Early steps of the conformational change of influenza virus hemagglutinin to a fusion active state: stability and energetics of the hemagglutinin

A conformational change of the homotrimeric glycoprotein hemagglutinin (HA) of influenza virus mediates fusion between the viral envelope and the endosome membrane. The conformational change of the HA ectodomain is triggered by the acidic pH of the endosome lumen. An essential step of the conformational change is the formation of an extended coiled-coil motif exposing the hydrophobic fusion peptide toward the target membrane. The structures of the neutral-pH, non-fusion active conformation of the HA ectodomain and of a fragment of the ectodomain containing the coiled-coil motif are known. However, it is not known by which mechanism protonation triggers the conformational change of the stable neutral-pH conformation of the ectodomain. Here, recent studies on the stability of the HA ectodomain at neutral pH, the energetics of the conformational change toward the fusion-active state and of the unfolding of the HA ectodomain are summarised. A model for the early steps of the conformational change of the HA ectodomain is presented. The model implicates that protonation leads to a partial dissociation of the distal domains of the HA monomers that is driven by electrostatic repulsion. The opening of the ectodomain enables water to enter the ectodomain. The interaction of water with respective sequences originally shielded from contact with water drives the formation of the coiled-coil structure.     

Restriction of de novo pyrimidine biosynthesis inhibits Th1 cell activation and promotes Th2 cell differentiation

Leflunomide, an inhibitor of de novo pyrimidine biosynthesis, has recently been introduced as a treatment for rheumatoid arthritis in an attempt to ameliorate inflammation by inhibiting lymphocyte activation. Although the immunosuppressive ability of leflunomide has been well described in several experimental animal models, the precise effects of a limited pyrimidine supply on T cell differentiation and effector functions have not been elucidated. We investigated the impact of restricted pyrimidine biosynthesis on the activation and differentiation of CD4 T cells in vivo and in vitro. Decreased activation of memory CD4 T cells in the presence of leflunomide resulted in impaired generation and outgrowth of Th1 effectors without an alteration of Th2 cell activation. Moreover, priming of naive T cells in the presence of leflunomide promoted Th2 differentiation from uncommitted precursors in vitro and enhanced Th2 effector functions in vivo, as indicated by an increase in Ag-specific Th2 cells and in the Th2-dependent Ag-specific Ig responses (IgG1) in immunized mice. The effects of leflunomide on T cell proliferation and differentiation could be antagonized by exogenous UTP, suggesting that they were related to a profound inhibition of de novo pyrimidine biosynthesis. These results indicate that leflunomide might exert its anti-inflammatory activities in the treatment of autoimmune diseases by preventing the generation of proinflammatory Th1 effectors and promoting Th2 cell differentiation. Moreover, the results further suggest that differentiation of CD4 T cells can be regulated at the level of nucleotide biosynthesis.     

The Perception of Dynamic and Static Facial Expressions of Happiness and Disgust Investigated by ERPs and fMRI Constrained Source Analysis

A recent functional magnetic resonance imaging (fMRI) study by our group demonstrated that dynamic emotional faces are more accurately recognized and evoked more widespread patterns of hemodynamic brain responses than static emotional faces. Based on this experimental design, the present study aimed at investigating the spatio-temporal processing of static and dynamic emotional facial expressions in 19 healthy women by means of multi-channel electroencephalography (EEG), event-related potentials (ERP) and fMRI-constrained regional source analyses. ERP analysis showed an increased amplitude of the LPP (late posterior positivity) over centro-parietal regions for static facial expressions of disgust compared to neutral faces. In addition, the LPP was more widespread and temporally prolonged for dynamic compared to static faces of disgust and happiness. fMRI constrained source analysis on static emotional face stimuli indicated the spatio-temporal modulation of predominantly posterior regional brain activation related to the visual processing stream for both emotional valences when compared to the neutral condition in the fusiform gyrus. The spatio-temporal processing of dynamic stimuli yielded enhanced source activity for emotional compared to neutral conditions in temporal (e.g., fusiform gyrus), and frontal regions (e.g., ventromedial prefrontal cortex, medial and inferior frontal cortex) in early and again in later time windows. The present data support the view that dynamic facial displays trigger more information reflected in complex neural networks, in particular because of their changing features potentially triggering sustained activation related to a continuing evaluation of those faces. A combined fMRI and EEG approach thus provides an advanced insight to the spatio-temporal characteristics of emotional face processing, by also revealing additional neural generators, not identifiable by the only use of an fMRI approach.     

The role of CD18 in phorbol ester-induced human monocyte-mediated cytotoxicity

Monocyte cell surface molecules play an important role in the regulation of monocyte function. To investigate the molecular basis of monocyte-mediated cytotoxicity, we tested the ability of a variety of mediators to stimulate human monocyte-mediated cytotoxicity. Phorbol myristic acetate (PMA) stimulated significant monocyte-mediated killing of tumor cells in an 18-hr indium-111 release assay. Five other cytoactive substances did not induce monocyte-mediated cytotoxicity. The acquisition of monocyte cytotoxicity was associated with nearly a twofold increase in surface expression of three CD18-bearing cell surface molecules (CD11a, CD11b, CD11c). The direct involvement of the CD18-bearing molecules in monocyte-mediated cytotoxicity was investigated using monoclonal antibody (MAb) inhibition. MAb recognizing the CD18 subunit significantly inhibited monocyte-mediated killing. The inhibition by anti-CD18 MAb could not be attributed to LFA-1 (CD11a) alone, suggesting that CR3 (CD11b) and p150,95 (CD11c) may also participate in monocyte-mediated cytotoxicity. In contrast, seven of eight other cell surface structures were not affected by PMA treatment, and MAb to all eight cell surface structures did not inhibit killing. These findings suggest that CD18-bearing molecules are upregulated with monocyte activation and may play a functional role in monocyte-mediated killing.     

Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone

Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched (“raft”) domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2–GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA’s raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells.     

On the correct use of ‘data’

Acknowledgement  Thanks are due to Drs. Paul Johnston (P.Johnston@ exeter.ac.uk) and E. William Beese (ebeese@t-online.de) for kindly providing dictionary definitions and for sharing their thoughts about the correct use of ‘data’.