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RESEARCH ARTICLE

Open=20 Access=20

Everything we publish is freely available online throughout the = world, for=20 you to read, download, copy, distribute, and use (with attribution) any = way you=20 wish. No permission required. Read a detailed = definition of=20 open access.

The Mannose Receptor Mediates Dengue Virus Infection of = Macrophages

Joanna L. Miller¹,=20 Barend J. M. deWet¹,=20 Luisa Martinez-Pomares²,=20 Catherine M. Radcliffe³^=C2=A4,=20 Raymond A. Dwek³,=20 Pauline M. Rudd³^=C2=A4,=20 Siamon Gordon¹^*=20

1 = Sir William=20 Dunn School of Pathology, University of Oxford, Oxford, United Kingdom, = 2 School of Molecular Medical = Sciences,=20 Institute of Infection, Immunity and Inflammation, University of = Nottingham,=20 Queen's Medical Centre, Nottingham, United Kingdom, 3 Glycobiology Institute, Department of = Biochemistry, University of Oxford, Oxford, United Kingdom

Macrophages (M=C3=98) and mononuclear phagocytes are major targets of = infection by=20 dengue virus (DV), a mosquito-borne flavivirus that can cause = haemorrhagic fever=20 in humans. To our knowledge, we show for the first time that the M=C3=98 = mannose=20 receptor (MR) binds to all four serotypes of DV and specifically to the = envelope=20 glycoprotein. Glycan analysis, ELISA, and blot overlay assays = demonstrate that=20 MR binds via its carbohydrate recognition domains to mosquito and human=20 cell=E2=80=93produced DV antigen. This binding is abrogated by = deglycosylation of the DV=20 envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 = cells=20 confers DV binding. Furthermore, DV infection of primary human M=C3=98 = can be blocked=20 by anti-MR antibodies. MR is a prototypic marker of alternatively = activated M=C3=98,=20 and pre-treatment of human monocytes or M=C3=98 with type 2 cytokines = (IL-4 or IL-13)=20 enhances their susceptibility to productive DV infection. Our findings = indicate=20 a new functional role for the MR in DV infection.

Funding. JLM and = BJMdW are=20 supported by the Pediatric Dengue Vaccine Innitiative, LMP is supported = by The=20 University of Nottingham, and glycan analysis was supported by the = Oxford=20 Glycobiology Institute Endowment.

Competing = interests. The=20 authors have declared that no competing interests exist.

Editor: Grant McFadden, University of = Florida,=20 United States of America

Citation: Miller JL, deWet=20 BJM, Martinez-Pomares L, Radcliffe CM, Dwek RA, et al. = (2008) The=20 Mannose Receptor Mediates Dengue Virus Infection of Macrophages. PLoS = Pathog=20 4(2): e17 doi:10.1371/journ= al.ppat.0040017

Received: July 6, 2007;=20 Accepted: December 17, 2007; = Published:=20 February 8, 2008

Copyright: =C2=A9 2008 Miller et al. = This is an=20 open-access article distributed under the terms of the Creative Commons=20 Attribution License, which permits unrestricted use, distribution, and=20 reproduction in any medium, provided the original author and source are=20 credited.

* To whom correspondence should be = addressed.=20 E-mail: siamon.gordon@path.ox.ac.uk

=C2=A4 Current address: = Dublin-Oxford Glycobiology=20 Laboratory, National Institute for Bioprocessing Research and Training,=20 University College Dublin, Dublin 4, Ireland

Author Summary

Dengue disease and its severe manifestations are a growing public = health=20 concern, with a third to half the world's population living in = dengue-endemic=20 areas. In recent years there have been significant advances in = understanding=20 dengue virus (DV) interactions with target cells such as macrophages, = dendritic=20 cells, hepatocytes, and endothelial cells. Interaction with and = infection of=20 these cells leads to the production of new virions as well as immune = mediators,=20 which can shape the course of the subsequent immune response. The = vascular=20 leakage associated with dengue haemorrhagic fever is believed to be = immune=20 mediated. Our work on the interaction of DV with human macrophages has = led to=20 two major findings; first, we have identified that the macrophage = mannose=20 receptor is important for mediating the infection of human macrophages = by DV,=20 and second, that the type 2 cytokines IL-4 and IL-13 enhance macrophage=20 susceptibility to DV infection. DV=E2=80=93receptor interactions are of = critical=20 importance for understanding not only the mechanisms of entry, but also = the=20 biology of infection and the pathogenesis. Understanding the = immunopathogenesis=20 of dengue disease is crucial to the development of both a safe dengue = vaccine=20 and therapeutic inhibitors of early DV replication.

Introduction

Dengue is the most prevalent mosquito-borne viral disease worldwide = and in=20 the past 40 years has undergone a global resurgence such that almost = half the=20 world's population are currently living at risk in dengue-endemic areas = [1= ].=20 There is a spectrum of disease severity following dengue virus (DV) = infection=20 that in its more severe forms results in dengue haemorrhagic fever (DHF) = and=20 shock syndrome. The resultant morbidity and mortality, and subsequent=20 considerable economic burden, make the development of a safe and = effective=20 vaccine imperative. DV pathogenesis is complex and multifactorial [2= ],=20 and macrophages (M=C3=98) are thought to play an important role in = disease both as=20 primary targets of viral infection and as a source of immunomodulatory=20 cytokines. The four serotypes of DV (DV1-DV4) bind to a number of = opsonic and=20 non-opsonic receptors on cells of the mononuclear phagocyte lineage = including=20 DC-SIGN [3= ,4= ],=20 glycosaminoglycans [5= ],=20 and when in complex with specific antibody, Fc and complement receptors = [6= ].

MR is a multi-domain protein that is composed of a cysteine-rich (CR) = domain=20 which has lectin activity and binds to sulphated sugars, a fibronectin = type-II=20 (FNII) domain that mediates binding to collagen [7= ]=20 and eight C-type-lectin-like domains (or carbohydrate-recognition = domains, CRD).=20 The fourth CRD mediates most of the specificity of these domains for = glycans=20 terminating in mannose, fucose and N-acetyl glucosamine. In addition to = many=20 endogenous ligands, MR binds to bacteria (e.g. Mycobacterium=20 tuberculosis), fungi (e.g. Pneumocystis carinii) and = viruses (e.g.=20 HIV). MR is constitutively internalized from the plasma membrane by=20 clathrin-mediated endocytosis and recycled back to the cell surface.=20 Intracellular targeting is mediated by a tyrosine-based motif in the = cytoplasmic=20 tail, although it contains no recognised signalling motifs (for a = comprehensive=20 review see [8= ]).=20 DC-SIGN, a lectin with similar sugar specificity to that of the MR, can = mediate=20 DV attachment to dendritic cells [3= ,4= ].=20 Even though DV binding to DC-SIGN on these cells is important for = attachment,=20 DC-SIGN-mediated viral endocytosis is not required for DV entry [9= ].

While the immune response to viruses is classically described as Th1=20 mediated, the literature in the case of DV suggests that this may not be = absolute. IgE (characteristic of a Th2 environment) has recently been = shown to=20 be elevated in the acute stages of DV infection [10]=20 and at defervesence [11].=20 A microarray study of whole blood gene expression during secondary DV = infection=20 has shown early upregulation of IL-13 transcripts in acute samples from = DHF=20 patients [12].=20 This suggests that a Th2 response may be occurring in patients at some = stages of=20 infection. Indeed, studies of IL-12, IL-13 and TGF=CE=B2 cytokine levels = in DHF=20 patients suggest that the DV response shifts from a Th1-dominant = response to a=20 Th2-biased response during disease progression [13,14].=20 The immune responses in infants/neonates differ qualitatively from those = of=20 adults, with the immature immune system having a bias towards Th2 rather = than=20 Th1 immune responses, presenting a particularly relevant challenge for = pediatric=20 DV vaccination [15].

M=C3=98 are profoundly influenced by the cytokine profile in their = immediate=20 environment. Functionally diverse subsets of alternatively or = classically=20 activated mononuclear phagocytes can develop in an immune response. = Exposure of=20 M=C3=98 to IL-4 or IL-13 elicits an =E2=80=98alternate type of = activation=E2=80=99, as opposed to the=20 classical activation induced by IFN=CE=B3 [16].=20 These alternatively activated cells have been implicated to have = regulatory=20 functions in cellular and humoral immunity by affecting the balance of = pro- and=20 anti-inflammatory reactions [17].=20 Protein expression studies and transcriptional profiling have shown that = IL-4=20 induces upregulation of several receptors, including the mannose = receptor (MR)=20 on monocytes and M=C3=98 [18,19].

In this study we show that MR binds to DV grown in mosquito cells and = to=20 recombinant mammalian cell=E2=80=93produced DV envelope glycoprotein. A = recombinant MR=20 fusion protein (CRD4=E2=80=937-Fc) was shown to recognize DV envelope = (E) protein in=20 ELISA and blot overlays, and binding was inhibited by mannose, fucose = and EDTA.=20 The presence of MR on transfected cells is sufficient to confer DV = binding. DV=20 infection of M=C3=98 was blocked by antibodies against the human MR = suggesting that=20 it is a novel functional receptor contributing to DV infection. We also = show=20 that pre-treatment of primary human monocytes with Th2 cytokines = (IL-4/IL-13),=20 which upregulate MR expression, increases their susceptibility to DV = infection=20 in vitro. Better understanding of receptor/s and entry pathways = mediating=20 infection in humans could be crucial to the design and safety of a = dengue=20 vaccine.

Results

Soluble MR Binds Mosquito Cell=E2=80=93Derived DV and Recombinant = Soluble E=20 Protein

The ability of MR to bind DV antigen produced in mosquito (C6/36) and = human=20 (293T) cells was examined. ELISA wells were coated with semi-purified=20 C6/36-grown DV2 or recombinant soluble E (sE) protein produced in the=20 endothelial kidney cell line 293T (see below for characterisation of = this=20 reagent) and probed with the entire extracellular region of the murine = MR=20 expressed with an HA tag or recombinant truncated forms of the murine MR = with=20 human Fc tags. MR-HA bound to purified mosquito cell=E2=80=93derived DV2 = (Figur= e=20 1A) and to sE (Figur= e=20 1B), and binding was mediated specifically by CRD4=E2=80=937 and not = the CR or the=20 FNII domains. Binding of CRD4=E2=80=937-Fc to both C6/36-grown DV2 (Figur= e=20 1C) and sE (Figur= e=20 1D) was inhibited by 2mM d-mannose, = 2mM l-fucose, and to a lesser extent 2mM d-galactose, and depended on the presence of = divalent=20 cations. This is consistent with the known sugar specificity and calcium = dependence of the MR CRD4=E2=80=937 domains.

Figure 1. MR Binding to DV Is = Mediated by the=20 CRD4=E2=80=937

(A) Binding of MR extracellular domain (MR-HA) and chimeric MR fusion = protein=20 constructs to mosquito cell=E2=80=93derived (C6/36) NGC strain DV2 and = (B) soluble E=20 protein (sE) produced in a human cell line, detected by ELISA. Closed = bars, DV=20 antigen; open bars, uncoated wells. The MR-HA protein was detected with = an=20 anti-murine MR antibody (MR5D3). Binding of the Fc fusion proteins to = the DV/sE=20 was detected with an anti-Fc antibody. (C) Inhibition of = CRD4=E2=80=937-Fc binding to=20 mosquito cell=E2=80=93derived semi-purified DV2 and (D) sE, detected by = ELISA as above.=20 (E) Binding of CRD4=E2=80=937-Fc to all four serotypes of DV, detected = with an anti-Fc=20 antibody. Strains examined were mosquito cell (C6/36)=E2=80=93derived = virus of DV1=20 (Hawaii), DV2 (NGC), DV3 (H-87) and DV4 (H-241), and Vero = cell=E2=80=93grown DV2=20 (16681). Differential binding to the serotypes may reflect coating = levels, as=20 indicated by (F) binding of rabbit anti-DV1=E2=80=934 antibody. Normal = rabbit=20 immunoglobulin (RIg) was included as a control and both were detected = with an=20 anti-rabbit antibody. The low levels of CRD4=E2=80=937-Fc binding to = C6/36-grown DV2 in=20 this panel reflect a shorter development time than in the other panels. = Data are=20 expressed as mean and SD of triplicate wells. Representative data from = two to=20 five independent experiments are shown. Recombinant fusion proteins = contain the=20 cysteine-rich (CR) domain, fibronectin type-II (FNII) domain, and = various=20 carbohydrate-recognition domains (CRD) of the MR.

We extended the study by investigating the binding of = CRD4=E2=80=937-Fc to mosquito=20 cell=E2=80=93derived virus of the other 3 DV serotypes (DV1, DV3 and = DV4) and to=20 mammalian (Vero) cell=E2=80=93grown DV2 by ELISA. CRD4=E2=80=937-Fc = bound to all four serotypes=20 of DV in a dose-dependent manner (Figur= e=20 1E). Binding of the CRD4=E2=80=937-Fc correlated with the different = coating levels=20 of these antigens as determined with an antibody against all 4 DV = serotypes (Figur= e=20 1F). In addition, we tested whether CRD4=E2=80=937-Fc and sMR-HA = interacted with=20 other flaviviruses. ELISA data showed that both CRD4=E2=80=937-Fc and = MR-HA both bound=20 to Japanese encephalitis virus (inactivated vaccine antigen) and = tick-borne=20 encephalitis virus (inactivated, mouse brain-grown) (data not = shown).

The specificity of MR CRD4=E2=80=937-Fc binding to DV sE was further = examined by blot=20 overlay. CRD4=E2=80=937 bound exclusively to a single band that migrated = at 52 kDa (Figur= e=20 2A, left-hand lane). This band was recognized by the anti-E protein=20 antibody, 3H5, when blots were stripped and reprobed (Figur= e=20 2B, left-hand lane), confirming that CRD4=E2=80=937 binds to DV = E-protein. CRD4=E2=80=937=20 did not bind sE deglycosylated with peptide:N-glycosidase F (PNGaseF) = (Figur= e=20 2A, right-hand lane), in contrast to 3H5 that bound to both the = native and=20 the deglycosylated forms of the protein (Figur= e=20 2B), indicating that CRD4=E2=80=937 binds specifically to N-linked = glycans on sE.=20 Binding of CRD4=E2=80=937 to sE was inhibited by the presence of either = 2mM d-mannose, 2mM l-fucose or=20 20mM EDTA. The presence of sE on these blots was confirmed by washing = blots and=20 reprobing in the absence of inhibitors (data not shown). In addition, = CRD4=E2=80=937-Fc=20 did not bind to unglycosylated domain III of DV E protein produced in = bacteria=20 in ELISA experiments (data not shown).

Figure 2. MR CRD4-7-Fc Binds = Specifically to=20 sE in Blot Overlay, and Binding Depends on N-Linked Sugars

sE and PNGaseF-treated sE were resolved by SDS-PAGE using 10% gels,=20 transferred to nitrocellulose membranes, and (A) probed with MR = CRD4=E2=80=937-Fc. (B)=20 Blots were subsequently stripped and reprobed with the anti-E protein = antibody,=20 3H5.

Given the interaction of DV with MR described above, it was important = to=20 characterise the glycans on the human cell=E2=80=93produced sE, = especially since we are=20 unaware of any similar analysis in the literature. This reagent is = valuable, as=20 its glycan modifications may more closely resemble the patterns found on = viral=20 particles produced during infection in the human host compared with = baculovirus=20 and E. coli produced molecules. DV E protein has two conserved = N-linked=20 glycosylation sites at Asn-67 and Asn-153. Deglycosylation of sE by = PNGaseF led=20 to a shift in apparent mobility on SDS-PAGE from 52 kDa to 46 kDa (the = predicted=20 molecular weight of sE is 45 kDa), indicating that the protein carries = N-linked=20 glycan modifications (Figur= e=20 3A and 3= B).=20 Conversely, digestion of sE by endoglycosidase H, which cleaves high = mannose=20 oligosaccharides, did not result in a mobility shift on SDS-PAGE (Figur= e=20 3B). RNAse B was deglycosylated by both enzymes under corresponding = reaction=20 conditions as a positive control (data not shown). A more specific = glycan=20 analysis by sequential digestion with sialidase, fucosidase and = mannosidases (Figur= e=20 3C) showed approximately 40% of the glycoforms were sialylated and = 25%=20 contained =CE=B11=E2=80=933,4 linked outer arm fucose. There was no = evidence of terminal=20 mannose. The glycans were also processed by weak anion exchange (WAX) = HPLC=20 before and after sialidase digestion. There were charged glycoforms = remaining=20 after sialidase digestion which may be sulphated (data not shown).

Figure 3. Production and = Characterisation of=20 Recombinant Soluble Dengue Virus E-Glycoprotein

(A) SDS-PAGE of sE protein preparation resolved on 10% gel and = stained with=20 Coomassie Brilliant Blue. Lane 1, sE preparation as eluted from = NiNTA-agarose.=20 Lane 2, PNGaseF-treated sE. Lane 3, PNGaseF alone.

(B) Western blot of sE before (Lane 1) and after treatment with = PNGaseF (Lane=20 2) or EndoH (Lane 3), resolved under reducing conditions by 10% = SDS-PAGE, and=20 probed with the anti-DV E monoclonal antibody, 3H5.

(C) NP HPLC chromatograms of the complete pool of 2AB labelled = glycans of sE=20 glycoprotein, together with sequential digestions. a) Complete glycan = pool,=20 undigested, with structural representation of A2G2S2. b) Glycans = digested with=20 Arthrobacter ureafaciens sialidase (abs), which releases = =CE=B12=E2=80=936 and 3=20 linked sialic acids. Boxed sections show peaks containing = =CE=B11=E2=80=933 or 4 linked=20 fucose residues, with structural representations of A2G2 and A2FG2. c) = Glycans=20 digested with abs and almond meal fucosidase (amf), which releases = =CE=B11=E2=80=933 and 4=20 linked fucose residues.

Key: A2, biantennary; G, galactose; F, fucose; S, sialic acid. Filled = square,=20 N-acetyl glucosamine; open circle, mannose; open diamond, = galactose;=20 filled star, sialic acid; open diamond with dot, fucose. The solid lines = are=20 =CE=B2-linkage, dotted lines are =CE=B1-linkage, and curved lines are = unknown=20 linkage.

Cell Surface MR Expression Confers DV Binding

To further evaluate MR as a potential DV receptor, we examined = binding of DV=20 to human MR-transfected 3T3 cells (3T3.hMR). As DC-SIGN has previously = been=20 shown to be an important attachment receptor for DV, DV binding to = 3T3.hMR cells=20 was compared with binding to 3T3 cells transfected with DC-SIGN. = Initially we=20 confirmed expression of the respective receptors on the 3T3 cell surface = (Figur= e=20 4A and 4= B).=20 To assess virus binding, cells were incubated with mosquito = cell=E2=80=93grown DV,=20 unbound virus was washed away and DV bound to the cells was detected = with the=20 anti-E protein antibody, 3H5 (Figur= e=20 4C=E2=80=934= E).=20 Histograms show a clear shift in fluorescent intensity indicating DV = binding to=20 cells transfected with either human MR or DC-SIGN. Similar data were = observed=20 using anti-pre-membrane glycoprotein (prM) antibody (2H2; data not = shown) to=20 detect bound DV. Thus, surface expression of human MR on transfected 3T3 = cells=20 was sufficient to confer DV binding.

Figure 4. DV Binds to Cells = Transfected with=20 Human MR or DC-SIGN

The expression levels of (A) MR and (B) DC-SIGN on cells transfected = with MR=20 (3T3.hMR), DC-SIGN (3T3.DC-SIGN), or vector only (3T3) were assessed by = flow=20 cytometery. Receptor expression was detected with 15=E2=80=932 = (anti-hMR) or 120507=20 (anti-DC-SIGN) antibodies (pale and dark blue lines). Matched isotype = control=20 (purple and green lines) and secondary antibody only (brown and red = lines)=20 staining is included. Binding of NGC DV2 to cells transfected with (C) = vector=20 only (3T3), (D) MR (3T3.hMR), and (E) DC-SIGN (3T3.DC-SIGN) for 90 min = on ice=20 was detected by flow cytometry. The histograms show the binding of = anti-DV2=20 antibody (3H5; brown and green lines) and isotype matched control = antibody (blue=20 and red lines). The relative fluorescence intensity was measured by = FACSCalibur=20 analysis and the data are normalised and presented as percent of = maximum.=20 Representative data from one of two independent experiments are = shown.

Type 2 Cytokines Enhance M=C3=98 Susceptibility to DV Infection

A human primary cell culture assay system was established in which we = examined the functional role of MR and the effects of cytokines on the=20 susceptibility of mononuclear phagocytes to DV infection. Monocytes were = purified from human PBMC fractions and cultured for 2 or 7 d to prepare=20 monocyte-derived M=C3=98 (MDM=C3=98) or differentiated into = monocyte-derived dendritic=20 cells (MDDC) prior to infection. The percentage of cells infected was = quantified=20 by microscopy by staining nuclei with DAPI and viral antigen with the = anti-DV2 E=20 protein monoclonal antibody, 3H5. MDDC were more susceptible to = infection by DV=20 (percent infected DC: 12.3% +/=E2=88=92 7.1) compared with either 2 or 7 = d=20 differentiated MDM=C3=98 in the absence of added cytokine (percent = infected 2 d MDM=C3=98:=20 1.8% +/=E2=88=92 0.7; 7 day MDM=C3=98: 1.1% +/=E2=88=92 0.3) from the = same donor (3 donors), using a=20 multiplicity of infection of 0.4. The presence of DV non-structural = protein in=20 infected 2 d MDM=C3=98 using anti-NS1 monoclonal antibodies (obtained = from Eva=20 Harris) suggested that active viral replication and de novo viral = protein=20 production, rather than mere uptake of viral antigen, was occurring = (data not=20 shown). Plaque assays on supernatants from infected primary 2 or 7 d = MDM=C3=98 cell=20 cultures confirmed the occurrence of productive infection, with viral = titres=20 increasing over time and reaching 10⁴ pfu/ml in cell = supernatants 2 d=20 after infection of 6 =C3=97 10⁶ cells.

Considering that MDDC, which were grown in an IL-4/GM-CSF cytokine = cocktail,=20 were infected to a higher degree than the MDM=C3=98, various cytokines = were tested=20 for their ability to alter the susceptibility of MDM=C3=98 to DV = infection by=20 pre-incubation with monocytes for 48 h prior to DV infection (see Tabl= e=20 S1 for full list and concentrations). Only the type 2 cytokines IL-4 = and=20 IL-13, which utilize a common receptor chain, had a substantial effect = on the=20 susceptibility of MDM=C3=98 to DV infection in culture (Figur= e=20 5A and 5= B),=20 increasing the percentage of infected cells from around 1% to between 4% = and 21%=20 (Figur= e=20 5C). An almost 6-fold average increase in the percentage of cells = infected=20 was observed for 8 independent donors (p =3D 0.005). IL-4 may also = contribute to=20 the enhanced degree of DV infection of MDDC, as GM-CSF alone did not = increase=20 the level of infection of MDM=C3=98 (Tabl= e=20 S1).

Figure 5. IL-4 and IL-13 Enhance = Monocyte and=20 M=C3=98 Susceptibility to DV Infection

Monocytes isolated from human peripheral blood were either treated = with IL-4=20 or IL-13 or were left untreated for 2 d (day 2 MDM=C3=98) prior to = infection with=20 dengue virus. Two days following infection, permeabilized cells were = stained=20 with antibody to dengue E protein (green) and nuclei stained with DAPI=20 (blue).

(A) Low power image. (B) Single DV-infected cell confocal image = showing DV=20 protein distributed throughout the cytoplasm. Image represents a single=20 x-y section through the middle of a cell. Fluorescence = images=20 are shown next to the corresponding transmission image. (C) The = percentage of=20 cells infected was counted by microscopy. Each line represents a single = donor.=20 (D) Monocytes were matured into MDM=C3=98 by 7 d incubation prior to = treatment with=20 IL-4 for 2 d (day 9 MDM=C3=98). Cells were infected with DV and stained, = and percent=20 infected cells calculated.

Neither the age of the cells nor the length of treatment altered the=20 enhancing effects of IL-4. Enhanced susceptibility to DV infection was = seen when=20 monocytes were allowed to differentiate into M=C3=98 over 7 d, and then = treated with=20 IL-4 for 48 h prior to DV infection (day 9 MDM=C3=98, Figur= e=20 5D). An 8-fold increase in the percentage of infected cells was = observed for=20 3 independent donors (p =3D 0.03). Alternatively, monocytes treated for = 2=E2=80=937 d with=20 IL-4 prior to infection with DV all showed similar heightened = susceptibility=20 (data not shown). This suggested that the increased number of infected = cells was=20 not due to maturation of the cells. A dose response experiment indicated = that=20 the enhancement of DV infection of MDM=C3=98 could be achieved with as = little as=20 1.5ng/ml IL-4 (data not shown).

The interaction of DV with primary cells appears to be = multifactorial, as we=20 found variability between donors and receptor expression level. Surface=20 expression of both MR and DC-SIGN is upregulated on MDM=C3=98 by IL-4 = treatment (Figur= e=20 6A and 6= B),=20 consistent with previous data for DC-SIGN on human monocytes [20]=20 and for MR on primary mouse M=C3=98 [19,21].=20 While the fold increase in surface MR levels following IL-4 treatment = (4.1=E2=80=93fold=20 +/=E2=88=921.3) parallels the fold increase in the percent of infected = cells (4.6-fold=20 +/=E2=88=92 1.4), from analysis of a limited number of donors no clear = correlation can=20 be drawn between the two. This is also true of the relationship between=20 upregulation of surface DC-SIGN expression (7.7-fold +/=E2=88=92 0.9) on = the same=20 IL-4-treated cells and increase in the percent of infected cells (mean = of 4=20 donors; data not shown).

Figure 6. IL-4 Treatment Enhances = Surface=20 Expression of MR and DC-SIGN on Human MDM=C3=98

The effects of 2 d IL-4 treatment (25ng/ml) of human monocytes on = surface=20 expression levels of (A) MR and (B) DC-SIGN were assessed by flow = cytometery.=20 Receptor expression was detected with 15=E2=80=932 (anti-hMR) or 120507 = (anti-DC-SIGN)=20 antibodies (black lines) on untreated cells (open histogram) and = IL-4-treated=20 cells (filled histogram). Matched isotype control (grey lines) staining = is=20 included. The relative fluorescence intensity was measured by = FACSCalibur=20 analysis and the data are normalised and presented as percent of = maximum.=20 Representative data from one of eight donors are shown.

Anti-MR Antibodies Block DV Infection in Human M=C3=98

The functional role of MR in DV infection of primary human M=C3=98 = was=20 investigated using a polyclonal anti-MR antibody to block infection. = This was=20 examined in the IL-4-treated MDM=C3=98 since these cells showed the = highest rate of=20 DV infection. Anti-MR antibody significantly blocked DV infection of=20 IL-4-treated MDM=C3=98 (p =3D 0.008) in all donors tested (6 donors; Figur= e=20 7A shows data from one representative donor). Normal goat serum = control did=20 not inhibit infection, suggesting that the MR may be a new functional = receptor=20 contributing to DV infection of human M=C3=98. Production of infectious = virus=20 (pfu/ml) by these cells at 2 d post infection was reduced by = 60%=E2=80=9395% with either=20 mannan or anti-human MR antibody (Figur= e=20 7C), indicating that attachment and/or entry via this receptor is = required=20 for productive infection. The goat anti-human MR antibody blocked = mannosylated=20 BSA-FITC binding to both IL-4-treated MDM=C3=98 and 3T3 cells = transfected with human=20 MR (Figu= re=20 S1).

Figure 7. Anti-MR and Anti-DC-SIGN = Antibodies=20 Inhibit the Ability of DV2 to Infect IL-4-Treated Monocytes

(A) IL-4-treated human monocytes were incubated in triplicate wells = with=20 medium alone (no block), 2mg/ml mannan, titrations of goat anti-human MR = antiserum, or with normal goat serum (NGS). Treated cells were infected = with=20 mosquito cell=E2=80=93grown 16681 DV2 at a multiplicity of infection of = 0.5 in the=20 presence of these inhibitors, incubated for 48 h and fixed.

(B) Monocytes were treated in triplicate wells, as above, including = blocking=20 with monoclonal antibodies specific for DC-SIGN (120507), DC-SIGNR = (120604), and=20 both DC-SIGN and DC-SIGNR (DC28 and 120612) or isotype controls (all at = 5ug/ml)=20 prior to infection with mosquito cell=E2=80=93grown 16681 DV2 at a = multiplicity of=20 infection of 0.04 in the presence of these inhibitors. Following = fixation, cells=20 were immunolabelled with anti-DV E protein monoclonal antibody 3H5 and = counted=20 using a fluorescent microscope, and percent of cells infected = calculated. Data=20 are expressed as mean and SD of triplicate wells. Representative data = from one=20 of multiple donors are shown.

(C) The titre of infectious virus in the cell supernatant at 48 h = post=20 infection was determined by plaque assay. Due to variation between = donors, the=20 titre at 48 h in the absence of block was normalised to 100. Each data = point is=20 the average of three infected wells, each plaqued in triplicate, and the = results=20 from six donors are shown in this graph (mean represented by a bar). The = p value=20 was calculated by unpaired, two-tailed t test.

Mannan, which blocks MR, DC-SIGN and other receptors with specificity = for=20 mannose, also blocked DV infection of MDM=C3=98 (Figur= e=20 7A and 7= B).=20 We tested the ability of several different anti-DC-SIGN antibodies to = block=20 infection of MDM=C3=98. Antibodies against DC-SIGN have been shown by = others to block=20 DV infection of DC [3= ,4= ].=20 Interestingly, some anti-DC-SIGN monoclonal antibodies also blocked DV = infection=20 of IL-4-treated MDM=C3=98 (Figur= e=20 7B), and to a similar degree to that observed blocking with mannan = or=20 anti-MR antibodies.

Discussion

We have shown for the first time that MR is a functional receptor for = DV=20 infection of human M=C3=98. Binding of the MR to DV surface = glycoproteins was=20 mediated via the lectin activity of the CRD binding to glycans on the DV = E=20 protein. Gain of function binding data showed that surface expression of = human=20 MR on 3T3 cells was sufficient to confer DV binding. Antibodies specific = for the=20 MR significantly blocked both infection of MDM=C3=98 and the production = of infectious=20 virus in these cells. FACS analysis showed surface MR expression = increased over=20 4-fold following IL-4 treatment of monocytes, corresponding with a = similar fold=20 increase in percent infected cells. Thus, the MR provides a potential = link=20 explaining the increase in M=C3=98 permissiveness to DV when stimulated = by IL-4 or=20 IL-13. We hypothesise that the MR may play a role in at least one of the = stages=20 of DV infection of human M=C3=98. The first stage of virus entry into a = cell is=20 attachment, and the mechanism by which MR enhances the efficiency of DV = entry=20 could be by increasing virus attachment, as suggested for DC-SIGN and = DV. As the=20 MR can be internalized by macropinocytosis, pinocytosis, = receptor-mediated=20 endocytosis and phagocytosis, its role could also be in increasing the = rate of=20 DV internalisation, the second stage of virus entry. Analogous to the = proposed=20 mechanism of Fc-receptor enhancement of DV-antibody complex = attachment/uptake in=20 antibody-dependent enhancement, the presence of receptors such as MR or = DC-SIGN,=20 which enhance virus attachment/entry, may play a significant role in = vivo.

Anti-DC-SIGN monoclonal antibodies were also able to block DV = infection of=20 MDM=C3=98, as has been seen previously in MDDC. The anti-DC-SIGN = antibodies that=20 blocked DV infection of IL-4 treated MDM=C3=98 to the greatest degree = (DC28 and=20 120612) are known to cross react with DC-SIGNR. We hypothesise that the=20 different specificities of the monoclonal antibodies may explain why = some but=20 not all block DV infection of M=C3=98. These findings corroborate data = by=20 Tassaneetrithep et al. [4= ]=20 examining blocking of DV infection of THP-1 cells transfected with = either=20 DC-SIGN or DC-SIGNR, where the same two anti-DC-SIGN antibodies (but not = others)=20 blocked infection. The observation that antibodies to either MR or = DC-SIGN can=20 inhibit infection to such an extensive degree suggests that DV is likely = to be=20 using both MR and DC-SIGN for entry on cells that express both. It is = difficult=20 to assess the relative (and possibly differing) roles of MR and DC-SIGN = in DV=20 infection of primary M=C3=98 or DC that express both; however, our = observation that=20 expression of MR on 3T3 cells confers DV binding suggests that MR can = mediate=20 direct recognition of DV by myeloid cells. DC-SIGN has been suggested to = function in DV attachment rather than internalisation, raising the = possibility=20 of another receptor being involved in the internalisation step of = infection. At=20 this stage the simplest hypothesis that explains the above findings in = primary=20 cells expressing both MR and DC-SIGN is that DC-SIGN is required for DV=20 attachment and MR for internalisation. Alternatively, these receptors = may=20 function simultaneously and even co-operatively throughout the = infectious=20 process. Further studies on the cellular expression, location in the = cell and=20 ligand specificity of these receptors may provide clues as to the = absolute roles=20 they play in DV infection.

MR is ideally poised to act as a DV entry receptor given its = constitutive=20 recycling to the cell surface and ability to promote ligand = internalisation via=20 both endocytic and phagocytic pathways. While DC-SIGN mainly localises = to the=20 plasma membrane [22],=20 85% of cellular MR is located within the endocytic pathway = [8= ]=20 as a large intracellular receptor pool from which internalised receptor = is=20 rapidly replaced. In addition to expression on M=C3=98, certain = subpopulations of DC,=20 including dermal DC in human skin, express the MR [22],=20 in which case it may be involved in antigen delivery for presentation = [23,24].=20 MR is expressed on M=C3=98 as well as cells lining venous sinuses in = human spleen [25]=20 and is therefore well located to act as a receptor for DV replication in = these=20 physiologically relevant target cells. Here it may have roles in = clearance or=20 adhesion, but also potentially in vascular leakage. A soluble form of MR = has=20 been found to be abundant in mouse [26]=20 and human plasma (unpublished observations, L. Martinez-Pomares) and its = CR=20 domain has targets in the spleen. Binding of ligands by soluble MR can = result in=20 the transport of MR ligands to the B cell follicles, which may lead to = clearance=20 or enhanced presentation of viral antigen depending on TLR = co-stimulation.=20 Soluble MR could also potentially play a role in protection of virus = from=20 complement activation. Further elucidation of the exact role of MR in = the=20 attachment/entry/infectivity of DV will be a fundamental step in gaining = a=20 better understanding of DV pathogenesis.

MR and DC-SIGN both contain lectin domains, but differ distinctly in = terms of=20 ligand specificity, with MR binding terminal mannose, fucose and = N-acetyl=20 glucosamine, and DC-SIGN binding mannose within high-mannose = oligosaccharides=20 and fucosylated glycans [8= ,27,28].=20 Glycosylation endows unique properties to glycoproteins and can play a=20 significant role in immunity. Recent observations using DV mutants in = one or=20 both of the N-linked glycosylation motifs have shown that N-linked = glycosylation=20 at Asn-67 is required for virus growth in mammalian cells [29].=20 In addition, new data by Mondotte and colleagues showed that DV lacking=20 carbohydrate at Asn-67 had reduced capacity to infect MDDC [30].=20 This, combined with the observation that MDDC express both DC-SIGN and = MR [22],=20 and our demonstration that the presence of MR alone is sufficient to = confer DV=20 binding to transfected cells, suggest that glycosylation at Asn-67 may = be=20 relevant for mediating MR binding, in addition to that of DC-SIGN. While = the sE=20 may contain sulphated glycans, it was not bound by the CR fusion protein = (Figur= e=20 1B), and so the CR domain of the MR is not expected to contribute to = the=20 binding of MR to DV. Our deglycosylation studies on sE show that it = bears either=20 complex or hybrid N-linked glycans. Terminal fucose is a reported ligand = of MR=20 and as such is the likely ligand on this source of DV antigen. A more = detailed=20 glycan analysis of DV grown in human M=C3=98 will be an important = challenge for the=20 future.

We expanded our study of the interaction of MR with DV by = demonstrating=20 binding of CRD4-7-Fc to all four serotypes of DV. Differences in = glycosylation=20 between DV serotypes, and more broadly between different flaviviruses, = may be=20 relevant for interaction with lectin receptors such as MR and DC-SIGN. = We showed=20 that MR can bind in ELISA to Japanese encephalitis virus and tick-borne=20 encephalitis virus, both of which are reported to have glycosylated = envelope=20 proteins.

In this report we have shown that the type 2 cytokines IL-4 and IL-13 = enhance=20 the susceptibility of MDM=C3=98 to DV infection. The mechanisms = resulting in=20 increased infection in response to IL-4 and IL-13 are unknown. Analysis = of=20 IL-4-treated human monocytes showed that these cells are characterised = by the=20 overexpression and enhanced function of several endocytic receptors, = including=20 scavenger and C-type lectin receptors [18].=20 Functional ligand binding and transcriptional profiling studies [19,31]=20 reveal that the MR is markedly upregulated on alternatively activated = M=C3=98.

A number of important conditions result in polarised activation of = M=C3=98=20 phenotype. Our findings make it highly relevant to understand the = clinical and=20 epidemiological significance of a Th2 environment on DV infection, = pathogenesis,=20 and enhancement and in the development of a desirable vaccine response. = It will=20 be of great interest to examine the broader context in which dengue = pathogenesis=20 occurs by considering the effects on DV disease of settings that induce = Th2=20 cytokines, including co-infection with parasites, the presence of immune = complexes and allergy (e.g. asthma). There are few studies into the = implications=20 of co-incidence of parasitic infections or allergic disease and dengue=20 infection. Guzman and colleagues showed significantly enhanced = replication of DV=20 in PBMC from asthmatic patients compared with controls [32].=20 During secondary dengue infection when anti-DV antibody is present, the = study of=20 DV interactions with M=C3=98 stimulated through Fc=CE=B3R ligation = (=E2=80=98type II' activated=20 M=C3=98), may be of particular relevance. Given the risk of = antibody-dependent=20 enhancement to vaccine trials, a Th2 environment (which is known to = influence=20 humoral responses and FcR expression [33,34])=20 may be a contributing factor to vaccine efficacy and DV pathogenesis and = as such=20 will require further examination.

Materials and Methods

Viruses and cell lines.

16681 and New Guinea C (NGC) strains of DV2 (both gifts from E. = Gould, Oxford=20 Centre for Ecology and Hydrology, UK) were propagated in the Aedes=20 albopictus=E2=80=93derived C6/36 cell line (a gift from Armed = Forces Research=20 Institute of Medical Sciences, Thailand). Virus titres were obtained by = plaque=20 assay on LLC-MK2 monkey kidney cells (a gift from Armed Forces Research=20 Institute of Medical Sciences, Thailand), as described previously [35].=20 16681 strain DV2 grown in Vero cells, precipitated with polyethylene = glycol,=20 purified on a sucrose gradient and inactivated with formaldehyde was = from=20 Biodesign. Hawaii strain DV1, NGC strain DV2, H-87 strain DV3 and H-241 = strain=20 DV4 grown in C6/36 cells were precipitated with 7% polyethylene glycol = and=20 inactivated with beta-propiolactone (Biodesign). Human 293T-HEK cells = (ATCC)=20 were maintained in Dulbecco's modified Eagle's medium (DMEM) = (Invitrogen)=20 supplemented with 2mM glutamine, 0.1mg/ml streptomycin, 100U/ml = penicillin and=20 10% heat inactivated fetal calf serum (FCS).

A stable NIH3T3 cell line expressing the human MR (3T3.hMR) was a = gift from=20 Gordon Brown (University of Cape Town, South Africa) and Philip Taylor=20 (University of Cardiff, UK), made using the protocol described = previously [21].=20 Human MR was amplified from cDNA derived from human mRNA. High = expressing clones=20 were selected after limiting dilution in 96 well plates. Control 3T3 = cells=20 expressing the vector only were prepared in parallel. Production of = stable=20 NIH3T3 expressing the murine MR (3T3.mMR) has been described previously = [21].=20 These NIH3T3 transfectants were maintained in DMEM (Invitrogen) = supplemented as=20 above and with 0.6mg/ml geneticin (Invitrogen). NIH3T3 cells transfected = with=20 DC-SIGN (3T3.DCSIGN) were obtained through the NIH AIDS Research and = Reference=20 Reagent Program (from Drs Thomas Martin and Vineet KewalRamani) and = maintained=20 in similar DMEM medium without geneticin.

Reagents.

Fc chimeric proteins (derived from the murine MR) and the=20 hemagglutinin-tagged form of MR (MR-HA) were prepared by Richard = Stillion as=20 described previously [7= ,36].=20 Mannan was from Saccharomyces cerevisiae (Sigma). Antibodies = were=20 specific for DV2 E protein (3H5; a gift from Dale Greiner), MR (goat = anti-hMR; a=20 kind gift from Philip Stahl (Washington University School of Medicine, = St.=20 Louis, MO), DC-SIGN (120507), DC-SIGNR (120604) and both DC-SIGN and = DC-SIGNR=20 (DC28 and 120612) (all from R&D Systems). The specificity of = anti-hMR=20 antibody is examined in Figu= re=20 S1.

Preparation of primary cells.

For the generation of MDM=C3=98 and MDDC, human PBMC were isolated = from buffy=20 coats (NHS Blood and Transport) by centrifugation over a Ficoll-PaqueTM = PLUS=20 (Amersham) gradient, according to standard protocols. Adherent = monocytes,=20 isolated as described previously [37],=20 were cultured in X-VIVO medium (BioWhittaker) with 1% heat-inactivated=20 autologous plasma to allow differentiation into MDM=C3=98. These cells = were >95%=20 macrophages phenotypically (CD14+, CD16-, CD86+, HLA-DR+, CD3=E2=88=92) = (data not=20 shown). For the generation of MDDC, recombinant human IL-4 (25ng/ml; = Peprotech)=20 and GM-CSF (50ng/ml; Peprotech) were added to monocytes in X-VIVO medium = (BioWhittaker) with 1% heat-inactivated autologous plasma and the cells = cultured=20 for 4 d. The use of human blood was approved by the central Oxford = University=20 research ethics committee (MSD/IDEC/C1/2006/32).

DV infection.

Monocytes were treated with recombinant human IL-4 (25ng/ml; = Peprotech) or=20 IL-13 (10ng/ml; Peprotech) for 2 d then infected with mosquito = cell-grown 16681=20 DV2 virus. After 1 h the viral supernatant was replaced with cell = culture medium=20 without cytokine and the cells incubated for 2 d then fixed with 4%=20 paraformaldehyde. In some experiments the IL-4-treated MDM=C3=98 were = incubated with=20 various blocking reagents for 40 min at 37=C2=B0C before the addition of = 16681 DV2=20 virus (in the presence of blocking agent).

Detection of MR fusion protein binding to DV antigen ELISA.

96-well Maxi-Sorp plates were coated overnight with DV antigens = (50ug/ml=20 C6/36-grown NGC DV2 and 20ug/ml sE, or as specified in Figur= e=20 1) in phosphate-buffered saline (PBS; 138mM NaCl, 2.7mM KCl, 8mM=20 Na₂HPO₄, 1.5mM KH₂PO₄, [pH = 7.4]) at=20 4=C2=B0C in triplicate. Wells were blocked with 0.5% immunoglobulin-free = BSA (Sigma)=20 before incubation with 15 ug/ml MR-HA, 2ug/ml human Fc-fusion MR domain = proteins=20 or 15ug/ml rabbit anti-DV1=E2=80=934 antibody for 2 h at room = temperature in Tris=20 buffered saline (TBS; 10mM TrisHCl, pH 7.4, 0.154 M NaCl, 0.05% Tween = 20)=20 containing 10mM CaCl₂. For competitive binding studies MR = fusion=20 proteins were pre-incubated for 30 min on ice in TBS containing 1M NaCl = and 10mM=20 CaCl₂ in the presence of 2mM d-mannose,=20 2mM l-fucose or 2mM galactose, or TBS = containing=20 10mM EDTA. The wells were washed 6 times and binding of the Fc-fusion = proteins=20 was detected by incubating the wells with an alkaline = phosphatase-conjugated=20 anti-human antibody (1:1,000 dilution), visualized using 1mg/ml = 4-Nitrophenyl=20 phosphate disodium salt hexahydrate in 100mM Tris, 100mM NaCl, 5mM=20 MgCl₂, pH9.5, and the absorbance was read at 405nm. The wells = with=20 HA-tagged protein were incubated with 10ug/ml of MR5D3 (rat anti-mouse = MR; [21])=20 for 1 h at room temperature followed by an alkaline = phosphatase-conjugated=20 anti-rat antibody (1:1,000 dilution) and visualized as above. The wells = with=20 rabbit anti-DV1=E2=80=934 were incubated with an alkaline = phosphatase-conjugated=20 anti-rabbit antibody (1:1,000 dilution) and visualized as above. Wells = coated=20 with mannan and SO₄3GalNAc-PAA (Lectinity) were included as a = positive control for proteins containing the CRD4=E2=80=937 and CR = domains respectively=20 (data not shown). We verified that DV2 ligands had coated all of the = wells=20 equally by using direct ELISA detection of DV antigens 3H5 (10ug/ml) = followed by=20 a goat anti-mouse alkaline phosphatase-labelled (1:1,000 dilution) = secondary=20 antibody and visualised as above.

Blot overlays.

CRD4=E2=80=937 binding to sE was investigated by blot overlay by = running 0.9ug sE=20 protein and 0.9ug sE protein deglycosylated with peptide: N-glycosidase = F (New=20 England Biolabs) on 10% SDS-PAGE, and subsequently transferring proteins = to=20 Hybond C-Extra nitrocellulose membranes (Pharmacia). Blots were blocked = for 1 h=20 in 0.5% skimmed milk powder in TBS containing 10mM CaCl₂=20 (blocking/washing solution). Blots were probed with 1ug/ml MR = CRD4=E2=80=937-Fc in the=20 absence or presence of either 2mM d-mannose, 2mM=20 l-fucose or 20mM EDTA for 2 h, and then = washed=20 three times with blocking solution. Binding was detected with 1ug/ml = horseradish=20 peroxidase-conjugated anti-human IgG antibody (Vector Laboratories) and=20 visualized by chemiluminescence. Blots probed in the presence of = inhibitors were=20 washed 3 times and reprobed with MR CRD4=E2=80=937-Fc in the absence of = inhibitor. Blots=20 were stripped by incubation in 63mM Tris, pH 6.7, 2% SDS and 100mM=20 2-mercaptoethanol at 50=C2=B0C for 30 min, re-blocked and probed with = the anti-DV=20 E-protein antibody, 3H5, at 10ug/ml. Binding was detected with 10ug/ml=20 horseradish peroxidase-conjugated anti-mouse IgG antibody and visualized = by=20 chemiluminescence.

Production of recombinant sE protein.

An open reading frame consisting of the last 20 aa of C-protein, the = entire=20 prM protein, and E protein truncated by 96 amino acids at the C-terminus = and=20 containing a hexahistidine tag was amplified by PCR from cDNA prepared = from DV2=20 strain 16681. The expression cassette was cloned in the mammalian = expression=20 vector pLEX [38]=20 and transfected into human 293T-HEK cells cultured in Optimem = (Invitrogen), and=20 sE was partially purified from the supernatants of these cultures by=20 Ni-chelation affinity chromatography (Figur= e=20 3A). The identity of the protein was confirmed by mass-spectrometric = analysis of peptides resulting from tryptic digest of the excised = SDS-PAGE band=20 (data not shown).

sE glycan analysis.

Glycans were released from approximately 25ug of recombinant soluble = dengue=20 virus E-glycoprotein and labelled by reductive amination with the = fluorophore=20 2-aminobenzamide [39,40].=20 The glycans were processed through NP-HPLC and the retention times for = the=20 individual glycans were converted to glucose units (GU) using a standard = dextran=20 curve [41].=20 These were then compared with a database of experimental values (http://glycobase.ucd.ie/cgi-bin/public/glycobase.cgi) and initial assignments made were = confirmed=20 following digestions of the glycans with an array of exoglycosidase = enzymes [40].

Flow cytometry.

FACS was performed according to conventional protcols at 4=C2=B0C in = the presence=20 of 2mM NaN₃. Non-specific binding sites on cells were blocked = with=20 PBS containing 5% heat-inactivated rabbit serum, 5% heat-inactivated = goat serum,=20 0.5% BSA and 5mM EDTA (blocking buffer) before the addition of primary=20 antibodies. Surface expressed MR and DC-SIGN was detected using 10ug/ml = 15=E2=80=932=20 (Serotec) and 120507 (R&D Systems) monoclonal antibodies, = respectively, and=20 was compared with an isotype control (Serotec). Surface bound DV was = detected=20 using 10ug/ml 3H5. The primary antibodies were detected using Alexafluor = 488-conjugated anti-mouse antibody (Molecular Probes) diluted 1:200 in = blocking=20 buffer. Cells were fixed with 1% paraformaldehyde in PBS before = analysis.=20 Binding was quantified on a FACSCalibur flow cytometer and data from = 10,000 cells were routinely acquired for each sample. Data = were=20 analysed using FlowJo software (Treestar). Percent of max represents the = number=20 of events normalised according to FlowJo algorithms. Fold increase in = receptor=20 expression was measured by geometric mean fluorescent intensity of = specific=20 receptor antibody staining for IL-4-treated cells divided by untreated=20 cells.

Cell binding assays.

Cells suspensions were prepared by scraping cells to preserve = receptor=20 expression at the cell surface. Mosquito cell-grown NGC DV2 (1.5 =C3=97=20 10⁶ pfu/ml) or media was incubated with cells (4 =C3=97 = 10⁶) at=20 a multiplicity of 0.35 infectious virions per cell for 80 min on ice. = Unbound=20 virus was washed away with cold media, the cells were fixed with 1%=20 paraformaldehyde in PBS, and surface bound virus detected with anti-DV = E-protein=20 antibody by flow cytometry as described above.

FITC-labelled, mannosylated or galactosylated BSA (5ug/ml; Sigma) was = incubated for 90 min at 37=C2=B0C with primary human MDM=C3=98 or 3T3 = transfectants plated=20 on tissue culture-treated plastic. For blocking studies cells were = pre-incubated=20 with mannan (2mg/ml), normal goat serum (NGS) or goat-anti hMR antibody = for 20=20 min at 37=C2=B0C. After incubation cells were washed with PBS, harvested = using PBS=20 containing 5mM EDTA and lidocaine (4mg/ml) and fixed in 2% = paraformaldehyde in=20 PBS. Binding was quantified by a FACSCalibur flow cytometer and analysed = using=20 FlowJo software.

Immunofluorescence microscopy.

Fixed, DV-infected cells were permeabilised with 0.5% Triton-X and = stained=20 with 10ug/ml 3H5 followed by an Alexafluor 488-labelled secondary = anti-mouse IgG=20 antibody (Molecular Probes) and the nuclei stained with DAPI. Stained = coverslips=20 were mounted in DakoCytomation fluorescent mounting medium (Dako), and = analyzed=20 using either CCD1 (Axioplan) and CCD camera (Spot) or a = META^TM=20 confocal microscope linked to LSM 510^TM software (Carl Zeiss=20 MicroImaging, Inc.). Confocal images were acquired sequentially using = the=20 multitrack configuration of the Zeiss META^TM to avoid = bleed-through=20 between fluorescence channels, and the appropriate controls with and = without=20 primary antibody were performed. Additional image processing was = performed using=20 Adobe Photoshop 7. The image is presented as single two-dimensional = x-y=20 sections and the corresponding transmission image. At least twelve = fields=20 (600=E2=80=931,000 individual cells) were counted by fluorescent = micrsocopy.

Statistical analysis.

Statstics were calculated using GraphPad PRISM (version 2.0; GraphPad = Software, San Diego, CA) and Microsoft Excel. Two-tailed Student's = t=20 tests were used to calculate p values.

Supporting Information

Figure=20 S1. Goat Anti-Human MR Antibody Specifically Blocks = Mannosylated=20 BSA-FITC Binding to Cells Expressing Human MR

(2.1 MB TIF)

Table=20 S1. Effects of Cytokines and Cytokine Combinations on DV = Infection=20 of Monocytes

(35 KB RTF)

Acknowledgments

We acknowledge Alexandre Akoulitchev and Ben Thomas at the Central = Proteomics=20 Facility of the Dunn School of Pathology for mass spectrometry analysis, = Eva=20 Harris and Jennifer Kyle for monoclonal antibodies to dengue virus and = helpful=20 advice, Richard Stillion for preparation of Fc chimeric and HA-tagged = mannose=20 receptor proteins, and Alan Barratt for recombinant domain III.

Author = contributions. JLM,=20 BJMdW, LMP, CMR, PMR, and SG conceived and designed the experiments. = JLM, BJMdW,=20 and CMR performed the experiments. JLM, BJMdW, CMR, PMR, and SG analyzed = the=20 data. LMP, CMR, RAD contributed reagents/materials/analysis tools. JLM, = BJMdW,=20 LMP, CMR, and SG wrote the paper.

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