DC-SIGN and L-SIGN can act as attachment receptors for alphaviruses and distinguish between mosquito cell- and mammalian cell-derived viruses

Abstract

C-type lectins such as DC-SIGN and L-SIGN, which bind mannose-enriched carbohydrate modifications of host and pathogen proteins, have been shown to bind glycoproteins of several viruses and facilitate either cis or trans infection. DC-SIGN and L-SIGN are expressed in several early targets of arbovirus infection, including dendritic cells (DCs) and cells of the reticuloendothelial system. In the present study, we show that DC-SIGN and L-SIGN can function as attachment receptors for Sindbis (SB) virus, an arbovirus of the Alphavirus genus. Human monocytic THP-1 cells stably transfected with DC-SIGN or L-SIGN were permissive for SB virus replication, while untransfected controls were essentially nonpermissive. The majority of control THP-1 cells were permissive when attachment and entry steps were eliminated through electroporation of virus transcripts. Infectivity for the DC-SIGN/L-SIGN-expressing cells was largely blocked by yeast mannan, EDTA, or a DC-SIGN/L-SIGN-specific monoclonal antibody. Infection of primary human DCs by SB virus was also dependent upon SIGN expression by similar criteria. Furthermore, production of virus particles in either C6/36 mosquito cells or CHO mammalian cells under conditions that limited complex carbohydrate content greatly increased SB virus binding to and infection of THP-1 cells expressing these lectins. C6/36-derived virus also was much more infectious for primary human DCs than CHO-derived virus. These results suggest that (i) lectin molecules such as DC-SIGN and L-SIGN may represent common attachment receptor molecules for arthropod-borne viruses, (ii) arbovirus particles produced in and delivered by arthropod vectors may preferentially target vertebrate host cells bearing these or similar lectin molecules, and (iii) a cell line has been identified that can productively replicate alphaviruses but is deficient in attachment receptors.

FIG. 1.

DC-SIGN/L-SIGN expression and SB virus infection of THP-1 cells. (A) Histogram of flow cytometric analysis of cell surface DC-SIGN/L-SIGN expression on THP-1 control and transfected cells. Control THP-1 cells were stained with MAb 612, and transfected cells were stained with MAb 612 or the isotype control MAb. (B) Infectivity of 39GFP-M (solid bars), 39GFP-C (hatched bars), or CHOK1-derived HS binding mutant (S1K70GFP) SB virus (open bars) for control or transfected THP-1 cells and electroporation efficiency of TR339GFP transcripts (crosshatched bars) for control THP-1 cells. *, S1K70 infectivity not measured. (C) Fluorescence micrographs (magnification, ×100) of THP-1 cells infected with 39GFP-M virus. Equal numbers of each THP-1 cell type were infected with 39GFP-M as described in Materials and Methods and then diluted 10-fold into 96-well plates, incubated for 12 h, and photographed on the fluorescence microscope. Bright-field observations confirmed that each view shown contained similar numbers of cells. The arrow indicates a rare GFP-positive cell in control THP-1 cells.

FIG. 2.

Effect of infection temperature on infectivity of 39GFP-M for THP-1 cells. Solid bars, 37°C; hatched bars, 4°C.

FIG. 3.

Infectivity of 39GFP-M (solid bars), 39-GFP-C (hatched bars), or 39GFP derived from THP-1 (crosshatched bars) or DF-1 (open bars) cells for control THP-1 or DC-SIGN/L-SIGN-expressing cells.

FIG. 4.

Growth of TR339 in cultures of transfected THP-1 cells. DC-SIGN-transfected cells (squares) or L-SIGN-transfected cells (circles) were infected with 39-M (open symbols) or 39-C (solid symbols) virus.

FIG. 5.

Infectivity of 39GFP-M (solid bars), 39GFP-C (hatched bars), 39GFP-L (crosshatched bars), or 39GFP-DMJ (open bars) virus for control THP-1 or DC-SIGN/L-SIGN-expressing cells.

FIG. 6.

Infectivity of 39GFP for cells in the presence of DC-SIGN/L-SIGN competitors or EDTA. (A) Infectivity of 39GFP-M virus for THP-1 cells expressing DC-SIGN (solid triangles) or L-SIGN (solid circles) in the presence of increasing concentrations of mannan. (B) Infectivity of 39GFP-M virus for THP-1 cells expressing DC-SIGN (solid symbols) or L-SIGN (open symbols) in the presence of increasing concentrations of MAb 612 (circles) or an isotype-matched control MAb (triangles). (C) Infectivity of 39GFP-M (solid bars), 39GFP-L (hatched bars), or 39GFP-DMJ (crosshatched bars) virus for THP-1 cells in the presence of 200 μg of mannan per ml, 20 μg of MAb 612 per ml, or 5 mM EDTA. The control for mannan- or EDTA-treated cells was untreated cells. The control for MAb 612 was 20 μg of isotype-matched MAb per ml.

FIG. 7.

Binding of radiolabeled TR339 or S1K70 virus to THP-1 control and DC-SIGN/L-SIGN-transfected cells. The viruses used were 39-C (open bars), 39-M (solid bars), 39-L (hatched bars), or 39-DMJ (crosshatched bars) and S1K70 (striped bars), which was derived from CHO cells. *, binding of TR339 derived from Lec1- and 1-DMJ-treated Pro5 CHO cells not tested on Δ35 THP-1 cells.

FIG. 8.

Flow cytometric analysis of cell surface DC-SIGN expression and infectivity of 39GFP viruses for primary human DCs. (A) Histogram showing MAb and isotype control antibody reactivity with primary human DCs. (B) Relative infectivity of 39GFP-M and 39GFP-C for human DCs and relative blocking of DC infection by 39GFP-M by 200 μg of mannan per ml, 5 mM EDTA, 20 μg of MAb 612 or isotype control antibody per ml. Approximately 30% of the cells infected with 39GFP-M were positive for GFP at 6 hpi.