Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

Evolution of circular permutations in multidomain proteins

Modular rearrangements play an important role in protein evolution. Functional modules, often tantamount to structural domains or smaller fragments, are in many cases well conserved but reoccur in a different order and across many protein families. The underlying genetic mechanisms are gene duplication, fusion, and loss of sequence fragments. As a consequence, the sequential order of domains can be inverted, leading to what is known as circularly permutated proteins. Using a recently developed algorithm, we have identified a large number of such rearrangements and analyzed their evolutionary history. We searched for examples which have arisen by one of the three postulated mechanisms: independent fusion/fission, "duplication/deletion," and plasmid-mediated "cut and paste." We conclude that all three mechanisms can be observed, with the independent fusion/fission being the most frequent. This can be partly attributed to highly mobile domains. Duplication/deletion has been found in modular proteins such as peptide synthases.     

MiGenes: a searchable interspecies database of mitochondrial proteins curated using gene ontology annotation

MOTIVATION: There has been an explosion of interest in the role of mitochondria in programmed cell death and other fundamental pathological processes underlying the development of human diseases. Nevertheless, the inventory of mitochondrial proteins encoded in the nuclear genome remains incomplete, providing an impediment to mitochondrial research at the interface with systems biology. We created the MiGenes database to further define the scope of the mitochondrial proteome in humans and model organisms including mice, rats, flies and worms as well as budding and fission yeasts. MiGenes is intended to stimulate mitochondrial research using model organisms. SUMMARY: MiGenes is a large-scale relational database that is automatically updated to keep pace with advances in mitochondrial proteomics and is curated to assure that the designation of proteins as mitochondrial reflects gene ontology (GO) annotations supported by high-quality evidence codes. A set of postulates is proposed to help define which proteins are authentic components of mitochondria. MiGenes incorporates >1160 new GO annotations to human, mouse and rat protein records, 370 of which represent the first GO annotation reflecting a mitochondrial localization. MiGenes employs a flexible search interface that permits batchwise accession number searches to support high-throughput proteomic studies. A web interface is provided to permit members of the mitochondrial research community to suggest modifications in protein annotations or mitochondrial status.     

Root Growth Effects on Soluble C and P in Manured and Non-manured Soils

There is limited research on relationships between root characteristics and soil chemical properties and processes. Because previous studies have shown specific C compounds may release previously sorbed P and make P more plant-available, crops which contribute to high soil C levels could play an important role in soil P cycling. The objectives of this study were to determine (1) whether rotation crops had different amounts of root growth, (2) whether different amounts of root growth among the crop species could be related to different levels of soluble soil C and (3) whether there were differences in P concentration among the soils under different crops that could be related to soluble C soil concentration. Roots and soil from potato (Solanum tuberosum L.), barley (Hordeum vulgare L.), soybean (Glycine max (L.) Merr.), and a forage consisting of alfalfa (Medicago sativa L.) and timothy (Phleum pretense L.) were sampled from the Aroostook Research Farm in Presque Isle, Maine, during the summers of 2003 and 2004 to determine root length density (RLD) and soluble C and P concentrations. Half of the sampled plots were amended with beef manure and half were not amended. Barley and forage consistently had higher RLD than potato or soybean crops. Barley and forage typically had higher concentrations of soluble soil C than potato or soybean, but the differences were significant at only three of the five sampling dates. RLD was significantly correlated to soluble C (r=0.56) only for amended soils on the August 2003 sampling date. For other dates r values were non-significant and ranged from 0.32 to 0.49. As with soil C, soluble soil P levels were typically higher in barley and forage than in potato or soybean crops. Significant differences were detected at four of the five sampling dates. Correlations between soluble C and soluble P were significant at two of the five sampling dates (r = 0.58 and 0.62) in amended soils and one of five sampling dates (r = 0.80) in unamended soils. Although the correlations between RLD and soluble C were not significant at every sampling date, the August 2003 data do suggest a possible effect of roots on soluble C. In addition, significant correlations between soluble C and soluble P at several sampling dates suggest a relationship between these parameters. Therefore cropping systems that include crops with higher amounts of root growth may promote increased soluble soil C levels and enhance P bioavailability.     

Force-controlled equilibria of specific vesicle-substrate adhesion

We have developed "vertical" magnetic tweezers capable of exerting controlled pico and subpico Newton forces. Using this apparatus, we apply a point-like force to a vesicle that is adhered by means of specific bonds between the vesicle-carrying oligosaccharide sialyl LewisX and the surface-grafted E-selectin. An exponential decrease of the bound vesicle area with the decay rate that is insensitive to the force and the composition of the system is observed. We measure an equilibrium under force that is associated with an increased binding in the center of the contact zone. We also show that the determination of the shape is potentially sufficient to determine the number of formed specific bonds.     

Seed banks on Attalea phalerata (Arecaceae) stems in the Pantanal wetland, Brazil

Background and Aims

Seeds can accumulate in the soil or elsewhere, such as on the stems of palms when these are covered by persistent sheaths. These sheaths could act as a safe site for some species. Here, we studied whether persistent sheaths of the palm Attalea phalerata (Arecaceae) are available sites for seed accumulation in the Pantanal wetland of Brazil. We also investigated whether the composition, richness and diversity of species of seeds in the persistent sheaths are determined by habitat (riparian forest and forest patches) and/or season (wet and dry).

Methods

All accumulated material was collected from ten persistent sheaths along the stems of 64 A. phalerata individuals (16 per habitat and 16 per season). The material was then individually inspected under a stereomicroscope to record seed species and number.

Key Results

Of the 640 sheaths sampled, 65 % contained seeds (n = 3468). This seed bank included 75 species belonging to 12 families, and was primarily composed of small, endozoochoric seeds, with a few abundant species (Cecropia pachystachya and Ficus pertusa). Moraceae was the richest family (four species) and Urticaceae the most abundant (1594 seeds). Stems of A. phalerata in the riparian forest had 1·8 times more seeds and 1·3 times more species than those in forest patches. In the wet season we sampled 4·1 times more seeds and 2·2 more species on palm stems than in the dry season. Richness did not differ between habitats, but was higher in the wet season. Abundance was higher in forest patches and in the wet season.

Conclusions

Attalea phalerata stems contain a rich seed bank, comparable to soil seed banks of tropical forests. As most of these seeds are not adapted to grow in flooding conditions, palm stems might be regarded as safe sites for seeds (and seedlings) to escape from the seasonal flooding of the Pantanal.     

Missense substitutions in the GAS1 protein present in holoprosencephaly patients reduce the affinity for its ligand,SHH

Holprosencephaly (HPE) is the most common disorder of the developing forebrain in humans, and is characterized by varying degrees of abnormal union of the cerebral hemispheres. These defects are typically co-associated with midline craniofacial anomalies. The combination of forebrain and craniofacial defects that comprise HPE can present along a broad and variable phenotypic spectrum. Both the SHH and NODAL signaling pathways play important roles in the pathogenesis of this disorder. Disruption of these pathways by chromosomal rearrangements, mutations in pathway-related genes and/or biochemical alterations are proposed to contribute to HPE in a large number of patients. Additional factors that are not yet fully delineated are also very likely to be involved in the pathogenesis and phenotypic heterogeneity of the disorder. Genetic loss of GAS1, a cell membrane receptor and positive regulator of SHH, has been demonstrated to contribute to the HPE phenotypic spectrum in animal models. We have evaluated the coding and flanking sequence of GAS1 in 394 patients who have clinical findings within the HPE phenotypic spectrum, and now report five novel missense sequence variants among five unrelated HPE probands. Finally, we tested the effect of these variants (as well as previously reported GAS1 variants) on the ability of GAS1 to bind to SHH. Here, we demonstrate that sequence variants in GAS1 can impair its physical interaction with SHH, suggesting a decrease in the SHH downstream signaling cascade as a pathogenic mechanism of disease.     

Indole-3-acetaldoxime-derived compounds restrict root colonization in the beneficial interaction between Arabidopsis roots and the endophyte Piriformospora indica

The growth-promoting and root-colonizing endophyte Piriformospora indica induces camalexin and the expression of CYP79B2, CYP79B3, CYP71A13, PAD3, and WRKY33 required for the synthesis of indole-3-acetaldoxime (IAOx)-derived compounds in the roots of Arabidopsis seedlings. Upregulation of the mRNA levels by P. indica requires cytoplasmic calcium elevation and mitogen-activated protein kinase 3 but not root-hair-deficient 2, radical oxygen production, or the 3-phosphoinositide-dependent kinase 1/oxidative signal-inducible 1 pathway. Because P. indica-mediated growth promotion is impaired in cyp79B2 cyp79B3 seedlings, while pad3 seedlings-which do not accumulate camalexin-still respond to the fungus, IAOx-derived compounds other than camalexin (e.g., indole glucosinolates) are required during early phases of the beneficial interaction. The roots of cyp79B2 cyp79B3 seedlings are more colonized than wild-type roots, and upregulation of the defense genes pathogenesis-related (PR)-1, PR-3, PDF1.2, phenylalanine ammonia lyase, and germin indicates that the mutant responds to the lack of IAOx-derived compounds by activating other defense processes. After 6 weeks on soil, defense genes are no longer upregulated in wild-type, cyp79B2 cyp79B3, and pad3 roots. This results in uncontrolled fungal growth in the mutant roots and reduced performance of the mutants. We propose that a long-term harmony between the two symbionts requires restriction of root colonization by IAOx-derived compounds.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.