Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.     

The encapsidated genome of Microplitis demolitor bracovirus integrates into the host Pseudoplusia includens

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism.     

Systematic analysis of a wasp parasitism arsenal

Parasitoid wasps are among the most diverse insects on earth with many species causing major mortality in host populations. Parasitoids introduce a variety of factors into hosts to promote parasitism, including symbiotic viruses, venom, teratocytes and wasp larvae. Polydnavirus‐carrying wasps use viruses to globally suppress host immunity and prevent rejection of developing parasites. Although prior results provide detailed insights into the genes viruses deliver to hosts, little is known about other products. RNAseq and proteomics were used to characterize the proteins secreted by venom glands, teratocytes and larvae from Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). These data revealed that venom glands and teratocytes secrete large amounts of a small number of products relative to ovaries and larvae. Venom and teratocyte products exhibited almost no overlap with one another or MdBV genes, which suggested that M. demolitor effector molecules are functionally partitioned according to their source. This finding was well illustrated in the case of MdBV and teratocytes. Many viral proteins have immunosuppressive functions that include disruption of antimicrobial peptide production, yet this study showed that teratocytes express high levels of the antimicrobial peptide hymenoptaecin, which likely compensates for MdBV‐mediated immunosuppression. A second key finding was the prevalence of duplications among genes encoding venom and teratocyte molecules. Several of these gene families share similarities with proteins from other species, while also showing specificity of expression in venom glands or teratocytes. Overall, these results provide the first comprehensive analysis of the proteins a polydnavirus‐carrying wasp introduces into its host.     

Widespread Genome Reorganization of an Obligate Virus Mutualist

The family Polydnaviridae is of interest because it provides the best example of viruses that have evolved a mutualistic association with their animal hosts. Polydnaviruses in the genus Bracovirus are strictly associated with parasitoid wasps in the family Braconidae, and evolved ∼100 million years ago from a nudivirus. Each wasp species relies on its associated bracovirus to parasitize hosts, while each bracovirus relies on its wasp for vertical transmission. Prior studies establish that bracovirus genomes consist of proviral segments and nudivirus-like replication genes, but how these components are organized in the genomes of wasps is unknown. Here, we sequenced the genome of the wasp Microplitis demolitor to characterize the proviral genome of M. demolitor bracovirus (MdBV). Unlike nudiviruses, bracoviruses produce virions that package multiple circular, double-stranded DNAs. DNA segments packaged into MdBV virions resided in eight dispersed loci in the M. demolitor genome. Each proviral segment was bounded by homologous motifs that guide processing to form mature viral DNAs. Rapid evolution of proviral segments obscured homology between other bracovirus-carrying wasps and MdBV. However, some domains flanking MdBV proviral loci were shared with other species. All MdBV genes previously identified to encode proteins required for replication were identified. Some of these genes resided in a multigene cluster but others, including subunits of the RNA polymerase that transcribes structural genes and integrases that process proviral segments, were widely dispersed in the M. demolitor genome. Overall, our results indicate that genome dispersal is a key feature in the evolution of bracoviruses into mutualists.     

Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions

Ep-CAM is a new type of cell adhesion molecule (CAM) which does not structurally resemble the members of the four major families (cadherins, integrins, selectins, and CAMs of the immunoglobulin superfamily) and mediates Ca(2+)-independent, homophilic adhesions. The extracellular domain of Ep-CAM consists of a cysteine-rich region, containing two type II epidermal growth factor (EGF)-like repeats, followed by a cysteine-poor region. We generated mutated Ep-CAM forms with various deletions in the extracellular domain. These deletion mutants, together with monoclonal antibodies recognizing different epitopes in the extracellular domain, were used to investigate the role of the EGF-like repeats in the formation of intercellular contacts mediated by Ep-CAM molecules. We established that both EGF-like repeats are required for the formation of Ep-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAM molecules at the cell-cell boundaries, and the anchorage of the Ep-CAM adhesion complex to F-actin via alpha-actinin. Deletion of either EGF-like repeat was sufficient to inhibit the adhesion properties of the molecule. The first EGF-like repeat of Ep-CAM is required for reciprocal interactions between Ep-CAM molecules on adjacent cells, as was demonstrated with blocking antibodies. The second EGF-like repeat was mainly required for lateral interactions between Ep-CAM molecules. Lateral interactions between Ep-CAM molecules result in the formation of tetramers, which might be the first and necessary step in the formation of Ep-CAM-mediated intercellular contacts.     

Analysis of the subgroup A avian sarcoma and leukosis virus receptor: the 40-residue, cysteine-rich, low-density lipoprotein receptor repeat motif of Tva is sufficient to mediate viral entry.

The genes encoding the receptor for subgroup A Rous sarcoma viruses (tva) were recently cloned from both chicken and quail cells (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993; J. A. T. Young, P. Bates, and H. E. Varmus, J. Virol. 67:1811-1816, 1993). Previous work suggested that only the extracellular domain of Tva interacts with the virus (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993). Tva is a small membrane-associated protein containing in its extracellular domain a 40-amino-acid region which is closely related to the low-density lipoprotein receptor (LDLR) repeat motif. To determine the region of the Tva extracellular domain responsible for viral receptor function, we created chimeric proteins containing various regions of the Tva extracellular domain fused with a murine CD8 membrane anchor. Analysis of these proteins demonstrates that any chimera containing the Tva LDLR repeat motif can specifically bind the envelope protein of subgroup A avian sarcoma and leukosis viruses. Furthermore, NIH 3T3 cell lines expressing these chimeric proteins were efficiently infected by subgroup A avian sarcoma and leukosis virus vectors. Our results demonstrate that the 40-residue-long LDLR repeat motif of Tva is responsible for viral receptor function.     

Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin

The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin- binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix.     

Characterization of multiple adhesive and counteradhesive domains in the extracellular matrix protein cytotactin

The extracellular matrix molecule cytotactin is a multidomain protein that plays a role in cell migration, proliferation, and differentiation during development. To analyze the structure-function relationships of the different domains of this glycoprotein, we have prepared a series of fusion constructs in bacterial expression vectors. Results obtained using a number of adhesion assays suggest that at least four independent cell binding regions are distributed among the various cytotactin domains. Two of these are adhesive; two others appear to be counteradhesive in that they inhibit cell attachment to otherwise favorable substrates. The adhesive regions were mapped to the fibronectin type III repeats II-VI and the fibrinogen domain. The morphology of the cells plated onto these adhesive fragments differed; the cells spread on the fibronectin type III repeats as they do on fibronectin, but remained round on the fibrinogen domain. The counteradhesive properties of the molecule were mapped to the EGF-like repeats and the last two fibronectin type III repeats, VII-VIII. The latter region also contained a cell attachment activity that was observed only after proteolysis of the cells. Several cell types were used in these analyses, including fibroblasts, neurons, and glia, all of which are known to bind to cytotactin. The different domains exert their effects in a concentration-dependent manner and can be inhibited by an excess of the soluble molecule, consistent with the hypothesis that the observed properties are mediated by specific receptors. Moreover, it appears that some of these receptors are restricted to particular cell types. For example, glial cells bound better than neurons to the fibrinogen domain and fibroblasts bound better than glia and neurons to the EGF fragment. These results provide a basis for understanding the multiple activities of cytotactin and a framework for isolating different receptors that mediate the various cellular responses to this molecule.     

Segmented Helical Structure of the Neck Region of the Glycan-Binding Receptor DC-SIGNR

Carbohydrate-recognition domains (CRDs) in the glycan-binding receptors DC-SIGN (dendritic-cell-specific intercellular adhesion molecule 1-grabbing nonintegrin; CD209) and DC-SIGNR (DC-SIGN-related receptor, also known as L-SIGN and variously designated CD209L and CD299) are projected from the membrane surface by extended neck domains containing multiple repeats of a largely conserved 23-amino-acid sequence motif. Crystals of a fragment of the neck domain of DC-SIGNR containing multiple repeats in which each molecule extends through multiple unit cells, such that the observed crystallographic asymmetric unit represents one repeat averaged over six repeats of the protein, have been obtained. The repeats are largely α-helical. Based on the structure and arrangement of the repeats in the crystal, the neck region can be described as a series of four-helix bundles connected by short, non-helical linkers. Combining the structure of the isolated neck domain with a previously determined overlapping structure of the distal end of the neck region with the CRDs attached provides a model of the almost-complete extracellular portion of the receptor. The results are consistent with previous characterization of the extended structure for the isolated neck region and the extracellular domain. The organization of the neck suggests how CRDs may be disposed differently in DC-SIGN compared with DC-SIGNR and in variant forms of DC-SIGNR assembled from polypeptides with different numbers of repeats in the neck domain.     

Localizing the adhesive and signaling functions of plakoglobin

Plakoglobin (PKG) is a major component of cell-cell adhesive junctions. It is also closely related to the Drosophila segment polarity gene product armadillo and can induce a WNT-like neural axis duplication (NAD) phenotype in Xenopus [Karnovsky and Klymkowsky, 1995].* To define the regions of PKG involved in cell adhesion and inductive signaling, we examined the behavior of mutated forms of PKG in Xenopus. Deletion of amino acids 22 through 39 (in the Xenopus. PKG sequence) increased the apparent stability of the polypeptide within the embryo and increased its ability to induce a WNT-like, NAD phenotype when expressed in the vegetal hemisphere. The N-terminal “head” and first 6 “ARM” repeats of PKG, or the C-terminal “tail” and the last 3 “ARM” repeats, could be removed without destroying the remaining polypeptide's ability to induce a NAD phenotype. The nuclear localization of mutant PKGs, however, was not strictly correlated with the ability to induce a NAD phenotype, i.e., some inactive polypeptides still accumulate in nuclei. Removal of PKG's head and first ARM repeat, which includes its α-catenin binding site, resulted in a polypeptide that, when expressed in the embryo, generated a dramatic cell adhesion defect. Removal of the next three ARM repeats abolished this adhesion defect, suggesting that the polypeptide no longer competes effectively with endogenous catenins for binding to cadherins. Expression of a form of PKG truncated after the 5th ARM repeat produced a milder cell adhesion defect, whereas expression of a polypeptide truncated after the 8th ARM repeat had little apparent effect on cellular adhesion. Based on these observations, we conclude that functions related to stability and cellular adhesion reside in the N-terminal region of the polypeptide, whereas the ability to induce a NAD phenotype lies within repeats 6–10 of the central region. The function(s) of the C-terminal domain of PKG remain uncertain at this time. Dev. Genet. 20:91–102, 1997. © 1997 Wiley-Liss, Inc.     

The structure of neurexin 1α reveals features promoting a role as synaptic organizer

α-neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The α-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how α-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1α extracellular domain (n1α) to 2.65 ?. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 ? structure of n1α carrying splice insert SS#3 in LNS4 reveals that SS#3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables α-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.     

Characterization of cadherin-24, a novel alternatively spliced type II cadherin

Cadherins comprise a superfamily of calcium-dependent cell-cell adhesion molecules. Within the superfamily are six subfamilies including type I and type II cadherins. Both type I and type II cadherins are composed of five extracellular repeat domains with conserved calcium-binding motifs, a single pass transmembrane domain, and a highly conserved cytoplasmic domain that interacts with beta-catenin and p120 catenin. In this study, we describe a novel cadherin, cadherin-24. It is a type II cadherin with a 781-codon open reading frame, which encodes a type II cadherin protein complete with five extracellular repeats containing calcium-binding motifs, a transmembrane domain, and a conserved cytoplasmic domain. Cadherin-24 has the unusual feature of being alternatively spliced in extracellular repeat 4. This alternative exon encodes 38 in-frame amino acids, resulting in an 819-amino-acid protein. Sequence analysis suggests the presence of beta-catenin and p120 catenin-binding sequences, and immunoprecipitation experiments confirm the ability of both forms of the novel cadherin to associate with alpha-catenin, beta-catenin, and p120 catenin. In addition, aggregation assays show that both forms of cadherin-24 mediate strong cell-cell adhesion.     

Tensin: A potential link between the cytoskeleton and signal transduction

Cytoskeletal proteins provide the structural foundation that allows cells to exist in a highly organized manner. Recent evidence suggests that certain cytoskeletal proteins not only maintain structural integrity, but might also be associated with signal transduction and suppression of tumorigenesis. Since the time of the discovery of tensin, a fair amount of data has been gathered which supports the notion that tensin is one such protein possessing these characteristics. In this review, we discuss recent studies that: (1) elucidate a role for tensin in maintenance of cellular structure and signal transduction; (2) implicate tensin as the anchor for actin filaments at the focal adhesion; (3) describe the phosphorylation of tensin; (4) describe potential targets for its Src homology region 2 domain; (5) describe the association between tensin and the nuclear protein p130; and (6) demonstrate that increased tensin expression in a cell line appears to reduce its transformation potential.     

The minimal essential unit for cadherin-mediated intercellular adhesion comprises extracellular domains 1 and 2

N-cadherin comprises five homologous extracellular domains, a transmembrane, and a cytoplasmic domain. The extracellular domains of N-cadherin play important roles in homophilic cell adhesion, but the contribution of each domain to this phenomenon has not been fully evaluated. In particular, the following questions remain unanswered: what is the minimal domain combination that can generate cell adhesion, how is domain organization related to adhesive strength, and does the cytoplasmic domain serve to facilitate extracellular domain interaction? To address these issues, we made serial constructs of the extracellular domains of N-cadherin and produced various cell lines to examine adhesion properties. We show that the first domain of N-cadherin alone on the cell surface fails to generate adhesive activity and that the first two domains of N-cadherin form the "minimal essential unit" to mediate cell adhesion. Cell lines expressing longer extracellular domains or N-cadherin wild type cells formed larger cellular aggregates than those expressing shorter aggregates. However, adhesion strength, as measured by a shearing test, did not reveal any differences among these aggregative cell lines, suggesting that the first two domains of N-cadherin cells generate the same strength of adhesive activity as longer extracellular domain cells. Furthermore, truncations of the first two domains of N-cadherin are also sufficient to form cisdimerization at an adhesive junction. Our findings suggest that the extracellular domains of N-cadherin have distinct roles in cell adhesion, i.e. the first two domains are responsible for homophilic adhesion activity, and the other domains promote adhesion efficiency most likely by positioning essential domains relatively far out from the cell surface.     

Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines.

Members of the Toll-like receptor (TLR) family mediate dorsoventral patterning and cellular adhesion in insects as well as immune responses to microbial products in both insects and mammals. TLRs are characterized by extracellular leucine-rich repeat domains and an intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor and plant disease resistance genes. Ten human TLRs have been cloned as well as RP105, a protein similar to TLR4 but lacking the intracellular signaling domain. However, only five TLRs have described functions as receptors for bacterial products (e.g., LPS, lipoproteins). To identify potential sites of action, we used quantitative real-time RT-PCR to examine systematically the expression of mRNAs encoding all known human TLRs, RP105, and several other proteins important in TLR functions (e.g., MD-1, MD-2, CD14, MyD88). Most tissues tested expressed at least one TLR, and several expressed all (spleen, peripheral blood leukocytes). Analysis of TLR expression in fractionated primary human leukocytes (CD4(+), CD8(+), CD19(+), monocytes, and granulocytes) indicates that professional phagocytes express the greatest variety of TLR mRNAs although several TLRs appear more restricted to B cells, suggesting additional roles for TLRs in adaptive immunity. Monocyte-like THP-1 cells regulate TLR mRNA levels in response to a variety of stimuli including phorbol esters, LPS, bacterial lipoproteins, live bacteria, and cytokines. Furthermore, addition of Escherichia coli to human blood ex vivo caused distinct changes in TLR expression, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation.     

早幼粒白血病蛋白核体与病毒感染

早幼粒白血病蛋白核体（promyelocytic leukaemia nuclear bodies,PML-NBs）是哺乳动物细胞中普遍存在的一种动态的细胞核亚结构,参与DNA损伤与修复、细胞衰老与凋亡、基因表达调控以及肿瘤发生与抑制等多种重要的细胞活动。研究表明,PML-NBs也是多种病毒入侵细胞的作用靶点。PML-NBs通过介导细胞固有免疫反应或者作为细胞干扰素信号通路元件参与宿主细胞的抗病毒防御活动。该文以几种DNA和RNA病毒为例,综述了在病毒感染过程中PML-NBs与病毒的相互作用以及这些相互作用的功能意义,从而揭示PML-NBs在抵御病毒感染和免疫反应中的重要作用,并提出运用病毒单分子实时示踪（Single-virus Tracking）这一新技术深入研究PML-NBs在病毒感染中作用的可行性。