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ABSTRACT
The ultimate levels of dengue virus fusion are thought to happen when the membrane-proximal stem drives the transmembrane anchor of the viral envelope protein (E) towards the fusion loop, buried within the goal cell membrane. Crystal constructions of E have lacked this important stem area. We expressed and crystallized soluble mutant types of the dengue virus envelope protein (sE) that embrace parts of the juxtamembrane stem. Their constructions characterize late-stage fusion intermediates. The proximal a part of the stem has each intra- and intermolecular interactions, so the chain “zips up” alongside the trimer seam. The penultimate interplay we detected includes the conserved residue F402, which has hydrophobic contacts with a conserved floor on area II. These interactions don’t require any larger-scale modifications in trimer packing. The strategies for expression and crystallization of sE containing stem reported right here might permit additional characterization of the ultimate levels of flavivirus fusion.
INTRODUCTION – “e protein dengue”
The membrane-spanning envelope glycoprotein protein (E) of flaviviruses is each the principal determinant of icosahedral virion meeting and the fusion catalyst for merging viral and goal cell membranes (Fig. 1) (1, 2). The E protein folds into three domains, a membrane-proximal stem, and a transmembrane anchor (Fig. 1A). Numerous crystal constructions have proven the association of the three folded domains in each a dimeric prefusion conformation (Fig. 1C) (3, 6) and a low-pH-induced postfusion trimer (Fig. 1F) (4, 7). The hydrophobic fusion loops, buried on the dimer interface within the prefusion construction (3, 5, 6), cluster into a big hydrophobic floor at one finish of the postfusion trimer (4, 7, 8). On this orientation, the fusion loops connect the virus to the goal cell membrane. The membrane-proximal stem has two predicted amphipathic helices that lie half-buried within the outer leaflet of the viral membrane (Fig. 1C) (9, 10). For fusion to happen, this stem should span the size of area II (Fig. 1F). A possible mannequin is that it “zips up” alongside the gaps between the clustered domains, bringing collectively the transmembrane anchor and the fusion loops, inducing deformation of their related membranes and resulting in membrane merger.
A number of research present the significance of the amino-terminal a part of the stem and counsel that it kinds contacts with area II because the fusion-inducing transition proceeds (11–13). Efforts to visualise it instantly on this conformation have failed, nonetheless, as a result of together with the stem residues in recombinant E usually results in instability or aggregation of any secreted product. We describe right here a way for producing sE that features parts of the juxtamembrane stem and report its crystallization and construction willpower. The construction reveals that the N-terminal a part of the stem zips up alongside the seam between adjoining domains II within the trimer. The remainder of the stem in our constructs is disordered. The association of the trimer core is similar as in earlier, stemless constructions. The outcomes are in line with a mannequin wherein the N-terminal (proximal) stem stabilizes a trimer with clustered fusion loops, whereas the central half (residues 404 to 421) has few, if any, contacts with the folded domains within the E trimer.
MATERIALS AND METHODS
RESULTS
“e protein dengue”