L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus

Abstract

Hepatitis C virus (HCV) infects nearly 3% of the population of the world and is a major cause of liver disease. However, the mechanism whereby the virus targets the liver for infection remains unknown, because none of the putative cellular receptors for HCV are both expressed specifically in the liver and capable of binding HCV envelope glycoproteins. Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) is a calcium-dependent lectin expressed on endothelial cells of liver and lymph nodes. Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a homologous molecule expressed on dendritic cells, binds HIV and promotes infection. By using a virus-binding assay, we demonstrate that L-SIGN and DC-SIGN specifically bind naturally occurring HCV present in the sera of infected individuals. Further studies demonstrate that binding is mediated by the HCV envelope glycoprotein E2 and is blocked by specific inhibitors, including mannan, calcium chelators, and Abs to the lectin domain of the SIGN molecules. Thus, L-SIGN represents a liver-specific receptor for HCV, and L-SIGN and DC-SIGN may play important roles in HCV infection and immunity.

Figure 1

Cell-surface expression of L-SIGN and DC-SIGN in stably transfected HeLa cell lines. Flow cytometry was performed by using the cross-reactive mAb 612X (line), which binds to both transfected cell lines, the L-SIGN-specific mAb 614L (dotted line), and DC-SIGN-specific mAb 507D (dashed line), which bind only to the HeLa-L-SIGN or HeLa-DC-SIGN transfectants, respectively. Binding of an isotype-matched control mAb is indicated by filled histogram, and one representative result out of three independent analyses is shown.

Figure 2

L-SIGN and DC-SIGN transfectants bind HCV-E2. L-SIGN (Top), DC-SIGN (Middle), and control HeLa cells (Bottom) were incubated with HCV-E2-coated beads prepared by using a panel of anti-E2 mAbs. Adhesion was quantified by fluorescence-activated cell sorter analysis in the presence of adherence buffer (filled bars) and was blocked by mannan (open bars). Different anti-E2 mAbs are indicated on the x axis, and the y axis represents the percentage of cells that have bound beads. One representative experiment out of three is shown.

Figure 3

Effect of mAbs or soluble ICAMs on adhesion of HCV-E2 to L-SIGN or DC-SIGN. HeLa cells expressing L-SIGN (filled bars) or DC-SIGN (open bars) were incubated with individual mAbs that bind the repeat region (DC6 and DC28) or the lectin-binding domain (612X, 604L, and 507D) or soluble ICAM-Fc conjugates as described. Binding of E2 beads was quantified by fluorescence with a FACScan machine, and results were normalized to isotype control (mIgG) levels. One representative data set from three experiments is shown.

Figure 4

L-SIGN and DC-SIGN bind to HCV virions from infected patients. HeLa transfectants or control cells were incubated with sera from three HCV RNA⁺ patients. HCV sera RNA titers (copies per ml) were no. 1, 850,000; no. 2, 242,000; and no. 3, 161,000 by COBAS MONITOR assay (Roche Molecular Systems). After washing, cells were lysed and RNA was extracted. HCV RNA was measured by a qualitative RT-PCR and Southern blot assay (A) or by a quantitative real-time PCR assay (B). Data are presented as fold increase above HeLa control cell binding for each matched sera, and absolute values (units/ml) are depicted for each sample. Binding to L-SIGN was inhibited by mannan. The cells were preincubated with mannan before addition of serum 2 as described previously. Bound HCV RNA was extracted and analyzed either by Southern blot (C) or quantitative real-time PCR (D).

Figure 5

Inhibition of HCV virion binding to L-SIGN and DC-SIGN by mannan. Cells were incubated with sera from three HCV RNA+ patients as described in Fig. 4. HCV sera RNA titers (copies per ml) were no. 4, 2.15 million; no. 5, 1.86 million; and no. 6, 1.16 million. Each serum was incubated with cells that had been pretreated with adherence buffer (filled bars) or mannan (open bars), and bound RNA was analyzed by real-time PCR as described. Fold increases above HeLa cell binding are indicated in the graph, and absolute levels (units/ml) are depicted (Inset).