A surprisingly simple electrostatic model explains bent vs. linear structures in M+-RG2 species (M = group 1 metal, Li–Fr; RG = rare gas, He–Rn)

It is found that a simple electrostatic model involving competition between the attractive dispersive interaction and induced-dipole repulsion between the two RG atoms performs extremely well in rationalizing the M+-RG2 geometries, where M = Group 1 metal and RG = rare gas. The Li+-RG2 and Na+-RG2 complexes have previously been found to exhibit quasilinear or linear minimum energy geometries, with the Na+-RG2 complexes having an additional bent local minimum [A. Andrejeva, A. M. Gardner, J. B. Graneek, R. J. Plowright, W. H. Breckenridge and T. G. Wright, J. Phys. Chem. A, 2013, 117, 13578]. In the present work, the geometries for M = K–Fr are found to be bent. A simple electrostatic model explains these conclusions and is able to account almost quantitatively for the binding energy of the second RG atom, as well as the form of the angular potential, for all thirty six titular species. Additionally, results of population analyses are presented together with orbital contour plots; combined with the success of the electrostatic model, the expectation that these complexes are all physically bound is confirmed

Human IFIT1 inhibits mRNA translation of rubulaviruses but not other members of the Paramyxoviridae family

This work was supported by The Welcome Trust (101788/Z/13/Z, 101792/Z/13/Z) and Medical research council grant (G1100110/1, MR/K024213/1).We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus (PIV) type 5, selectively inhibiting the translation of PIV5 mRNAs. Here we report that whilst PIV2, PIV5 and mumps virus (MuV) are sensitive to IFIT1, non-rubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV) and canine distemper virus (CDV) are resistant. The IFIT1-sensitivity of PIV5 was not rescued by co-infection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. Whilst the translation of PIV2, PIV5 and MuV mRNAs were directly inhibited by IFIT1, the translation of PIV3, SeV and CDV mRNAs were not. Using purified human mRNA capping enzymes we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5’ guanosine nucleoside cap (which need not be N7-methylated) and that this sensitivity is partly abrogated by 2’ O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2’ O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2’ -O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed. Importance Paramyxoviruses cause a wide variety of diseases and yet most of their genes encode for structural proteins and proteins involved in their replication cycle. Thus the amount of genetic information that determines the type of disease paramyxoviruses cause is relatively small. One factor that will influence disease outcomes is how they interact with innate host cell defences, including the interferon (IFN) system. Here we show that different paramyxoviruses interact in distinct ways with cells in a pre-existing IFN-induced antiviral state. Strikingly, all the rubulaviruses tested were sensitive to the antiviral action of ISG56/IFIT1, whilst all the other paramyxoviruses tested were resistant. We developed novel in vitro biochemical assays to investigate the mechanism of action of IFIT1, demonstrating that the mRNAs of rubulaviruses can be directly inhibited by IFIT1 and that this is at least partially because their mRNAs are not correctly methylated.Publisher PDFPeer reviewe

Cytoplasmic glycoengineering of Apx toxin fragments in the development of Actinobacillus pleuropneumoniae glycoconjugate vaccines.

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression

Vibrations of the S1 state of fluorobenzene-h5 and fluorobenzene-d5 via resonance-enhanced multiphoton ionization (REMPI) spectroscopy

We report resonance-enhanced multiphoton ionization spectra of the isotopologues fluorobenzeneh5 and fluorobenzene-d5. By making use of quantum chemical calculations, the changes in the wavenumber of the vibrational modes upon deuteration are examined. Additionally, the mixing of vibrational modes both between isotopologues and also between the two electronic states is discussed. The isotopic shifts lead to dramatic changes in the appearance of the spectrum as vibrations shift in and out of Fermi resonance. Assignments of the majority of the fluorobenzene-d5 observed bands are provided, aided by previous results on fluorobenzene-h5

Resonance-enhanced multiphoton ionization (REMPI) spectroscopy of bromobenzene and its perdeuterated isotopologue: assignment of the vibrations of the S0, S1 and D0+ states of bromobenzene and the S0 and D0+ states of iodobenzene

We report vibrationally-resolved spectra of the S1 S0 transition of bromobenzene using resonance-enhanced multiphoton ionization (REMPI) spectroscopy. We study bromobenzene-h5 as well as its perdeuterated isotopologue, bromobenzene-d5. The form of the vibrational modes between the isotopologues and also between the S0 and S1 electronic states are discussed for each species, allowing assignment of the bands to be achieved and the activity between states and isotopologues to be established. Vibrational bands are assigned utilizing quantum chemical calculations, previous experimental results and isotopic shifts. Previous work and assignments of the S1 spectra are discussed. Additionally, the vibrations in the ground state cation, D0+, are considered, since these have also been used by previous workers in assigning the excited neutral state spectra. We also examine the vibrations of iodobenzene in the S0 and D0+ states and comment on previous assignments of these. In summary, we have been able to assign corresponding vibrations across the whole monohalobenzene series of molecules, in the S0, S1 and D0+ states, gaining insight into vibrational activity and vibrational couplings

LGP2 plays a critical role in sensitizing mda-5 to activation by double-stranded RNA.

The DExD/H box RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene-5 (mda-5) sense viral RNA in the cytoplasm of infected cells and activate signal transduction pathways that trigger the production of type I interferons (IFNs). Laboratory of genetics and physiology 2 (LGP2) is thought to influence IFN production by regulating the activity of RIG-I and mda-5, although its mechanism of action is not known and its function is controversial. Here we show that expression of LGP2 potentiates IFN induction by polyinosinic-polycytidylic acid [poly(I:C)], commonly used as a synthetic mimic of viral dsRNA, and that this is particularly significant at limited levels of the inducer. The observed enhancement is mediated through co-operation with mda-5, which depends upon LGP2 for maximal activation in response to poly(I:C). This co-operation is dependent upon dsRNA binding by LGP2, and the presence of helicase domain IV, both of which are required for LGP2 to interact with mda-5. In contrast, although RIG-I can also be activated by poly(I:C), LGP2 does not have the ability to enhance IFN induction by RIG-I, and instead acts as an inhibitor of RIG-I-dependent poly(I:C) signaling. Thus the level of LGP2 expression is a critical factor in determining the cellular sensitivity to induction by dsRNA, and this may be important for rapid activation of the IFN response at early times post-infection when the levels of inducer are low

Theoretical study of M+ RG2: (M+= Ca, Sr, Ba and Ra; RG= He–Rn)

Ab initio calculations were employed to investigate M+ RG2 species, where M+ = Ca, Sr, Ba and Ra and RG= He–Rn. Geometries have been optimized, and cuts through the potential energy surfaces containing each global minimum have been calculated at the MP2 level of theory, employing triple-ζ quality basis sets. The interaction energies for these complexes were calculated employing the RCCSD(T) level of theory with quadruple-ζ quality basis sets. Trends in binding energies, De, equilibrium bond lengths, Re, and bond angles are discussed and rationalized by analyzing the electronic density. Mulliken, natural population, and atoms-in-molecules (AIM) population analyses are presented. It is found that some of these complexes involving the heavier Group 2 metals are bent whereas others are linear, deviating from observations for the corresponding Be and Mg metal-containing complexes, which have all previously been found to be bent. The results are discussed in terms of orbital hybridization and the different types of interaction present in these species

Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies

<p>Abstract</p> <p>Background</p> <p>Mumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly) of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT) of the other variant.</p> <p>Results</p> <p>VWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln₁₀₃Pro (Pro¹⁰³) and the Gly¹⁵⁶insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3), STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5) V protein structure in complex with STAT1-STAT2 heterodimer. <it>In silico </it>analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W¹⁷⁴, W¹⁷⁸, W¹⁸⁹) and Glu⁹⁵residue close to the Arg⁴⁰⁹and Lys⁴¹⁵of the nuclear localization signal (NLS) of STAT2, leaving exposed STAT1 Lys residues (K⁸⁵, K⁸⁷, K²⁹⁶, K⁴¹³, K⁵²⁵, K⁶⁷⁹, K⁶⁸⁵), which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2.</p> <p>Conclusions</p> <p>Our results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to inactivate both the IFN signaling pathway and antiviral response due to differences in their finest molecular interaction with STAT proteins.</p

Identification of DNA-Damage DNA-Binding Protein 1 as a Conditional Essential Factor for Cytomegalovirus Replication in Interferon-γ-Stimulated Cells

The mouse cytomegaloviral (MCMV) protein pM27 represents an indispensable factor for viral fitness in vivo selectively, antagonizing signal transducer and activator of transcription 2 (STAT2)-mediated interferon signal transduction. We wished to explore by which molecular mechanism pM27 accomplishes this effect. We demonstrate that pM27 is essential and sufficient to curtail the protein half-life of STAT2 molecules. Pharmacologic inhibition of the proteasome restored STAT2 amounts, leading to poly-ubiquitin-conjugated STAT2 forms. PM27 was found in complexes with an essential host ubiquitin ligase complex adaptor protein, DNA-damage DNA-binding protein (DDB) 1. Truncation mutants of pM27 showed a strict correlation between DDB1 interaction and their ability to degrade STAT2. SiRNA-mediated knock-down of DDB1 restored STAT2 in the presence of pM27 and strongly impaired viral replication in interferon conditioned cells, thus phenocopying the growth attenuation of M27-deficient virus. In a constructive process, pM27 recruits DDB1 to exploit ubiquitin ligase complexes catalyzing the obstruction of the STAT2-dependent antiviral state of cells to permit viral replication

Human Metapneumovirus Inhibits IFN-β Signaling by Downregulating Jak1 and Tyk2 Cellular Levels

Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN) signaling by an unidentified mechanism. In this study, we showed that infection of airway epithelial cells with hMPV decreased cellular level of Janus tyrosine kinase (Jak1) and tyrosine kinase 2 (Tyk2), due to enhanced proteosomal degradation and reduced gene transcription. In addition, hMPV infection also reduced the surface expression of type I IFN receptor (IFNAR). These inhibitory mechanisms are different from the ones employed by respiratory syncytial virus (RSV), which does not affect Jak1, Tyk2 or IFNAR expression, but degrades downstream signal transducer and activator of transcription proteins 2 (STAT2), although both viruses are pneumoviruses belonging to the Paramyxoviridae family. Our study identifies a novel mechanism by which hMPV inhibits STAT1 and 2 activation, ultimately leading to viral evasion of host IFN responses