The Ebola Viral Protein 35 N-Terminus Is a Parallel Tetramer.

Members of Mononegavirales, the order that includes nonsegmented negative sense RNA viruses (NNSVs), encode a small number of multifunctional proteins. In members of the Filoviridae family, virus protein 35 (VP35) facilitates immune evasion and functions as an obligatory cofactor for viral RNA synthesis. VP35 functions in a manner orthologous to that of phosphoproteins from other NNSVs. Although the critical roles of Ebola viral VP35 (eVP35) in immune evasion and RNA synthesis are well-appreciated, a complete understanding of its organization and its role in carrying out its many functions has yet to be fully realized. In particular, we currently lack information about the role of the oligomerization domain within eVP35. To address this limitation, we report here an investigation of the oligomer structure of eVP35 using hybrid methods that include multiangle light scattering, small-angle X-ray scattering, and cross-linking coupled with mass spectrometry to determine the shape and orientation of the eVP35 oligomer. Our integrative results are consistent with a parallel tetramer in which the N-terminal regions that are required for RNA synthesis are all oriented in the same direction. Furthermore, these results define a framework for targeting the symmetric tetramer for structure-based antiviral discovery.

Structural basis for human respiratory syncytial virus NS1-mediated modulation of host responses.

Human respiratory syncytial virus (hRSV) is a major cause of morbidity and mortality in the paediatric, elderly and immune-compromised populations1,2. A gap in our understanding of hRSV disease pathology is the interplay between virally encoded immune antagonists and host components that limit hRSV replication. hRSV encodes for non-structural (NS) proteins that are important immune antagonists3-6; however, the role of these proteins in viral pathogenesis is incompletely understood. Here, we report the crystal structure of hRSV NS1 protein, which suggests that NS1 is a structural paralogue of hRSV matrix (M) protein. Comparative analysis of the shared structural fold with M revealed regions unique to NS1. Studies on NS1 wild type or mutant alone or in recombinant RSVs demonstrate that structural regions unique to NS1 contribute to modulation of host responses, including inhibition of type I interferon responses, suppression of dendritic cell maturation and promotion of inflammatory responses. Transcriptional profiles of A549 cells infected with recombinant RSVs show significant differences in multiple host pathways, suggesting that NS1 may have a greater role in regulating host responses than previously appreciated. These results provide a framework to target NS1 for therapeutic development to limit hRSV-associated morbidity and mortality.

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide.

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.

Ebola virus VP35 interaction with dynein LC8 regulates viral RNA synthesis.

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

O-linked protein glycosylation in Mycoplasma.

Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high-resolution mass spectrometry. O-linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O-linked modification, and glucose was inferred by (13) C-labelling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.

Rhamnose biosynthesis in mycoplasmas requires precursor glycans larger than monosaccharide.

Despite the apparent absence of genes coding for the known pathways for biosynthesis, the monosaccharide rhamnose was detected in the d configuration in Mycoplasma pneumoniae and Mycoplasma pulmonis, and in both the d and l configurations in Mycoplasma arthritidis. Surprisingly, the monosaccharide glucose was not a precursor for rhamnose biosynthesis and was not incorporated at detectable levels in glucose-containing polysaccharides or glycoconjugates. In contrast, carbon atoms from starch, a polymer of glucose, were incorporated into rhamnose in each of the three species examined. When grown in a serum-free medium supplemented with starch, M. arthritidis synthesized higher levels of rhamnose, with a shift in the relative amounts of the d and l configurations. Our findings suggest the presence of a novel pathway for rhamnose synthesis that is widespread in the genus Mycoplasma.

Uranyl adsorption at the muscovite (mica)/water interface studied by second harmonic generation.

Uranyl adsorption at the muscovite (mica)/water interface was studied by second harmonic generation (SHG). Using the nonresonant χ(3) technique and the Gouy-Chapman model, the initial surface charge density of the mica surface was determined to be -0.022(1) C/m(2) at pH 6 and in the presence of dissolved carbonate. Under these same conditions, uranyl adsorption isotherms collected using nonresonant χ(3) experiments and resonantly enhanced SHG experiments that probe the ligand-to-metal charge transfer bands of the uranyl cation yielded a uranyl binding constant of 3(1) × 10(7) M(-1), corresponding to a Gibbs free energy of adsorption of -52.6(8) kJ/mol, and a maximum surface charge density at monolayer uranyl coverage of 0.028(3) C/m(2). These results suggest favorable adsorption of uranyl ions to the mica interface through strong ion-dipole or hydrogen interactions, with a 1:1 uranyl ion to surface site ratio that is indicative of monovalent cations ((UO(2))(3)(OH)(5)(+), (UO(2))(4)(OH)(7)(+), UO(2)OH(+), UO(2)Cl(+), UO(2)(CH(3)COO(-))(+)) binding at the interface, in addition to neutral uranyl species (UO(2)(OH)(2) and UO(2)CO(3)). This work provides benchmark measurements to be used in the improvement of contaminant transport modeling.

Exponential sensitivity and speciation of Al(III), Sc(III), Y(III), La(III), and Gd(III) at fused silica/water interfaces.

The binding contants, adsorption free energies, absolute adsorbate number densities, and interfacial charge densities of Al(III), Sc(III), Y(III), La(III), and Gd(III) interacting with fused silica/water interfaces held at pH 4 were determined using second harmonic generation and the Eisenthal χ((3)) technique. By examining the relationship between the measured adsorption free energies and the electric double layer interfacial potential at multiple electrolyte concentrations, we elucidate the charge state and possible binding pathways for each ion at the fused silica surface. Al(III) and Sc(III) ions are found to bind to the fused silica surface as fully hydrated trivalent species in a bidentate geometry. In contrast, the Y(III), La(III), and Gd(III) ions are each shown to adsorb to the silica surface in a decreased charge state, but the extent and mode of binding varies with each ion. By quantifying the exponential sensitivity of the surface coverage of the adsorbed ions to their charge state directly at the fused silica/water interface, we provide benchmarks for theory calculations describing the interactions of metal ions with oxide interfaces in geochemistry and hope to improve the prediction of trivalent metal ion transport through groundwater environments.

Divalent metal cation speciation and binding to surface-bound oligonucleotide single strands studied by second harmonic generation.

The binding of Sr(II), Ca(II), Mg(II), Ba(II), Mn(II), Zn(II), and Cd(II) to silica/water interfaces functionalized with A(15)T(6) oligonucleotides was quantified at pH 7 and 10 mM NaCl using the Eisenthal χ((3)) technique. The binding free energies range from -31.1(6) kJ/mol for Ba(II) to -33.8(4) kJ/mol for Ca(II). The ion densities were found to range from 2(1) ions/strand for Zn(II) to 11(1) ions/strand for Cd(II). Additionally, we quantified Mg(II) binding in the presence of varying background electrolyte concentrations which showed that the binding free energies changed in a linear fashion from -39.3(8) to -27(1) kJ/mol over the electrolyte concentration range of 1-80 mM, respectively. An adsorption free energy versus interfacial potential analysis allowed us to elucidate the speciation of the bound Mg(II) ions and to identify three possible binding pathways. Our findings suggest that Mg(II) binds as a fully hydrated divalent cation, most likely displacing DNA-bound Na ions. These measurements will serve as a benchmark for computer simulations of divalent metal cation/DNA interactions for geochemical and biosensing applications.

Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers.

This article reports nonlinear optical measurements that quantify, for the first time directly and without labels, how many Mg(2+) cations are bound to DNA 21-mers covalently linked to fused silica/water interfaces maintained at pH 7 and 10 mM NaCl, and what the thermodynamics are of these interactions. The overall interaction of Mg(2+) with adenine, thymine, guanine, and cytosine is found to involve -10.0 ± 0.3, -11.2 ± 0.3, -14.0 ± 0.4, and -14.9 ± 0.4 kJ/mol, and nonspecific interactions with the phosphate and sugar backbone are found to contribute -21.0 ± 0.6 kJ/mol for each Mg(2+) ion bound. The specific and nonspecific contributions to the interaction energy of Mg(2+) with oligonucleotide single strands is found to be additive, which suggests that within the uncertainty of these surface-specific experiments, the Mg(2+) ions are evenly distributed over the oligomers and not isolated to the most strongly binding nucleobase. The nucleobases adenine and thymine are found to bind only three Mg(2+) ions per 21-mer oligonucleotide, while the bases cytosine and guanine are found to bind eleven Mg(2+) ions per 21-mer oligonucleotide.

Fewer essential genes in mycoplasmas than previous studies suggest.

Here, we describe mutants of Mycoplasma pulmonis that were obtained using a minitransposon, Tn4001TF1, which actively transposes but is then unable to undergo subsequent excision events. Using Tn4001TF1, we disrupted 39 genes previously thought to be essential for growth. Thus, the number of genes required for growth has been overestimated. This study also revealed evidence of gene duplications in M. pulmonis and identified chromosome segregation proteins that are dispensable in mycoplasmas but essential in Bacillus subtilis.

Interactions of Al(III), La(III), Gd(III), and Lu(III) with the fused silica/water interface studied by second harmonic generation.

The interactions of the trivalent metal cations Al(III), La(III), Gd(III), and Lu(III) with the silica/water interface were studied using the nonlinear optical technique of second harmonic generation (SHG). Specifically, the Eisenthal chi(3) technique was used to quantify the thermodynamics of trivalent ion adsorption to the bare fused silica surface. SHG adsorption isotherms were measured and fit with the triple layer surface complexation model to obtain adsorption free energies, binding constants, and interfacial charge densities. The adsorption free energy for Al(III) was found to be -37.2(5) kJ/mol, while the adsorption free energies for the three trivalent lanthanide cations ranged from -29.9(9) to -32.2(7) kJ/mol. Despite identical ionic charges, the metals under investigation displayed different affinities for the fused silica/water interface, and this finding is analyzed and interpreted in the context of size-dependent metal cation properties and metal ion speciation. The thermodynamic results from this work are valuable benchmarks for computer simulations of trivalent metal transport in the environment.

Genome of Mycoplasma arthritidis.

The genomes of several species of mycoplasma have been sequenced. Most of these species rely on the glycolytic pathway for energy production, with the one exception of Ureaplasma, a species that breaks down urea as its principle source of acquiring energy. Several species, including as Mycoplasma arthritidis, are nonglycolytic and can use arginine as their source of energy. Described here are the genome sequence and a transposon library of M. arthritidis. The genome of 820,453 bp is typical in size for a mycoplasma and contains two large families of genes that are predicted to code for phase-variable proteins. The transposon library was constructed using a minitransposon that inserts stably into the mycoplasma genome. Of the 635 predicted coding regions, 218 were disrupted in a library of 1,100 members. Dispensable genes included the gene coding for the MAM superantigen and genes coding for ribosomal proteins S15, S18, and L15.