A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology.

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro. Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)- carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline\u27s unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics andNMRexperiments.Wetherefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis. © 2019 Singh et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc

Structural analysis of glutathionyl hemoglobin using native mass spectrometry

Glutathionylation is an example of reversible post-translation modification of proteins where free and accessible cysteine residues of proteins undergo thiol-disulfide exchange with oxidized glutathione (GSSG). In general, glutathionylation occurs under the condition of elevated oxidative stress in vivo. In human hemoglobin, Cys93 residue of beta globin chain was found to undergo this oxidative modification. Glutathionyl hemoglobin (GSHb) was reported to act as a biomarker of oxidative stress under several clinical conditions such as chronic renal failure, iron deficiency anemia, hyperlipidemia, diabetes mellitus, Friedreich's ataxia, atherosclerosis. Previously we showed that the functional abnormality associated with six-fold tighter oxygen binding of GSHb supposedly attributed to the conformational transition of the deoxy state of GSHb towards oxy hemoglobin like conformation. In the present study, we investigated the structural integrity and overall architecture of the quaternary structure of GSHb using native mass spectrometry and ion mobility mass spectrometry platforms. The dissociation equilibrium constants of both tetramer/dimer (K-d1) and dimer/monomer equilibrium (K-d2) was observed to increase by 1.91 folds and 3.64 folds respectively. However, the collision cross-section area of the tetrameric hemoglobin molecule remained unchanged upon glutathionylation. The molecular dynamics simulation data of normal human hemoglobin and GSHb was employed to support our experimental findings

Two Consecutive Prolines in the Fusion Peptide of Murine β-Coronavirus Spike Protein Predominantly Determine Fusogenicity and May Be Essential but Not Sufficient to Cause Demyelination

Combined in silico, in vitro, and in vivo comparative studies between isogenic-recombinant Mouse-Hepatitis-Virus-RSA59 and its proline deletion mutant, revealed a remarkable contribution of centrally located two consecutive prolines (PP) from Spike protein fusion peptide (FP) in enhancing virus fusogenic and hepato-neuropathogenic potential. To deepen our understanding of the underlying factors, we extend our studies to a non-fusogenic parental virus strain RSMHV2 (P) with a single proline in the FP and its proline inserted mutant, RSMHV2 (PP). Comparative in vitro and in vivo studies between virus strains RSA59(PP), RSMHV2 (P), and RSMHV2 (PP) in the FP demonstrate that the insertion of one proline significantly resulted in enhancing the virus fusogenicity, spread, and consecutive neuropathogenesis. Computational studies suggest that the central PP in Spike FP induces a locally ordered, compact, and rigid structure of the Spike protein in RSMHV2 (PP) compared to RSMHV2 (P), but globally the Spike S2-domain is akin to the parental strain RSA59(PP), the latter being the most flexible showing two potential wells in the energy landscape as observed from the molecular dynamics studies. The critical location of two central prolines of the FP is essential for fusogenicity and pathogenesis making it a potential site for designing antiviral

Molecular insights of inhibition in sickle hemoglobin polymerization upon glutathionylation: hydrogen/deuterium exchange mass spectrometry and molecular dynamics simulation-based approach

In sickle cell anemia, polymerization of hemoglobin in its deoxy state leads to the formation of insoluble fibers that result in sickling of red blood cells. Stereo-specific binding of isopropyl group of beta Val6, the mutated amino-acid residue of a tetrameric sickle hemoglobin molecule (HbS), with hydrophobic groove of another HbS tetramer initiates the polymerization. Glutathionylation of beta Cys93 in HbS was reported to inhibit the polymerization. However, the mechanism of inhibition in polymerization is unknown to date. In our study, the molecular insights of inhibition in polymerization were investigated by monitoring the conformational dynamics in solution phase using hydrogen/deuterium exchange-based mass spectrometry. The conformational rigidity imparted due to glutathionylation of HbS results in solvent shielding of beta Val6 and perturbation in the conformation of hydrophobic groove of HbS. Additionally, molecular dynamics simulation trajectory showed that the stereo-specific localization of glutathione moiety in the hydrophobic groove across the globin subunit interface of tetrameric HbS might contribute to inhibition in polymerization. These conformational insights in the inhibition of HbS polymerization upon glutathionylation might be translated in the molecularly targeted therapeutic approaches for sickle cell anemia

Test of light-lepton universality in τ\tau decays with the Belle II experiment

International audienceWe present a measurement of the ratio $R_\mu = \mathcal{B}(\tau^-\to \mu^-\bar\nu_\mu\nu_\tau) / \mathcal{B}(\tau^-\to e^-\bar\nu_e\nu_\tau)$ of branching fractions $\mathcal{B}$ of the $\tau$ lepton decaying to muons or electrons using data collected with the Belle II detector at the SuperKEKB $e^+e^-$ collider. The sample has an integrated luminosity of 362 fb$^{-1}$ at a centre-of-mass energy of 10.58 GeV. Using an optimised event selection, a binned maximum likelihood fit is performed using the momentum spectra of the electron and muon candidates. The result, $R_\mu = 0.9675 \pm 0.0007 \pm 0.0036$, where the first uncertainty is statistical and the second is systematic, is the most precise to date. It provides a stringent test of the light-lepton universality, translating to a ratio of the couplings of the muon and electron to the $W$ boson in $\tau$ decays of $0.9974 \pm 0.0019$, in agreement with the standard model expectation of unity