannan, EGTA, antibodies to DC-SIGN (AZN-D1 and AZN-D2), CD46 (13/42), or CD150 (5C6) or with a combination of the antibodies. Data for one representative experiment of three are shown. Error bars represent standard deviations of triplicates. *, P < 0.05; **, P < 0.01 (versus medium control).

FIG. 6.

DC-SIGN enhances the infection of measles virus in cis. (A) Raji-DC-SIGN cells express CD46, CD150, and DC-SIGN. Open histograms represent isotype controls, and filled histograms indicate specific antibody staining. The mean fluorescence intensity of staining is depicted. Expression is shown in log scale and ranges from 10⁰ to 10⁴. (B) Raji cells and Raji-DC-SIGN cells were infected with different dilutions of MV WTF. MV H expression was measured at 48 h postinfection to determine the level of infection. Data for one representative experiment of two are shown. Error bars represent standard deviations of triplicates.

FIG. 7.

DC-SIGN enhances MV infection of immature DCs. (A) High expression levels of maturation markers CD83 and CD86 on mature DCs indicate a mature phenotype. The expression of the different MV receptors was measured. Open histograms represent isotype controls, and filled histograms indicate specific antibody staining. The mean fluorescence intensity of specific staining is depicted. Expression is shown in log scale and ranges from 10⁰ to 10⁴. (B) Immature and mature DCs were infected with MV WTF (MOI, 0.3 and 1) or a mock control for 48 h. To determine the contribution of DC-SIGN, a specific antibody (AZN-D2) or mannan was used. An antibody (L7) or carbohydrate control (glucitol) was used as a control. With immature DCs, the role of CD150 could be determined by using a specific blocking antibody (5C6). To determine the level of infection, MV H expression was measured by flow cytometry. Percentages represent the numbers of infected cells. (C) Immature and mature DCs were infected with MV EDeGFP (MOI, 0.25) or a mock control for 48 h. Infection was determined by measuring GFP using flow cytometry. The MV receptors DC-SIGN, CD46, and CD150 were specifically blocked by antibodies (AZN-D2, 13/42, and 5C6, respectively) or a combination of the three antibodies to determine their roles in MV infection. Data for one representative experiment of three are shown. Error bars represent standard deviations of triplicates. *, P < 0.05; **, P < 0.01 (versus controls).