The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.The first step in viral entry is the stable attachment of the virion to the surface of a new target cell, a process that can be inefficient for many viruses (16,34,62,73). Cellular proteins that facilitate productive infection by increasing the efficiency of virus binding, but whose presence is not absolutely required for viral entry, are often referred to as attachment factors (4). Two of the most extensively studied attachment factors are the lectins DC-SIGN (CD209) (18, 29) and DC-SIGNR (L-SIGN) (CD209L) (7,67,78). Both are tetrameric type II transmembrane proteins containing calcium-dependent (C-type) carbohydrate recognition domains (CRDs) (55). DC-SIGN is highly expressed in monocyte-derived dendritic cells (MDDCs) in vitro (29) and at lower levels (86) in vivo in subsets of macrophages (45,53,79) and dendritic cells (23,29,40,80,86). DC-SIGNR is expressed on microvascular endothelial cells, especially in the liver sinusoids and lymph nodes (7,67,81). By facilitating virion attachment, DC-SIGN and DC-SIGNR [henceforth referred to collectively as DC-SIGN(R)] can greatly increase the susceptibility of permissive cells to infection by a wide array of enveloped viruses or allow nonpermissive cells to capture and transmit these viruses to target cells in trans (3,17,35,47,52,60,76,84).Viruses that bind to DC-SIGN(R) appear to do so via highmannose, N-linked glycans on their glycoproteins (44,48,51). This fact is readily explained by crystallographic studies demonstrating that mannose-rich oligosaccharides fit into elongated binding sites in the CRDs of DC-SIGN(R) (24). In addition to recognizing viral...

The two lectin receptors, CLEC-2 and Dectin-1, have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In this study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signaling by immunoreceptor tyrosine-based activation motif receptors, including Src, Syk, and Tec family kinases, and on phospholipase C␥. Strikingly, mutation of either Src homology (SH) 2 domain of Syk blocks signaling by CLEC-2 despite the fact that it has only a single YXXL motif. Furthermore, signaling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of immunoreceptor tyrosine-based activation motif receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same four amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2 domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signaling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif that requires the tandem SH2 domains of Syk but is only partially dependent on the SLP-76/BLNK family of adapters.The C-type lectin superfamily of transmembrane proteins consists of at least 70 members in the human genome (1). The superfamily can be divided into "classical" C-type lectins, which contain a carbohydrate recognition domain and bind sugars in a calcium-dependent manner, and the "nonclassical" C-type lectin-like proteins, which contain a C-type lectin-like domain, homologous to a carbohydrate recognition domain, but lacks the consensus sequence for binding sugars and calcium (2). Protein ligands for a number of classical and nonclassical C-type lectin receptors have been described.C-type lectin-like receptor 2 (CLEC-2) 6 is a type II transmembrane protein and a nonclassical C-type lectin (3). The C-type lectin-like domain in CLEC-2 is supported by a 41-amino acid neck region, a single transmembrane domain, and 31-amino acid cytoplasmic domain (3). CLEC-2 mRNA has been identified in liver and in blood cells, mostly of myeloid origin, including monocytes, granulocytes, and dendritic cells (3). Recently, we have identified expression of CLEC-2 in platelets and have shown that it functions as a receptor for the snake venom toxin rhodocytin (also known as aggretin), which elicits powerful platelet activation (4). Rhodocytin, however, also binds to several other platelet receptors (5, 6), making it unclear whether CLEC-2 is sufficient to mediate activation alone and thereby hampering analysis of the mechanism of activation.The cytosolic domain of CLEC-2 con...

West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E).West Nile virus (WNV) is an arthropod-borne virus classified in the Japanese encephalitis antigenic complex of the family Flaviviridae (9, 20, 32). The natural transmission cycle of WNV involves mosquitoes and birds, with humans and other mammals as incidental hosts (8,27). Phylogenetic analysis of WNV strains reveals the presence of two closely related but nonetheless distinct virus groups termed lineage I and lineage II (6). Lineage I strains (which include Kunjin viruses) are distributed worldwide (10,33) and are responsible for all major human outbreaks to date, including the current WNV epidemic in North America (35). In contrast, lineage II WNV strains are restricted to central and southern Africa and do not appear to be as pathogenic as lineage I isolates (6,7,35).WNV contains a single-stranded, plus-sense RNA genome that is translated as a single polyprotein (9). Cleavage of the polyprotein by viral and cellular proteases liberates the viral integral membrane proteins premembrane (prM) and envelope (E), as well as the capsid and seven nonstructural proteins (51). Flavivirus prM and E proteins form heterodimers in the endoplasmic reticulum (ER), where they facilitate virus budding into the ER (2, 40), although one report suggests that the WNV Sarafend strain buds at the plasma membrane (47). As particles travel through the secretory pathway, the bulk of the prM ectodomain is removed by endoproteolysis during transit through the trans-Golgi network (63). Cleavage of prM enables E protein to form head-to-tail homodimers, which form a lattice-like structure covering the surface of the mature, 50 nM diameter virus particle (44). During the process of viral entry, the E protein interacts with an unidentified cell surface receptor(s), followed by uptake into endosomes where the E protein undergoes conformational changes at mildly acid pH, resulting in fusion between the viral and cellular membranes (16,26).All WNV isolates contain a highly conserved N-linked glycosylation site within the ectodomain region of prM that is released during the final stages of particle maturation. In contrast, an N-linked glycosylation site in the E protein at residue 154 is present in many but not all lineage I strains. This site is also present in some lineage II strains, though others contain a 4-amino-acid deletion that ablates this N-linked glycosylation site (1, 6). Interestingly, many of the WNV isolates associated with significant human outbreaks, including the current North American epidemic, contain the N-linked glycosylation site in E (22,35,54). In addition, N-linked glycosylation of the WNV E protein may be associated with altered viral growth in vitro (56) and neuroinvasiveness in a murine model (3,5,61). Nlinked glycosylation of the E protein in WNV and other flaviviruses has been linked to alterations in pH sensitivity (5, 36) and virus yield (65) and likely plays a significant role in the interaction between dengue virus and DC-S...

Summary Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of for this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter Natural Resistance-Associated Macrophage Protein (NRAMP), as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were non-permissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multi-pass membrane proteins for infection.

Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography-tandem mass spectrometry (µLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient : strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels. Fourteen proteins were selected for analysis ; this group included those whose expression was common between both strains as well as unique for each strain. The proteins were identified by µLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project ; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.