The chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcepsilonRIgamma chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.     

Enteropathogenic Escherichia coli Tir recruits cellular SHP-2 through ITIM motifs to suppress host immune response

Immune responses to pathogens are regulated by immune receptors containing either an immunoreceptor tyrosine-based activation motif (ITAM) or an immunoreceptor tyrosine-based inhibitory motif (ITIM). The important diarrheal pathogen enteropathogenic Escherichia coli (EPEC) require delivery and insertion of the bacterial translocated intimin receptor (Tir) into the host plasma membrane for pedestal formation. The C-terminal region of Tir, encompassing Y483 and Y511, shares sequence similarity with cellular ITIMs. Here, we show that EPEC Tir suppresses the production of inflammatory cytokines by recruitment of SHP-2 and subsequent deubiquitination of TRAF6 in an ITIM dependent manner. Our findings revealed a novel mechanism by which the EPEC utilize its ITIM motifs to suppress and evade the host innate immune response, which could lead to the development of novel therapeutics to prevent bacterial infection.     

Systematic identification of immunoreceptor tyrosine-based inhibitory motifs in the human proteome

Immunoreceptor tyrosine-based inhibitory motifs (ITIMs) are short sequences of the consensus (ILV)-x-x-Y-x-(LV) in the cytoplasmic tail of immune receptors. The phosphorylation of tyrosines in ITIMs is known to be an important signalling mechanism regulating the activation of immune cells. The shortness of the motif makes it difficult to predict ITIMs in large protein databases. Simple pattern searches find ITIMs in approximately 30% of the protein sequences in the RefSeq database. The majority are false positive predictions. We propose a new database search strategy for ITIM-bearing transmembrane receptors based on the use of sequence context, i.e. the predictions of signal peptides, transmembrane helices (TMs) and protein domains. Our new protocol allowed us to narrow down the number of potential human ITIM receptors to 109 proteins (0.7% of RefPep). Of these, 36 have been described as ITIM receptors in the literature before. Many ITIMs are conserved between orthologous human and mouse proteins which represent novel ITIM receptor candidates. Publicly available DNA array expression data revealed that ITIM receptors are not exclusively expressed in blood cells. We hypothesise that ITIM signalling is not restricted to immune cells, but also functions in diverse solid organs of mouse and man.     

禽白血病病毒J亚群env基因的克隆和序列分析

应用多聚酶链反应（PCR）的方法扩增出ADOL-4817毒株的囊膜蛋白env基因,并克隆进大肠杆菌。经核酸序列分析证明,env基因的大小为1746 bp,其中gp85和gp37由1554 bp组成,可翻译成517个氨基酸,分子量为57.7 D。根据糖基化位点N-X-S/T的特点,发现ADOL-4817的env蛋白有15个潜在的糖基化位点。同源性分析证明,ADOL-4817的env基因与其它ALV-J的env基因序列同源性为88.8%～92.4%,而与外源性ALVs的相应序列的同源性仅为40.5%～51.4%,然而,与内源性的EAV-HP毒株的类env基因的同源性高达91.2%;另外,ADOL-4817毒株的gp37在C末端多了13个氨基酸。这些结果提示,ALV-J的env基因存在广泛的变异性,env基因可能来源于内源性和外源性ALVs的重组。     

Surface stability and immunogenicity of the human immunodeficiency virus envelope glycoprotein: role of the cytoplasmic domain

The effects of two functional domains, the membrane-proximal YXXPhi motif and the membrane-distal inhibitory sequence in the long cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env), on immunogenicity of the envelope protein were investigated. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env and a truncated Env with 50 amino acids in the cytoplasmic domain to delete the membrane distal inhibitory sequence for surface expression. Additional genes were generated in which the tyrosine residue in the YXXPhi motif was changed into a serine. Pulse-chase radioactive labeling and immunoprecipitation studies indicated that both domains can mediate endocytosis of the HIV Env, and removal of both domains is required to enhance HIV Env protein surface stability. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the mutant Env exhibiting enhanced surface stability induced significantly higher levels of antibody responses against the HIV Env protein. Our results suggest that the HIV Env cytoplasmic domain may play important roles in virus infection and pathogenesis by modulating its immunogenicity.     

Impact of natural polymorphism within the gp41 cytoplasmic tail of human immunodeficiency virus type 1 on the intracellular distribution of envelope glycoproteins and viral assembly

The motifs involved in the various functions of the human immunodeficiency virus type 1 (HIV-1) gp41 cytoplasmic tail (CT), particularly those related to the intracellular trafficking and assembly of envelope glycoproteins (Env) onto core particles, have generally been assessed with a restricted panel of T-cell laboratory-adapted virus strains. Here, we investigated gp41 CT sequences derived from individuals infected with HIV-1 viruses of various subtypes. We identified four patients harboring HIV variants with a natural polymorphism in the membrane-proximal tyrosine-based signal Y(712)SPL or the Y(802)W(803) diaromatic motif, which are two major determinants of Env intracellular trafficking. Confocal microscopy showed that the intracellular distribution of Env with a mutation in the tyrosine or diaromatic motif differed from that of Env with no mutation in these motifs. Surprisingly, the gp41 CTs of the primary viruses also had differential effects on the intracellular distribution of Env, independently of mutations in the tyrosine or diaromatic motifs, suggesting the involvement of additional determinants. Furthermore, analyses of virus replication kinetics indicated that the effects of mutations in the tyrosine or diaromatic motifs on viral replication depended on the gp41 CT context. These effects were at least partly due to differences in the efficiency of Env incorporation into virions. Thus, polymorphisms in primary HIV-1 gp41 CTs at the quasispecies or subtype level can influence the intracellular distribution of Env, its incorporation into virions, and viral replication capacity.     

The Rous sarcoma virus Env glycoprotein contains a highly conserved motif homologous to tyrosine-based endocytosis signals and displays an unusual internalization phenotype

The cytoplasmic domains of retroviral transmembrane (TM) glycoproteins contain conserved sequence motifs that resemble tyrosine-based (YXXO-type) endocytosis signals. We have previously described a mutant Rous sarcoma virus (RSV) Env protein, Env-mu26, with an L165R mutation in the membrane-spanning domain (MSD) of TM, that exhibited dramatically decreased steady-state surface expression (G. L. Davis and E. Hunter, J. Cell Biol. 105:1191-1203, 1987; P. B. Johnston, J. Y. Dong, and E. Hunter, Virology 206:353-361, 1995). We now demonstrate that the tyrosine of the Y(190)RKM motif in the RSV TM cytoplasmic domain is crucial for the mu26 phenotype and is part of an efficient internalization signal in the context of a mutant MSD. In contrast, despite the presence of the Y(190)RKM motif, wild-type RSV Env is constitutively internalized at a slow rate (1.1%/min) more characteristic of bulk uptake during membrane turnover than of active clustering into endocytic vesicles. The mu26 mutation and two MSD mutations that abrogate palmitoylation of TM resulted in enhanced Env endocytosis indicative of active concentration into coated pits. Surprisingly, an Env-Y190A mutant was apparently excluded from coated pits since its uptake rate of 0.3%/min was significantly below that expected for the bulk rate. We suggest that in RSV Env an inherently functional endocytosis motif is silenced by a counteracting determinant in the MSD that acts to prevent clustering of Env into endocytic vesicles. Mutations in either the cytoplasmic tail or the MSD that inactivate one of the two counteracting signals would thus render the remaining determinant dominant.     

Segregation of EAV-HP Ancient Endogenous Retroviruses within the Chicken Population

Avian leukosis virus subgroup J (ALV-J), an exogenous avian retrovirus, is thought to have evolved by recombination with the highly identical env gene of the endogenous avian retrovirus EAV-HP. Embryonic expression of EAV-HP env has been suggested to be associated with the induction of immunological tolerance, a feature observed in a significant proportion of meat-type chickens infected with ALV-J. In support of this hypothesis, we demonstrate that EAV-HP loci, some of which could be associated with tolerance, are still segregating within the chicken population.     

The highly conserved C-terminal dileucine motif in the cytosolic domain of the human immunodeficiency virus type 1 envelope glycoprotein is critical for its association with the AP-1 clathrin adaptor [correction of adapter

Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adaptor [corrected] proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adaptor [corrected] complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN- and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD-AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env-AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.     

Lipid rafts facilitate the interaction of PECAM-1 with the glycoprotein VI-FcR gamma-chain complex in human platelets

Glycoprotein (GP) VI, the main signaling receptor for collagen on platelets, is expressed in complex with the FcR gamma-chain. The latter contains an immunoreceptor tyrosine-based activation motif, which becomes phosphorylated, initiating a signaling cascade leading to the rapid activation and aggregation of platelets. Previous studies have shown that signaling by immunoreceptor tyrosine-based activation motif-containing receptors is counteracted by signals from receptors with immunoreceptor tyrosine-based inhibitory motifs. Here we show, by immunoprecipitation, that the GPVI-FcR gamma-chain complex associates with the immunoreceptor tyrosine-based inhibitory motif-containing receptor, PECAM-1. In platelets stimulated with collagen-related peptide (CRP-XL), tyrosine phosphorylation of PECAM-1 precedes that of the FcR gamma-chain, implying direct regulation of the former. The GPVI-FcR gamma-chain complex and PECAM-1 were present in both lipid raft and soluble fractions in human platelets; this distribution was unaltered by activation with CRP-XL. Their association occurred in lipid rafts and was lost after lipid raft depletion using methyl-beta-cyclodextrin. We propose that lipid raft clustering facilitates the interaction of PECAM-1 with the GPVI-FcR gamma-chain complex, leading to the down-regulation of the latter.     

我国1999—2003年间ALV—J野毒株gp85基因变异趋势

通过接种鸡胚成纤维细胞（CEF）、间接免疫荧光试验（IFA）和聚合酶链式反应（PCR）,连续五年从全国各地的送检病料中分离到14株J亚群白血病病毒（ALV-J）。为了动态观察ALV-J囊膜表面结构蛋白（GP85）的变异情况,对这14株野毒株的囊膜糖蛋白基因（env）进行了克隆和测序,将它们与HPRS-103株的GP85的氨基酸序列进行了比较,结果表明：ALV-J的囊膜表面结构蛋白发生了很大的变异,而且这些变异主要集中在高变区hr1、hr2和vr3;这些野毒株GP85的氨基酸序列的同源性在86．6％～100％之间（从同一鸡场中分离到的两株ALV-J即BJ00302与BJ0303的同源性为100％,其它毒株之间的同源性均小于100％）;有义突变与沉默突变的比例显示这3个高变区极有可能是免疫选择压作用的位点。     

Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41

A previous study from our laboratory reported a preferential conservation of arginine relative to lysine in the C-terminal tail (CTT) of HIV-1 envelope (Env). Despite substantial overall sequence variation in the CTT, specific arginines are highly conserved in the lentivirus lytic peptide (LLP) motifs and are scarcely substituted by lysines, in contrast to gp120 and the ectodomain of gp41. However, to date, no explanation has been provided to explain the selective incorporation and conservation of arginines over lysines in these motifs. Herein, we address the functions in virus replication of the most conserved arginines by performing conservative mutations of arginine to lysine in the LLP1 and LLP2 motifs. The presence of lysine in place of arginine in the LLP1 motif resulted in significant impairment of Env expression and consequently virus replication kinetics, Env fusogenicity, and incorporation. By contrast, lysine exchanges in LLP2 only affected the level of Env incorporation and fusogenicity. Our findings demonstrate that the conservative lysine substitutions significantly affect Env functional properties indicating a unique functional role for the highly conserved arginines in the LLP motifs. These results provide for the first time a functional explanation to the preferred incorporation of arginine, relative to lysine, in the CTT of HIV-1 Env. We propose that these arginines may provide unique functions for Env interaction with viral or cellular cofactors that then influence overall Env functional properties.     

Structure-based sequence alignment of type-II restriction endonucleases

The type-II restriction endonucleases generally do not share appreciable amino acid sequence homology. The crystal structures of restriction endonucleases EcoRI and BamHI have shown these enzymes to possess striking 3D-structural resemblance, i.e., they have a similar overall fold and similar active sites, though they possess <23% sequence identity. Structural superimposition of EcoRI, BamHI, EcoRV, and PvuII based on active site residues led to sequence alignments which showed nine possible sequence motifs. EcoRV and PvuII show a more similar pattern than EcoRI and BamHI suggesting that they belong to a different subgroup. The motifs are characterized by charged and/or hydrophobic residues. From other studies on the structure of these endonucleases, three of the motifs could be implicated in DNA binding, three in forming the active site and one in dimer formation. However, the motifs were not identifiable by regular sequence alignment methods. It is found that motif IX in BamHI is formed by reverse sequence order and the motif IX in PvuII is formed from the symmetry related monomer of the dimer. The inter-motif distance is also quite different in these cases. Of the nine motifs, motif III has been earlier identified as containing the PD motif involving one of the active site residues. These motifs were used in a modified profile analysis procedure to identify similar regions in eight other endonuclease sequences for which structures are not known.     

The membrane-proximal tyrosine-based sorting signal of human immunodeficiency virus type 1 gp41 is required for optimal viral infectivity

The membrane-proximal tyrosine-based sorting motif in the cytoplasmic domain of the human immunodeficiency virus type 1 Env glycoprotein is important for endocytosis from the plasma membrane, basolateral targeting of viral budding in polarized epithelial cells, and polarized budding from a localized region of the lymphocyte plasma membrane. To study the role of the Env sorting motif (Y712XXL) in infectivity, the incorporation of Env into virions, and viral entry, we disrupted the motif with a tyrosine-to-alanine substitution. To investigate the relationship between the Env sorting motif and the enhancement of infectivity by Nef, the EnvY712A substitution was made in both Nef-positive and Nef-negative backgrounds. In spreading infections, including those using primary lymphocytes, the growth of the Y712A mutant was as impaired as Nef-negative virus, and the EnvY712A/Delta-Nef combination mutant was almost completely defective. In single-round infections using CD4-positive HeLa cells, the EnvY712A mutation impaired infectivity, and Nef retained the ability to enhance the infectivity in the context of EnvY712A. EnvY712 and Nef were required for the optimal infectivity of virions produced from either HEK293T or MT4 cells, but these sequences were required for the optimal incorporation of Env only when virions were produced from MT4 cells. Despite the wild-type levels of Env in viruses produced from 293T cells, the entry of the EnvY712A and Delta-Nef mutants into target cells was impaired. We conclude that the membrane-proximal tyrosine-based sorting motif of gp41 Env is, like Nef, important for optimal viral infectivity and, in the case of MT4 T cells, virion incorporation of Env. Nef does not require the Y712XXL motif to enhance viral infectivity. The finding that EnvY712 and Nef each affect the efficiency of viral entry independently of the Env content of virions suggests that both viral proteins are involved in trafficking events that influence morphogenesis to produce maximally fusogenic virus.     

Effect of suppressive DNA on CpG-induced immune activation

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate a strong innate immune response. This stimulation can be abrogated by either removing the CpG DNA or adding inhibitory/suppressive motifs. Suppression is dominant over stimulation and is specific for CpG-induced immune responses (having no effect on LPS- or Con A-induced activation). Individual cells noncompetitively internalize both stimulatory and suppressive ODN. Studies using ODN composed of both stimulatory and suppressive motifs indicate that sequence recognition proceeds in a 5'-->3' direction, and that a 5' motif can block recognition of immediately 3' sequences. These findings contribute to our understanding of the immunomodulatory activity of DNA-based products and the rules that govern immune recognition of stimulatory and suppressive motifs.     

New I-type lectins of the CD 33-related siglec subgroup identified through genomics

Siglecs are sialic-acid-binding proteins of the Ig superfamily that are involved in cell-cell interactions and signalling. In recent years, several novel siglecs that are highly related to CD33/Siglec-2 have been identified through genomics and functional screens. In addition to their distinct sialic-acid-binding properties, most of these novel siglecs bear tyrosine-based signalling motifs that are typically found in inhibitory receptors of the immune system. The restricted expression patterns of CD33-related siglecs in the haemopoietic and immune systems suggests that they are involved in regulating leucocyte activation during inflammatory and immune responses.     

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2

B and T lymphocytes express receptors providing positive and negative co-stimulatory signals. We recently identified a novel co-stimulatory molecule, B and T lymphocyte attenuator (BTLA), which exerts inhibitory effects on B and T lymphocytes. The cytoplasmic domain of murine and human BTLA share three conserved tyrosine-based signaling motifs, a Grb-2 recognition consensus, and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of the cytoplasmic domain of BTLA induced the association with the protein tyrosine phosphatases SHP-1 and SHP-2. Association of SHP-1 and SHP-2 to other receptors can involve recruitment to either a single receptor ITIM or to two receptor ITIMs. Here, we analyzed the requirements of BTLA interaction with SHP-1 and SHP-2 in a series of murine and human BTLA mutants. For human BTLA, mutations of either Y257 or Y282, but not Y226, abrogated association with both SHP-1 and SHP-2. For murine BTLA, mutation of either Y274 or Y299, but not Y245, also abrogated association with both SHP-1 and SHP-2. These results indicate that for both murine and human BTLA, association with SHP-1 or SHP-2 requires both of conserved ITIM motifs and does not involve the conserved Grb-2 consensus. Thus, similar to the bisphosphoryl tyrosine-based activation motif (BTAM) by which the Grb-2 associated binder (Gab1), PDGF receptor, and PECAM-1 recruit SHP-2, BTLA also relies on dual ITIMs for its association with the phosphatases SHP-1 and SHP-2.