Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism.DOI: http://dx.doi.org/10.7554/eLife.00333.001

The final stages of dengue virus fusion are thought to occur when the membrane-proximal stem drives the transmembrane anchor of the viral envelope protein (E) toward the fusion loop, buried in the target cell membrane. Crystal structures of E have lacked this essential stem region. We expressed and crystallized soluble mutant forms of the dengue virus envelope protein (sE) that include portions of the juxtamembrane stem. Their structures represent late-stage fusion intermediates. The proximal part of the stem has both intra-and intermolecular interactions, so the chain "zips up" along the trimer seam. The penultimate interaction we detected involves the conserved residue F402, which has hydrophobic contacts with a conserved surface on domain II. These interactions do not require any larger-scale changes in trimer packing. The techniques for expression and crystallization of sE containing stem reported here may allow further characterization of the final stages of flavivirus fusion. The membrane-spanning envelope glycoprotein protein (E) of flaviviruses is both the principal determinant of icosahedral virion assembly and the fusion catalyst for merging viral and target cell membranes ( Fig. 1) (1, 2). The E protein folds into three domains, a membrane-proximal stem, and a transmembrane anchor (Fig. 1A). Various crystal structures have shown the arrangement of the three folded domains in both a dimeric prefusion conformation ( Fig. 1C) (3, 6) and a low-pHinduced postfusion trimer ( Fig. 1F) (4, 7). The hydrophobic fusion loops, buried at the dimer interface in the prefusion structure (3, 5, 6), cluster into a large hydrophobic surface at one end of the postfusion trimer (4,7,8). In this orientation, the fusion loops attach the virus to the target cell membrane. The membrane-proximal stem has two predicted amphipathic helices that lie half-buried in the outer leaflet of the viral membrane ( Fig. 1C) (9,10). For fusion to take place, this stem must span the length of domain II (Fig. 1F). A likely model is that it "zips up" along the gaps between the clustered domains, bringing together the transmembrane anchor and the fusion loops, inducing deformation of their associated membranes and leading to membrane merger.Several studies show the importance of the amino-terminal part of the stem and suggest that it forms contacts with domain II as the fusion-inducing transition proceeds (11-13). Efforts to visualize it directly in this conformation have failed, however, because including the stem residues in recombinant E generally leads to instability or aggregation of any secreted product. We describe here a method for producing sE that includes portions of the juxtamembrane stem and report its crystallization and structure determination. The structure shows that the N-terminal part of the stem zips up along the seam between adjacent domains II in the trimer. The rest of the stem in our constructs is disordered. The arrangement of the trimer core is the same as in previous, stemless structures. The results are consistent wi...

In this study, three recombinant mojastin peptides (Moj-WN, Moj-NN, and Moj-DM) were produced and compared functionally. Recombinant Moj peptides were purified as GST-fusions. GST-Moj-WN and GST-Moj-NN inhibited ADP-induced platelet aggregation in platelet rich plasma. The GST-Moj-WN had an IC50 of 160 nM, while the GST-Moj-NN had an IC50 of 493 nM. The GST-Moj-DM did not inhibit platelet aggregation. All three GST-Moj peptides inhibited SK-Mel-28 cell adhesion to fibronectin. The GST-Moj-WN inhibited the binding of SK-Mel-28 cells to fibronectin with an IC50 of 11 nM, followed by the GST-Moj-NN (IC50 of 28 nM), and the GST-Moj-DM (IC50 of 46 nM). The GST-Moj peptides' ability to induce apoptosis on SK-Mel-28 cells was determined using Annexin-V-FITC and nuclear fragmentation assays. Cells were incubated with 5 μM GST-Moj peptides for 24 h. At 5 μM GST-Moj-DM peptide, 13.56% +/- 2.08 of treated SK-Mel-28 cells were in early apoptosis. The GST-Moj-DM peptide also caused nuclear fragmentation as determined by fluorescent microscopy and Hoechst staining. The GST-Moj-WN and GST-Moj-NN peptides failed to induce apoptosis. We characterized the SK-Mel-28 integrin expression, as the first step in determining r-Moj binding specificity. Our results indicate that SK-Mel-28 cells express αvβ3, αv, α6, β1, and β3 integrin receptors.

Bipolar disorder (BD) is a common, recurring psychiatric illness with unknown pathogenesis. Recent studies suggest that microRNA (miRNA) levels in brains of BD patients are significantly altered, and these changes may offer insight into BD pathology or etiology. Previously, we observed significant alterations of miR-29c levels in extracellular vesicles (EVs) extracted from prefrontal cortex (Brodmann area 9, BA9) of BD patients. In this study, we show that EVs extracted from the anterior cingulate cortex (BA24), a crucial area for modulating emotional expression and affect, have increased levels of miR-149 in BD patients compared to controls. Because miR-149 has been shown to inhibit glial proliferation, increased miR-149 expression in BA24-derived EVs is consistent with the previously reported reduced glial cell numbers in BA24 of patients diagnosed with either familial BD or familial major depressive disorder. qPCR analysis of laser-microdissected neuronal and glial cells from BA24 cortical samples of BD patients verified that the glial, but not neuronal, population exhibits significantly increased miR-149 expression. Finally, we report altered expression of both miR-149 and miR-29c in EVs extracted from brains of Flinders Sensitive Line rats, a well-validated animal model exhibiting depressive-like behaviors and glial (astrocytic) dysfunction. These findings warrant future investigations into the potential of using EV miRNA signatures as biomarkers to further enhance the biological definition of BD. © 2017 Wiley Periodicals, Inc.