Functional Analysis of Cell Surface-Expressed Hepatitis C Virus E2 Glycoprotein

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to "native" E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry.     

Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).     

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.     

Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

The human CD81 (hCD81), the most recently proposed receptor of hepatitis C virus (HCV), can especifically bind to HCV envelope glycoprotein2 (E2). In this study, hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library (PVⅢ9aaCys). Eighteen of the 75 clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay (ELISA) and competitive inhibition test. Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA. Sequence comparison of the motif showed no amino acid homology with the native HCV E2. The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells, and induce HCV E2 specific immune response in vivo. These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules. Our findings cast new light on developing HCV receptor antagonists.     

Structural characterization of peptide fragments from hCD81-LEL.

The solution conformation of two peptides [1: PSGSNIISNLFKED; 2: GSSTLTALTTSVLKNNL] from human CD81 (hCD81) large extra-cellular loop (LEL) with known importance in the hepatitis C virus glycoprotein E2 (HCV-E2) binding interaction was characterized using circular dichroism spectroscopy. In addition, the solution structure of peptide 1 that contains a phenylalanine residue (F186 in hCD81) known to be critical in the binding interaction with HCV-E2 was determined using 1D and 2D 1H NMR spectroscopy. Both peptides are unstructured in water but begin forming significant helical conformation following the addition of 20% or more trifluoroethanol (v/v), a result consistent with their alpha-helical conformation found in the native protein. The CD data recorded as a function of pH and NaCl concentration are consistent with stabilization of the helical structure from electrostatic forces for both peptides. Peptide 1 is able to block the binding interaction of recombinant HCV-E2 (rHCV-E2) to hCD81 expressed on Molt-4 T cells at high concentrations (3.5 mM), a low affinity that we attributed to the random coil structure in water.     

Structural biology of human CD81, a receptor for hepatitis C virus

Human CD81, which is belonged to tetraspanin family, has been previously identified as a receptor for the hepatitis C virus envelope E 2 glycoprotein. The crystal structure of the human CD81 long extracellular domain, binding site for E 2 glycoprotein, is presented here at 1.6 A resolution. The tertiary structure of CD81-LEL, which is composed of five alpha-helices, is resemble for a mushroom-shaped molecules (stalk and head subdomains) and forms a dimer in the crystallographic asymmetric unit. The two disulfide bridges, which are conserved all the tetraspanin and are necessary for CD 81-HCV interaction, are stabilizing the conformation of the head domain. This head domain is solvent exposed surface region and is locating the amino acid residues which are essential for the E 2 binding. The hydrophobic cluster in this head domain may suggest that the presence of a docking site for a low complementary surface cavity in the partner E 2 glycoprotein. We proposed that the dimer structure may be important in the interactions of HCV E 2 glycoprotein and also the viral protein may occur in dimeric aggregation on the HCV envelope. This common structural motif of the tetraspanin provides the first insight onto the mechanism of HCV binding to human cell and may be targets for structure-based antiviral drug.     

Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein

The E2 protein of hepatitis C virus (HCV) is believed to be a virion surface glycoprotein that is a candidate for inclusion in an antiviral vaccine. A truncated soluble version of E2 has recently been shown to interact with CD81, suggesting that this protein may be a component of the receptor for HCV. When expressed in eukaryotic cells, a significant proportion of E2 forms misfolded aggregates. To analyze the specificity of interaction between E2 and CD81, the aggregated and monomeric forms of a truncated E2 glycoprotein (E2(661)) were separated by high-pressure liquid chromatography and analyzed for CD81 binding. Nonaggregated forms of E2 preferentially bound CD81 and a number of conformation-dependent monoclonal antibodies (MAbs). Furthermore, intracellular forms of E2(661) were found to bind CD81 with greater affinity than the extracellular forms. Intracellular and secreted forms of E2(661) were also found to differ in reactivity with MAbs and human sera, consistent with differences in antigenicity. Together, these data indicate that proper folding of E2 is important for its interaction with CD81 and that modifications of glycans can modulate this interaction. Identification of the biologically active forms of E2 will assist in the future design of vaccines to protect against HCV infection.     

Identification and expression of human CD81 gene on murine NIH/3T3 cell membrane

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). In this study, eukaryotic expression vector pCDM8-hCD81 containing hCD81 cDNA and pSV2neo helper plasmid was used to cotransfect with lipofectamine into murine fibroblast cell line NIH/3T3 to establish an hCD81-expressing cell line. Resistant cell clones were obtained 20 days after the selection with neomycin (600 micro/ml) and then cultured as monoclones. The expression of the transfected hCD81 gene in the cells was verified by RT-PCR and flow cytometry analyses. One of the selected cell clones showed obvious expression of hCD81 and was named NIH/3T3-hCD81. Competitive inhibition tests indicated that the binding of monoclonal anti-hCD81 (JS-81) to NIH/3T3-hCD81 cells was inhibited by recombinant HCV E2 protein, suggesting that the expressed hCD81 molecules on NIH/3T3-hCD81 cells maintain natural conformation of binding to HCV E2. The transfected NIH/3T3-hCD81 cells should be of great potential value in studies on HCV attachment and onset of infection.     

Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2

The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.     

Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner

We recently reported that retroviral pseudotypes bearing the hepatitis C virus (HCV) strain H and Con1 glycoproteins, genotype 1a and 1b, respectively, require CD81 as a coreceptor for virus-cell entry and infection. Soluble truncated E2 cloned from a number of diverse HCV genotypes fail to interact with CD81, suggesting that viruses of diverse origin may utilize different receptors and display altered cell tropism. We have used the pseudotyping system to study the tropism of viruses bearing diverse HCV glycoproteins. Viruses bearing these glycoproteins showed a 150-fold range in infectivity for hepatoma cells and failed to infect lymphoid cells. The level of glycoprotein incorporation into particles varied considerably between strains, generally reflecting the E2 expression level within transfected cells. However, differences in glycoprotein incorporation were not associated with virus infectivity, suggesting that infectivity is not limited by the absolute level of glycoprotein. All HCV pseudotypes failed to infect HepG2 cells and yet infected the same cells after transduction to express human CD81, confirming the critical role of CD81 in HCV infection. Interestingly, these HCV pseudotypes differed in their ability to infect HepG2 cells expressing a panel of CD81 variants, suggesting subtle differences in the interaction of CD81 residues with diverse viral glycoproteins. Our current model of HCV infection suggests that CD81, together with additional unknown liver specific receptor(s), mediate the virus-cell entry process.     

Identification of the hepatitis C virus E2 glycoprotein binding site on the large extracellular loop of CD81

The binding of hepatitis C virus glycoprotein E2 to the large extracellular loop (LEL) of CD81 has been shown to modulate human T-cell and NK cell activity in vitro. Using random mutagenesis of a chimera of maltose-binding protein and LEL residues 113 to 201, we have determined that the E2-binding site on CD81 comprises residues Ile(182), Phe(186), Asn(184), and Leu(162). These findings reveal an E2-binding surface of approximately 806 A(2) and potential target sites for the development of small-molecule inhibitors of E2 binding.     

Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding

Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.     

猪瘟病毒囊膜糖蛋白E2中和表位的定位及其免疫反应性分析

猪瘟病毒(CSFV)囊膜结构糖蛋白E2(gp55)是激发保护性免疫应答的主要抗原蛋白。E^ms和E2与细胞表面受体的相互作用介导病毒对细胞的感染过程。采用抗CSFV中和性单克隆抗体c24／10,淘选噬菌体展示的12肽随机肽库,结合噬菌体拟位免疫反应性分析结果,对CSFV E2蛋白中和表位进行定位。结果表明：F2蛋白的SPTTLR基序(832～837位氨基酸)构成CSFV特异性线性中和表位,基序的第一、二、三位氨基酸是表位与单克隆抗体c24／10结合所必需的氨基酸,也是表位的关键性氨基酸。     

Binding of hepatitis C virus E2 glycoprotein to CD81 does not correlate with species permissiveness to infection

Hepatitis C virus (HCV) glycoprotein E2 binds to human cells by interacting with the CD81 molecule, which has been proposed to be the viral receptor. A correlation between binding to CD81 and species permissiveness to HCV infection has also been reported. We have determined the sequence of CD81 from the tamarin, a primate species known to be refractory to HCV infection. Tamarin CD81 (t-CD81) differs from the human molecule at 5 amino acid positions (155, 163, 169, 180, and 196) within the large extracellular loop (LEL), where the binding site for E2 has been located. Soluble recombinant forms of human CD81 (h-CD81), t-CD81, and African green monkey CD81 (agm-CD81) LEL molecules were analyzed by enzyme-linked immunosorbent assay for binding to E2 glycoprotein. Both h-CD81 and t-CD81 molecules were able to bind E2. Competition experiments showed that the two receptors cross-compete and that the t-CD81 binds with stronger affinity than the human molecule. Recently, h-CD81 residue 186 has been characterized as the critical residue involved in the interaction with E2. Recombinant CD81 mutant proteins were expressed to test whether human and tamarin receptors interacted with E2 in a comparable manner. Mutation of residue 186 (F186L) dramatically reduced the binding capability of t-CD81, a result that has already been demonstrated for the human receptor, whereas the opposite mutation (L186F) in agm-CD81 resulted in a neat gain of binding activity. Finally, the in vitro data were confirmed by detection of E2 binding to cotton-top tamarin (Saguinus oedipus) cell line B95-8 expressing endogenous CD81. These results indicate that the binding of E2 to CD81 is not predictive of an infection-producing interaction between HCV and host cells.     

Structural characterization of recombinant human CD81 produced in Pichia pastoris

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.     

Mapping the intermedilysin-human CD59 receptor interface reveals a deep correspondence with the binding site on CD59 for complement binding proteins C8alpha and C9

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.     

Mapping a region of hepatitis C virus E2 that is responsible for escape from neutralizing antibodies and a core CD81-binding region that does not tolerate neutralization escape mutations

Understanding the interaction between broadly neutralizing antibodies and their epitopes provides a basis for the rational design of a preventive hepatitis C virus (HCV) vaccine. CBH-2, HC-11, and HC-1 are representatives of antibodies to overlapping epitopes on E2 that mediate neutralization by blocking virus binding to CD81. To obtain insights into escape mechanisms, infectious cell culture virus, 2a HCVcc, was propagated under increasing concentrations of a neutralizing antibody to isolate escape mutants. Three escape patterns were observed with these antibodies. First, CBH-2 escape mutants that contained mutations at D431G or A439E, which did not compromise viral fitness, were isolated. Second, under the selective pressure of HC-11, escape mutations progressed from a single L438F substitution at a low antibody concentration to double substitutions, L438F and N434D or L438F and T435A, at higher antibody concentrations. Escape from HC-11 was associated with a loss of viral fitness. An HCV pseudoparticle (HCVpp) containing the L438F mutation bound to CD81 half as efficiently as did wild-type (wt) HCVpp. Third, for HC-1, the antibody at a critical concentration completely suppressed viral replication and generated no escape mutants. Epitope mapping revealed contact residues for CBH-2 and HC-11 in two regions of the E2 glycoprotein, amino acids (aa) 425 to 443 and aa 529 to 535. Interestingly, contact residues for HC-1 were identified only in the region encompassing aa 529 to 535 and not in aa 425 to 443. Taken together, these findings point to a region of variability, aa 425 to 443, that is responsible primarily for viral escape from neutralization, with or without compromising viral fitness. Moreover, the region aa 529 to 535 is a core CD81 binding region that does not tolerate neutralization escape mutations.     

Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells

The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage.     

Adaptation of Hepatitis C Virus to Mouse CD81 Permits Infection of Mouse Cells in the Absence of Human Entry Factors

Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.     

Characterization of the CD200 receptor family in mice and humans and their interactions with CD200

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.