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 (
DC-SIGN (CD209) and its homolog L-SIGN (also called DC-SIGN-R, CD209L) belong to the C-type (calcium-dependent) lectin family. This large group of proteins which includes the mannose receptor, DEC-205 or langerin, is specialized in the recognition of carbohydrate structures present on cellular and viral proteins and is implicated in several processes such as cell adhesion and antigen presentation (
DC-SIGN and L-SIGN share nearly 77% amino acid identity and are closely related in global architecture. Both lectins are type II transmembrane proteins composed of a short cytoplasmic tail responsible for signalling and internalization, a transmembrane region, a neck domain consisting of eight repeat regions of 23 amino acids and a carbohydrate recognition domain (CRD) which binds carbohydrate ligands in a calcium-dependent manner ( 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.
DC-SIGN was originally cloned for its ability to bind and internalize the heavily glycosylated human immunodeficiency virus (HIV) gp120 protein (
* Viral envelope protein(s) bound by DC-SIGN or L-SIGN. Virus family Virus Mechanisms Viral envelope protein(s)* DNA
CMV
gB RNA
SARS
Spike
Ebola
GP Marburg
GP
Dengue
E HCV
E1/E2
HIV-1/-2
gp120 SIV
gp120
Sindbis
E1 or E2 (?)
DC-SIGN and L-SIGN are endocytic receptors and their cytoplasmic tails carry putative internalisation signals such as a dileucine (LL) motif (which is present in both DC-SIGN and L-SIGN) and a tri-acidic cluster that is believed to be involved in intracellular trafficking (
The contribution of DC-SIGN and L-SIGN to viral transmission and dissemination in vivo is currently unknown. Their role as principal attachment factors for a broad range of enveloped viruses and their restricted expression in anatomical site of virus exposure suggest that these two lectins dictate viral tropism for DCs and endothelial cells and consequently may influence viral pathogenesis. DCs are sentinel cells that capture pathogens entering skin or mucosal tissues and then migrate to the lymph nodes where they present processed antigens to T-cells, initiating adaptive immune responses. By interacting with DC-SIGN, viruses that are transmitted sexually (such as HIV) or through introduction into human skin by an insect vector (such as dengue virus or Sindbis virus) may hijack DC function either to modulate the immune response or to assure their dissemination from peripheral tissues to lymphoid organs (
In this chapter, we will provide general protocols to study the molecular interactions between viruses and DC-SIGN or L-SIGN and to investigate the functions of these two lectins in viral infection and transmission. We will first describe the methods used to obtain human dermal-like DCs and cells expressing DC-SIGN and L-SIGN. We next present protocols to produce soluble viral envelope proteins using a Semliki forest virus (SFV) vector (
All of the products used for cell culture are purchased from Invitrogen (RPMI 1640, Glasgow’s modified Eagle’s medium [GMEM], fetal calf serum [FCS], penicillin/streptomycin, HEPES, and tryptose phosphate broth) except cysteine/methionine-free Dulbecco’s modified Eagle’s medium (DMEM) (ICN Biomedicals). Phycoerythrin (PE)-conjugated mouse monoclonal antibodies (mAbs) directed against DC-SIGN (FAb161P), L-SIGN (FAb162P), or both lectins (FAb1621P) were purchased from R&D Systems. The anti-DC-SIGN mAb clone 1B10 (IgG2a, κ) has been developed in our laboratory and previously described ( HeLa and 293T cells are maintained in DMEM supplemented with 10% FCS and antibiotics (100 µg/mL−1 streptomycin and 100 U/mL−1 penicillin). Raji cells are grown in RPMI containing 10% FCS and antibiotics. BHK is cultured in GMEM with 5% FCS, 1% penicillin/streptomycin, 20 m Cell lines expressing DC-SIGN or L-SIGN are generated by transduction with the retroviral TRIP ΔU3 vector (a gift from Pierre Charneau, Pasteur Institute, France) encoding either DC-SIGN or L-SIGN (
Human peripheral blood mononuclear cells (PBMC) are isolated from healthy donors by density gradient centrifugation through Ficoll-Paque Plus (Amersham Biosciences). Lysis buffer: dissolve 8.3 g of NH4Cl and 1 g of NaHCO3 in 1 L of water complemented with 1 mL of EDTA (100 m MACS buffer: phosphate-buffered saline (PBS) containing 2 m Filters (cell strainer, 40 µm) used to eliminate cell aggregates are purchased from Falcon. Monocytes are negatively selected using FCR blocking and biotin antibodies (Ab) cocktail and anti-biotin magnetic beads (Miltenyi Biotec). Recombinant human interleukin (IL)-4 and recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) are purchased from PeproTech and Gentaur respectively.
Soluble viral glycoproteins are produced using the SFV vector in BHK mammalian cells. SFV shuts off the cellular translation machinery and so transduced cells produce only the viral protein of interest, which is secreted and accumulates in the supernatant of infected cells. This allows the production of large amounts of soluble proteins with a high degree of purity. The SFV expression vector was originally described in reviews ( pSFV-helper2 and pSFV-Δ Restriction enzymes Spe I and Sph I are purchased from New England Biolabs. The DNA purification kit (QIAquick PCR purification kit) is purchased from QIAgen. SP6 RNA polymerase and RNAsin are purchased from Roche Applied Science and Promega, respectively. 0.4-cm electroporation cuvets are purchased from Eurogentec. TNE buffer: 12 m Chymotrypsin and aprotinin are purchased from Sigma Aldrich. Peroxidase-conjugated mAb and diaminobenzidine (DAB) solution kit are obtained from Vector Laboratories (ABCYS Biologie). DAB solution is prepared by adding four drops of DAB solution, two drops of H2O2 solution, two drops of nickel solution, and two drops of buffer solution to 5 mL of water. [35S] cysteine and methionine (Pro-Mix 35S) are obtained from Amersham Biosciences. 1-deoxymannojirimycin hydrochloride (DMJ) and swainsonine are purchased from Calbiochem and Sigma respectively. These molecules specifically inhibit α-manosidase I and II and permit the production of proteins with mannose carbohydrate residues in mammalian cells ( Glycoproteins are concentrated through columns (Biomax, Millipore) with an appropriate molecular weight cut-off for the particular protein being produced. Endoglycosidase H (EndoH) and peptide: 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. 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. Production of HIV gp120DMJ and binding to DC-SIGN.
Buffer A: PBS containing 1% BSA, 0.2% γ-globulin, 0.1% sodium azide, 1 m Buffer B: serum-free RPMI containing 1 m Mannan, EDTA (pH ∼8.0) and EGTA (pH ∼8.0) are all purchased from Sigma Aldrich. Mannan stock solution is dissolved in water (5 mg/mL−1) and can be stored at 4°C for several months. Scintillation solution is obtained from Wallac (optiphase supermix solution).
Wild-type viruses or viral particles carrying the reporter genes firefly luciferase (Luc) or green fluorescent protein (GFP) can both be used to study DC-SIGN and L-SIGN-mediated
Fresh blood (450 mL) is completed to 600 mL with PBS. Prepare 20 50-mL tubes containing 15 mL of Ficoll. Slowly add 30 mL of blood into each tube, taking care to avoid mixing, then centrifuge for 20 min at 850 Aspirate the plasma and transfer the PBMCs (white ring) into new tubes containing 20 mL of PBS (two rings per tube). Complete each tube to 50 mL with PBS, and then centrifuge for 10 min at 300 Remove the supernatant and resuspend the pellet in 5 mL of lysis buffer for 4 min. Add 20 mL of PBS 2% FCS and centrifuge at 300
Isolated PBMCs are passed through a filter placed inside a 50-mL tube (to eliminate cell aggregates) and the filter is rinsed twice with 5 mL of MACS buffer. Centrifuge at 300 Resuspend the cells at up to 107 cells per 30 µL of MACS buffer and add 10 µL each of FCR blocking reagent and biotin antibody cocktail per 107 cells. Incubate for 10 min at 4°C. Add 30 µL of MACS buffer and 20 µL of anti-biotin microbeads per 107 cells and incubate for 15 min at 4°C. Complete to 50 mL with MACS buffer and centrifuge 10 min at 300 Resuspend the cells at 108 cells per 500 µL of MACS buffer. Wash the magnetized column with 3 mL of MACS buffer, then pass cells through the column. Rinse the column three times with 3 mL of MACS buffer and collect the eluate containing monocytes. Cells are cultured at 106 cells/mL−1 in RPMI with 10% FCS, 1% penicillin/streptomycin, 50 ng/mL−1 IL-4, and 100 ng/mL−1 GM-CSF for 6 d. GM-CSF and IL-4 are added every 2 d. Differentiation of monocyte-derived DCs is assessed by FACS analysis (CD14 negative and CD1a- and DC-SIGN-positive).
Digest 2 µg of pSFV-helper2 and pSFV-Δ pSFV-helper2 RNA is mixed with equal quantities of pSFV-Δ The RNA-cell mixture is subjected to two 0.4-ms pulses at 830 V and 25 µfarads in a Bio-Rad gene pulser and plated in 75 cm2 flasks in 15 mL of GMEM medium containing 5% FCS. Supernatants containing recombinant defective SFV particles are harvested 24 h later and cleared by centrifugation (850 The pellet is covered with 200 µL of TNE buffer and incubated at 4°C for 1 h in a sealed tube to resuspend. The virus is stored in 20-µL aliquots at −80°C.
Before infection, virus aliquots are activated by a chymotrypsin treatment (0.5 mg/mL−1 chymotrypsin, 1 m For infection, BHK cells (5 × 105 cells seeded in six-well plates 24 h before infection) are washed with serum-free GMEM and incubated with dilutions of viral particles in GMEM complemented with 2% FCS (500 µL of dilutions 10−2 to 10−7) at 37°C. One hour later, 2 mL of GMEM complemented with 5% FCS are added. At 7 h postinfection, cells are washed with serum-free GMEM medium and fixed with cold methanol for 5 min at −20°C follow by three washes with PBS. Cells are incubated with 500 µL of primary antibody (directed against the soluble glycoprotein of interest) diluted in PBS for 1 h at room temperature or overnight at 4°C. Cells are washed twice with PBS prior to incubation with the secondary peroxidase-conjugated antibody (1/200 in 700 µL PBS per well) for 30 min at room temperature. Cells are then washed three times in PBS and incubated in DAB solution for 2 to 10 min and then rinsed three times in PBS. Stained cells are counted with a micrometric objective to determine the virus titer.
Defective SFV particles are activated and cells are infected as described under At 6 h postinfection, cells are washed five times in serum-free GMEM to eliminate BSA and then maintained in serum-free GMEM for protein production. For the production of radiolabeled protein, cells are starved for 1 h in serum- and methionine/cysteine-free DMEM prior to addition of 100 µCi/mL−1 [35S] cysteine and methionine. Synthesis of proteins is continued up to 24 h postinfection in the presence or absence of α-mannosidase I and II inhibitors DMJ (1 m The glycosylation pattern of the proteins produced can be verified by treatment with endoglycosidase H (endoH; 2 mU, Roche) or Peptide:
Binding assays are performed in 96-well plates using 5 × 105 DC-SIGN- or L-SIGN-expressing cells in 100 µL of buffer A. Cells are pelleted by centrifugation (300 Binding was carried out for 2 h at 4°C with gentle agitation. Unbound radioactivity is removed by three washes with 200 µL of buffer A and cell pellets are resuspended in 25 µL of buffer A prior to addition of 175 µL of scintillate solution. Cell-associated radioactivity is counted in a 1450 Microbeta Trilux β counter (Wallac). To assess the specificity of interactions between viral glycoproteins and DC-SIGN or L-SIGN, cells are preincubated for 30 min at 4°C with mannan (a CRD competitor), the neutralizing anti-DC-SIGN mAb (1B10) or anti L-SIGN mAb (mAb1621) (each at 20 µg/mL−1), or the calcium chelator EDTA (5 m For internalization assays, 35S-labeled proteins are bound to parental or DC-SIGN-or L-SIGN-expressing cells as described above except that the buffer A is replaced by the buffer B. Cells are washed three times and incubated for 30 min either at 4°C in 100 µL of cold buffer B or at 37°C in 100 µl of preheated buffer B to initiate endocytosis. To quantify glycoprotein internalization, cells maintained either at 4°C or 37 °C are treated with 200 µL RMPI containing 20 m 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).
DC-SIGN- or L-SIGN-expressing cells and their parental counterpart (105 cells) are exposed to viral particles for 2 h at 37°C at varying MOI in FSC-free medium supplemented with 1% penicillin/streptomycin, pH approx 7.5. Cells are washed three times with complete medium to remove excess virus and incubated at 37°C. Viral replication is evaluated 2 to 3 d later, depending on the readout used (
DC-SIGN- or L-SIGN-expressing cells and their parental counterpart (105 cells) are incubated with viral particles at a high MOI for 2 h at 37°C in FCS-free medium supplemented with 1% penicillin/streptomycin, pH approx 7.5 (
DC-SIGN- or L-SIGN-expressing cells and their parental counterpart (105 cells) are incubated with viral particles at a low MOI (insufficient to directly infect target cells) for 2 h at 37°C and immediately co-cultured with target cells without washing. As an important control, viral particles are incubated with medium alone and 2 h later, transferred to target cells.
This assay permits to determine if viral particles bound to DC-SIGN or L-SIGN remain infectious for several days. The protocol is similar to that described under
Human primary LSECs expressing L-SIGN are difficult to obtain. For this reason, we use cell lines expressing L-SIGN and we describe only the generation of monocyte-derived DCs that constitutively express DC-SIGN. Alternatively, the SFV particle production can be bypassed. The electroporation of BHK cells with pSFV-Δ The quality of transcribed RNA is verified by agarose gel (1%) electrophoresis. Transcribed RNAs can be stored at −20°C before electroporation. Defective SFV particles are used at a MOI varying from 25 to 100. Some adjustments may be required depending on the gene of interest. The number of cells infected correlates with the quantity of protein produced and the efficiency of protein secreted. For information, the best production we observed is for HCV envelope protein E2 ( DC-SIGN- or L-SIGN-mediated viral internalization can also be investigated by confocal microscopy using purified viral glycoproteins ( Optionally, the soluble glycoprotein can be purified by immuno-affinity when a tag peptide is introduced into its sequence. Several commercial tag peptides are available such as the Flag tag peptide which we used ( Read-out is specific for the virus studied. We generally use wild-type viruses or viral particles carrying a reporter gene (GFP or Luc). For example, for HIV or HCV, we use single-cycle pseudotyped viral particles that are generated by co-transfecting 293T cells with an HIV-1 NL ΔEnv Luc or GFP (a HIV provirus lacking the Env gene and carrying the Luc or GFP gene in the place of Nef) and a cDNA plasmid encoding either HIV or HCV envelope proteins DC-SIGN- or L-SIGN-expressing cells used for infection
While this chapter was in press, four new studies were published concerning the interactions between DC-SIGN and viruses, and the role of DC-SIGN in HIV dissemination (