The C type lectins DC-SIGN and L-SIGN: receptors for viral glycoproteins

Abstract

DC-SIGN and L-SIGN are C-type lectins that recognize carbohydrate structures present on viral glycoproteins and function as attachment factors for several enveloped viruses. DC-SIGN and L-SIGN enhance viral entry and facilitate infection of cells that express the cognate entry receptor (cis-infection). They are also able to capture viruses and transfer viral infections to other target cells (trans-infection). In this chapter, we will give an overview of protocols used to produce soluble viral glycoproteins at high levels and to study the molecular basis of viruses/DC-SIGN and L-SIGN binding and internalization. We will also describe techniques to investigate the molecular mechanisms by which DC-SIGN or L-SIGN spread viral infections.

Fig. 1.

Structure of DC-SIGN and L-SIGN proteins. The C-type lectins DC-SIGN and L-SIGN are type II transmembrane proteins. Their cytoplasmic tails contain internalization signals (di-leucine, tyrosine, and tri-acidic) which are involved in internalization of the lectin. The extracellular domain is composed of a carbohydrate recognition domain (CRD) and a neck domain (conserved in the case of DC-SIGN, variable for L-SIGN) implicated in the oligomerization of these lectins. The oligomerization is probably important for the orientation and subsequently for the function of the CRDs.

Fig. 2.

Semliki forest virus (SFV) expression vector. The SFV vector is composed of two RNAs which are electroporated into BHK cells. New synthesized particles incorporate only the RNA coding for nonstructural proteins and the protein of interest (NS, nonstructural and S, structural) because it is the only one with an encapsidation signal. The furin site of the SFV envelope protein is replaced by a chymotrypsin site so the particles can be activated by chymotrypsin digestion.

Fig. 3.

Carbohydrate maturation in mammalian cells. Proteins with NXS or NXT sites that pass through the endoplasmic reticulum can be potentially glycosylated. Glycoproteins are sensitive to EndoH until they are modified by α1,2-mannosidase II. Swainsonine and 1-deoxymannojirimycin hydrochloride (DMJ) block maturation steps of glycoprotein carbohydrates. These drugs permit to produce mannosylated glycoproteins in mammalian cells. 1, internal tri-mannose branch recognized by DC-SIGN; 2, external tri-mannose branch; ER, endoplasmic reticulum; GDP, guanosine biphosphate; UDP, uridine biphosphate.

Fig. 4.

Production of HIV gp120^DMJ and binding to DC-SIGN. (A) Soluble HIV gp120 is produced in BHK cells in the presence or absence of mannosidase inhibitors (1 mM DMJ and 5 µM swainsonine) (HIV gp120^DMJ or HIV gp120, respectively) as described under Subheading 3.2.3. Secreted proteins were subjected to digestion with EndoH or PNGase F and analyzed by Western blot. Only HIV g120^DMJ is sensitive to EndoH confirming its high mannosylated glycosylation. (B) ³⁵S-labeled HIV gp120^DMJ and HIV gp120 (20 nM) are bound to Raji and Raji-DC-SIGN cells for 2 h at 4°C. Cells are washed three times before measuring cell-associated radioactivity. Note that only HIV gp120^DMJ, which carries only mannosylated N-glycans, binds to DC-SIGN.

Fig. 5.

DC-SIGN induces internalization of viral glycoproteins. HeLa and HeLa-DC-SIGN cells are incubated for 2 h at 4°C with ³⁵S-labeled hepatitis C virus (HCV) E2 glycoprotein. Cells are extensively washed to eliminate unbound material and incubated for 30 min either at 4°C or 37°C. Cells are treated with EDTA or mock treated to distinguish internalized (EDTA-resistant) from cell surface bound HCV-E2 glycoprotein (EDTA-sensitive).