An arginine switch in the species B adenovirus knob determines high-affinity engagement of cellular receptor CD46

Adenoviruses (Ads) are icosahedral, nonenveloped viruses with a double-stranded DNA genome. The 51 known Ad serotypes exhibit profound variations in cell tropism and disease types. The number of observed Ad infections is steadily increasing, sometimes leading to fatal outcomes even in healthy individuals. Species B Ads can cause kidney infections, hemorrhagic cystitis, and severe respiratory infections, and most of them use the membrane cofactor protein CD46 as a cellular receptor. The crystal structure of the human Ad type 11 (Ad11) knob complexed with CD46 is known; however, the determinants of CD46 binding in related species B Ads remain unclear. We report here a structural and functional analysis of the Ad11 knob, as well as the Ad7 and Ad14 knobs, which are closely related in sequence to the Ad11 knob but have altered CD46-binding properties. The comparison of the structures of the three knobs, which we determined at very high resolution, provides a platform for understanding these differences and allows us to propose a mechanism for productive high-affinity engagement of CD46. At the center of this mechanism is an Ad knob arginine that needs to switch its orientation in order to engage CD46 with high affinity. Quantum chemical calculations showed that the CD46-binding affinity of Ad11 is significantly higher than that of Ad7. Thus, while Ad7 and Ad14 also bind CD46, the affinity and kinetics of these interactions suggest that these Ads are unlikely to use CD46 productively. The proposed mechanism is likely to determine the receptor usage of all CD46-binding Ads.     

Localization of regions in CD46 that interact with adenovirus

A variety of pathogens use CD46, a ubiquitously expressed membrane protein that regulates complement activation, as a cellular attachment receptor. While the CD46 binding sites of several pathogens, including measles virus, Neisseria gonorrhea, and human herpesvirus 6, have been described, the region of CD46 responsible for adenovirus binding has not been determined. In this study, we used competition experiments with known CD46 ligands, CD46-specific antibodies, and a set of CD46 mutants to localize the binding domain for the group B adenovirus serotype 35 (Ad35). Our results show that Ad35 competes with measles virus for binding to CD46 but not with complement protein C3b. We further show that this interaction is a protein-protein interaction and that N glycosylations do not critically contribute to infection with Ad35 fiber-containing Ad vectors. Our data demonstrate that the native conformation of the CCP2 domain is crucial for Ad35 binding and that the substitution of amino acids at positions 130 to 135 or 152 to 156 completely abolishes the receptor function of CD46. These regions localize to the same planar face of CD46 and likely form an extended adenovirus binding surface, since no single amino acid substitution within these areas eliminates virus binding. Finally, we demonstrate that the infection with a virus possessing human group B serotype Ad11 fibers is also mediated by the CCP2 domain. This information is important to better characterize the mechanisms of the receptor recognition by adenovirus relative to other pathogens that interact with CD46, and it may help in the design of antiviral therapeutics against adenovirus serotypes that use CD46 as a primary cellular attachment receptor.     

CD46 is a cellular receptor for all species B adenoviruses except types 3 and 7

The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7.     

Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob

Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a K_D (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.     

In vitro and in vivo properties of adenovirus vectors with increased affinity to CD46

Gene transfer vectors containing adenovirus (Ad) serotype 35 (Ad35) fibers have shown promise for cancer and stem cell gene therapy. In this study, we attempted to improve the in vitro and in vivo infection properties of these vectors by increasing their affinity to the Ad35 fiber receptor CD46. We constructed Ad vectors containing either the wild-type Ad35 fiber knob (Ad5/35) or Ad35 knob mutants with 4-fold- and 60-fold-higher affinity to CD46 (Ad5/35+ and Ad5/35++, respectively). In in vitro studies with cell lines, the higher affinities of Ad5/35+ and Ad5/35++ to CD46 did not translate into correspondingly higher transduction efficiencies, regardless of the CD46 receptor density present on cells. However, in vivo, in a mouse model with preestablished CD46(high) liver metastases, intravenous injection of Ad5/35++ resulted in more-efficient tumor cell transduction. We conclude that Ad5/35 vectors with increased affinity to CD46 have an advantage in competing with non-CD46-mediated sequestration of vector particles after intravenous injection.     

CD46 is a cellular receptor for group B adenoviruses

Group B adenoviruses, a subgenus of human Adenoviridae, are associated with a variety of often-fatal illnesses in immunocompromised individuals, including bone marrow transplant recipients and cancer and AIDS patients. Recently, group B adenovirus derivatives have gained interest as attractive gene therapy vectors because they can transduce target tissues, such as hematopoietic stem cells, dendritic cells and malignant tumor cells, that are refractory to infection by commonly used adenoviral vectors. Whereas many adenoviruses infect cells through the coxsackievirus and adenovirus receptor (CAR), group B adenoviruses use an alternate, as-yet-unidentified cellular attachment receptor. Using mass spectrometric analysis of proteins interacting with a group B fiber, we identified human CD46 as a cellular attachment receptor for most group B adenoviruses. We show that ectopic expression of human CD46 rendered nonhuman cells susceptible to infection with group B viruses in vitro and in vivo. In addition, both siRNA-mediated knockdown of CD46 and a soluble form of CD46 blocked infection of human cell lines and primary human cells. The discovery that group B adenoviruses use CD46, a ubiquitously expressed complement regulatory protein, as a cellular attachment receptor elucidates the diverse clinical manifestation of group B virus infections, and bears directly on the application of these vectors for gene therapy.     

The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.     

Epitopes and hemagglutination binding domain on subgenus B:2 adenovirus fibers.

The adenovirus fiber serves as a ligand between the virus and the host cell receptor and manifests hemagglutination (HA) activity and antigenic domains. We have screened both the antigenic and immunogenic epitopes on the adenovirus fibers of subgenus B:2 by using recombinant fiber proteins (rfibers) expressed in Escherichia coli, synthesized peptides (P1 to P8), and the corresponding antisera. The results indicated that P4 (amino acids [aa] 201 to 220), P5 (aa 231 to 250), and P7 (aa 275 to 295) presented both antigenic and immunogenic epitopes in adenovirus type 11 prototype (Ad11p), Ad34a, and Ad11a fibers. P6 (aa 251 to 270) presented both epitopes in Ad11a fiber but only an antigenic epitope in other fibers. The C-terminal 20 amino acids of the fiber, corresponding to P8, manifested an epitope of low-level immunogenicity. P5, localized at the N-terminal aa 231 to 250, displayed an epitope that reacted with fibers of all the members of subgenus B analyzed. The rfibers of Ad11p and Ad34a displayed HA activity with monkey erythrocytes, though those of Ad11a did not. Mutagenesis of the rfibers revealed that neither the fragment replacements, 11p20211a, llp26011a,and 11a28011p, nor the Ad11p rfiber with the substitutions of Tyr-260-->H (Tyr260H)and Arg279Q displayed HA activity. The Ad11a fiber knob was sensitive to proteolytic digestion, whereas that of Ad11p was resistant. The results demonstrated that the decisive HA binding domain was presented at aa 260 to 280 and was conformation dependent. Nearby amino acids, aa 283 and 284, may also affect the HA function.     

Structural variations in the style and pollen presentation mechanisms ofBlumea species in India

The 22 species ofBlumea occurring in India have been investigated for structural variations of style and the pollen presentation mechanisms. The typically gynomonecious capitulum, with several series of female florets on the periphery and fewer bisexual florets in the centre, presents diverse patterns in the structure and behaviour of anthers, styles and stigmas. Thus, the different pollen presentation mechanisms show that inbreeding, outbreeding, or both mechanisms may be operating within a single capitulum. Based on the total number of florets in a capitulum, the species have been grouped into 4 categories.     

Avidity binding of human adenovirus serotypes 3 and 7 to the membrane cofactor CD46 triggers infection

The species B human adenoviruses (HAdVs) infect cells upon attaching to CD46 or desmoglein 2 (DSG-2) by one or several of their 12 fiber knob trimers (FKs). To test whether DSG-2 and CD46 simultaneously serve as virus receptors for adenovirus type 3 (Ad3), we performed individual and combined CD46/DSG-2 loss-of-function studies in human lung A549 and 16HBE14o cells. Our results suggest that in these cells, DSG-2 functions as a major attachment receptor for Ad3, whereas CD46 exerts a minor contribution to virus attachment and uptake in the range of ～10%. However, in other cells the role of CD46 may be more pronounced depending on, e.g., the expression levels of the receptors. To test if avidity allows Ad3/7 to use CD46 as a receptor, we performed gain-of-function studies. The cell surface levels of ectopically expressed CD46 in CHO or human M010119 melanoma cells lacking DSG-2 positively correlated with Ad3/7 infections, while Ad11/35 infections depended on CD46 but less on CD46 levels. Antibody-cross-linked soluble CD46 blocked Ad3/7/11/35 infections, while soluble CD46 alone blocked Ad11/35 but not Ad3/7. Soluble Ad3/7-FKs poorly inhibited Ad3/7 infection of CHO-CD46 cells, illustrating that Ad3/7-FKs bind with low affinity to CD46. This was confirmed by Biacore studies. Ad3/7-FK binding to immobilized CD46 at low density was not detected, unlike that of Ad11/35-FK. At higher CD46 densities, however, Ad3/7-FK bound to CD46 with only 15-fold-higher dissociation constants than those of Ad11/35-FK. These data show that an avidity mechanism for Ad3/7 binding to CD46 leads to infection of CD46-positive cells.     

Physical association of moesin and CD46 as a receptor complex for measles virus.

Recently, two cellular membrane proteins, the membrane cofactor protein CD46 and the membrane-organizing external spike protein, moesin, have been identified to be functionally associated with measles virus (MV) infectivity of cells. We investigated the functional consequences of binding of monoclonal antibodies to both molecules individually and combined on MV attachment, fusion, and plaque formation and the putative direct physical interaction of moesin and CD46. We found that antibodies to moesin or CD46 separately inhibited MV-cell interactions to a high percentage in the plaque test, by approximately 85 and 75%, respectively. The inhibition by combinations of antibodies was additive at low concentrations and complete at high concentrations. This indicates that similar sites of interaction were blocked by steric hindrance. Furthermore, antimoesin antibodies blocked the infection of CD46-negative mouse cell lines with MV. Chemical cross-linking of cell surface proteins indicated the close proximity of CD46 and moesin in the membrane of human cells, and coimmunoprecipitation of moesin with CD46 suggested their physical interaction. Immunohistochemically by electron microscopy, CD46 and moesin were found to be localized at sites of the cellular membrane where MV particles adsorbed. These data support a model of direct interaction of CD46 and moesin in the cellular membrane and suggest that this complex is functionally involved in the uptake of MV into cells.     

转座子引起的猪ktn1基因结构变异及其与生产性能的关联分析

为探明转座子对猪的ktn1基因及其侧翼区结构变异的贡献,从全基因组测序(WGS)数据库中获取14个猪基因组中的ktn1基因序列和侧翼序列,通过ClustalX多序列比对和RepeatMasker转座子注释,全面解析转座子对ktn1的影响。通过PCR检测到一个SINEA1转座子插入多态,在苏姜猪群体中与相关性状进行关联分析。结果显示,ktn1基因及其侧翼区中含有至少77个转座子片段,其中绝大部分(98.70%)为SINE类转座子,并鉴定到9个小结构变异和4个由转座子引起的大结构变异,表明转座子是基因变异的重要来源。其中一个SINEA1插入多态引起的结构变异,在不同品种中呈现丰富的多态性,且无插入个体(SINE-/-)苏姜猪的断奶窝重((64.20±10.6) kg)比纯合有插入个体(SINE+/+)((74.14±9.0) kg)和杂合有插入个体(SINE+/-)((69.71±7.7) kg)轻(P<0.05),表明基于转座子插入多态研发分子标记具有可行性,提示转座子插入多态分子标记在分子辅助育种中具有较强的应用潜力。     

There are two different species B adenovirus receptors: sBAR,common to species B1 and B2 adenoviruses,and sB2AR,exclusively used by species B2 adenoviruses

Unlike most adenovirus (Ad) serotypes, the species B Ads do not use the coxsackie-adenovirus receptor as an attachment receptor. The species B attachment receptor(s) has not yet been identified and is also poorly characterized. Species B Ads can be further divided into species B1 and B2 Ads, and these display different organ tropisms, suggesting a difference in receptor usage. We have studied the receptor interactions of the species B1 serotypes 3p and 7p and the species B2 serotypes 11p and 35 and characterized the properties of the species B receptor(s). Reciprocal blocking experiments using unlabeled Ad11p or Ad3p virions to block the binding to A549 cells of (35)S-labeled 3p, 7p, 11p, and 35 showed that only Ad11p virions efficiently blocked the binding of all the species B Ads studied (> or =70%). Thus, there is apparently a common species B Ad receptor (sBAR). However, Ad3p virions only partially (< or =30%) blocked the binding of Ad11p and Ad35 to A549 cells. Binding experiments after trypsin treatment of the cells confirmed that the species B2 serotypes address at least two different receptors on A549 and J82 cells, since sBAR is trypsin sensitive but the species B2 Ad receptor (sB2AR) is not. Both receptors are proteins or glycoproteins, since binding of all species B serotypes was abolished after proteinase K or subtilisin treatment of A549 or J82 cells. Furthermore, binding of the species B serotypes to sBAR was abolished with EDTA and restored with Ca(2+), whereas the binding of Ad11p and Ad35 to SB2AR was independent of divalent cations.     

Human-like immune responses in CD46 transgenic mice

Neisseria meningitidis is a major cause of sepsis and/or meningitis. These bacteria normally cause disease only in humans, however, mice expressing human CD46 are susceptible to meningococcal disease. To explain the sensitivity of CD46 transgenic mice to meningococci, we evaluated early immune responses. Stimulation of TNF, IL-6, and IL-10 was stronger in CD46 transgenic mice compared with nontransgenic mice, and resembled human responses. In CD46 transgenic mice, bacterial clearance in blood started at later time points, and neutrophil numbers in blood were lower compared with nontransgenic mice. Further, elevated levels of activated microglia cells and cyclooxygenase-2 were observed in brain of infected CD46 transgenic mice. Intraperitoneal administration of meningococci lead to increased levels of macrophages only in the i.p. cavity of CD46 transgenic mice. Most of the responses were impaired or absent using LPS-deficient meningococci, showing the importance of LPS in the early immune response to meningococcal infection. Taken together, these data demonstrate that responses in mice expressing human CD46 mimic human meningococcal disease in many aspects, and demonstrate novel important links between CD46 and the innate immune system.     

Structural variations within the transferrin binding site on transferrin-binding protein B, TbpB

Pathogenic bacteria acquire the essential element iron through specialized uptake pathways that are necessary in the iron-limiting environments of the host. Members of the Gram-negative Neisseriaceae and Pasteurellaceae families have adapted to acquire iron from the host iron binding glycoprotein, transferrin (Tf), through a receptor complex comprised of transferring-binding protein (Tbp) A and B. Because of the critical role they play in the host, these surface-exposed proteins are invariably present in clinical isolates and thus are considered prime vaccine targets. The specific interactions between TbpB and Tf are essential and ultimately might be exploited to create a broad-spectrum vaccine. In this study, we report the structure of TbpBs from two porcine pathogens, Actinobacillus pleuropneumoniae and suis. Paradoxically, despite a common Tf target, these swine related TbpBs show substantial sequence variation in their Tf-binding site. The TbpB structures, supported by docking simulations, surface plasmon resonance and hydrogen/deuterium exchange experiments with wild-type and mutant TbpBs, explain why there are structurally conserved elements within TbpB homologs despite major sequence variation that are required for binding Tf.