Impact of polymorphisms in the DC-SIGNR neck domain on the interaction with pathogens

Abstract

The lectins DC-SIGN and DC-SIGNR augment infection by human immunodeficiency virus (HIV), Ebolavirus (EBOV) and other pathogens. The neck domain of these proteins drives multimerization, which is believed to be required for efficient recognition of multivalent ligands. The neck domain of DC-SIGN consists of seven sequence repeats with rare variations. In contrast, the DC-SIGNR neck domain is polymorphic and, in addition to the wild type (wt) allele with seven repeat units, allelic forms with five and six sequence repeats are frequently found. A potential association of the DC-SIGNR genotype and risk of HIV-1 infection is currently under debate. Therefore, we investigated if DC-SIGNR alleles with five and six repeat units exhibit defects in pathogen capture. Here, we show that wt DC-SIGNR and patient derived alleles with five and six repeats bind viral glycoproteins, augment viral infection and tetramerize with comparable efficiency. Moreover, coexpression of wt DC-SIGNR and alleles with five repeats did not decrease the interaction with pathogens compared to expression of each allele alone, suggesting that potential formation of hetero-oligomers does not appreciably reduce pathogen binding, at least under conditions of high expression. Thus, our results do not provide evidence for diminished pathogen capture by DC-SIGNR alleles with five and six repeat units. Albeit, we cannot exclude that subtle, but in vivo relevant differences remained undetected, our analysis suggests that indirect mechanisms could account for the association of polymorphisms in the DC-SIGNR neck region with reduced risk of HIV-1 infection.

Fig. 1

Schematic representation of the DC-SIGNR alleles analyzed. The neck domain of wt DC-SIGNR contains seven and a half repeat units (RU). Each repeat unit can be subdivided into two sequence blocks, indicated in grey and white. The allele F10 is wt, while allele 125 contains six and alleles C57 and 1411 contain five repeat units, respectively.

Fig. 2

DC-SIGNR alleles with five and six repeat units are efficiently expressed at the cell surface. Published wt DC-SIGN and the indicated patient derived DC-SIGNR alleles were transiently expressed in 293T cells and surface expression analyzed by FACS using a DC-SIGN/DC-SIGNR-crossreactive monoclonal antibody. The black-filled histograms indicate staining of control cells, the white-filled histograms show staining of lectin expressing cells. Similar results were obtained in two independent experiments.

Fig. 3

DC-SIGNR alleles with variations in the repeat region bind viral glycoproteins. (A) The indicated DC-SIGNR alleles or empty vector were transiently transfected into 293T cells and the cells incubated with concentrated supernatants containing comparable amounts of the indicated glycoproteins fused to the Fc portion of human immunoglobulin or the Fc portion of human immunoglobulin alone. Glycoprotein binding was analyzed by FACS. The grey-filled histograms indicate staining of control transfected cells, the black lines show staining of lectin expressing cells. Similar results were obtained in an independent experiment. Ig, Immunoglobulin. (B) The geometric mean channel fluorescence values measured in panel A are shown. An independent experiment yielded similar results.

Fig. 4

Wild type DC-SIGNR and DC-SIGNR alleles with five and six repeat units augment viral infectivity with comparable efficiency. (A) Enhancement of filovirus glycoprotein and SARS-CoV-S dependent infection. The indicated lectins were transiently expressed on 293T cells, the cells infected with lentiviral reporter viruses bearing the indicated glycoproteins and luciferase activities in cellular lysates determined. The results of a representative experiment performed in quadruplicates are shown, error bars indicate standard deviation (SD). Similar results were obtained in two independent experiments. (B) Augmentation of HIV infection. The indicated lectins were transiently expressed on 293T cells, the cells pulsed with replication competent HIV-1 reporter virus, washed, cocultivated with CEMx174 5.25 target cells and luciferase activities in the cocultures determined. The results of a representative experiment carried out in quadruplicates are shown and were confirmed in two independent experiments. Error bars indicate SD.

Fig. 5

DC-SIGNR alleles with five or six repeat units multimerize. The DC-SIGNR alleles were transiently expressed in 293T cells, the cells lysed under non-reducing and reducing conditions, the lysates separated by SDS-gelelectrophoresis and lectin expression detected by Western blot. The results were confirmed in two independent experiments. Lane 1, 7RU (F10); lane 2, 6RU (125); lane 3, 5RU (C57); lane 4, 5RU (1411), lane 5, control. The arrows indicate bands of the size expected for DC-SIGNR multimers.

Fig. 6

Coexpression of wt DC-SIGNR and DC-SIGNR alleles with five repeat units does not diminish augmentation of viral infectivity. (A) Augmentation of EBOV-GP dependent infection. The indicated lectins were transiently expressed on 293T cells, the cells infected with lentiviral reporter viruses bearing the indicated glycoproteins and luciferase activities in cell lysates determined. The results of a representative experiment performed in quadruplicates are shown, similar results were obtained in two independent experiments. Error bars indicate SD. (B) Augmentation of HIV-1 transfer. The indicated lectins were transiently expressed on 293T cells, the cells incubated with replication competent HIV-1 NL4-3 reporter virus, washed and cocultivated with CEMx174 5.25 target cells and luciferase activities in the cocultures determined. The average of six independent experiments performed with two separate plasmid preparations are shown. Error bars indicate standard error of the mean.