Structure of a Dengue Virus Envelope Protein Late-Stage Fusion Intermediate

Abstract

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...

Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination

“…However, the inclusion of Pr-M in subunit vaccines has been questioned, since recent evidence indicates that anti-Pr antibodies can enhance infection by promoting internalization of immature viruses (Dejnirattisai et al, 2010;Rodenhuis-Zybert et al, 2010). Also, the stem and anchor regions of E were shown to affect E expression in the absence of viral infection (Klein et al, 2013).…”

Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination

“…The DENV envelope (E) glycoprotein is required for viral binding and entry into cells (8,9). E protein is also the main target of neutralizing antibodies (10).…”

The Membrane-Proximal “Stem” Region Increases the Stability of the Flavivirus E Protein Postfusion Trimer and Modulates Its Structure

“…In crystal structures of dengue virus trimers containing N-terminal portions of the stem, only the first stem residues (up to amino acid 404 [TBEV numbering]) exhibited intra-and intermolecular interactions with DII, whereas the remaining residues were disordered (14). A loop in DIII, however, was in a different conformation in these structures compared to the stemless trimer (14). This may be due to crystallizing of the trimers at different pHs (14) but could also indicate that the presence of the (25,26).…”

“…H2 has been resolved in the 3.5-Å structure of dengue virus only recently (12). With the exception of the first few amino acids following the C terminus of the E ectodomain (13,14), details of the stem in the context of the postfusion trimer are presently unknown. In X-ray crystallographic analyses of truncated dengue virus sE trimers that included N-terminal portions of the stem, most of the stem parts were disordered (14).…”

“…With the exception of the first few amino acids following the C terminus of the E ectodomain (13,14), details of the stem in the context of the postfusion trimer are presently unknown. In X-ray crystallographic analyses of truncated dengue virus sE trimers that included N-terminal portions of the stem, most of the stem parts were disordered (14). The objective of our work was to generate experimental evidence for interactions of the stem with the core of the postfusion E trimer.…”