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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.

SUBMITTER: Klein DE

PROVIDER: S-EPMC3571469 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Structure of a dengue virus envelope protein late-stage fusion intermediate.

Klein Daryl E DE Choi Jason L JL Harrison Stephen C SC

Journal of virology 20121212 4

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 pa ...[more]

PMID: 23236058

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Project description:The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV) in three countries, provides key insights in developing strategies for tackling ZIKV, which has caused global panic to microcephaly and Guillain-Barre Syndrome. Dengue virus (DENV), a member of the family Flaviviridae, the causal agent of the self-limiting Dengue fever and the potentially fatal hemorrhagic fever/dengue shock syndrome, has been a scourge in tropical countries for many centuries. The recently solved structure of mature ZIKV (PDB ID:5IRE) has provided key insights into the structure of the envelope (E) and membrane (M) proteins, the primary target of neutralizing antibodies. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes, and also enumerates residue pairs that undergo significant polarity reversal. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. These secondary structures are conserved in the related Dengue virus (DENV), and possibly rationalize isolation techniques particle adsorption on magnetic beads coated with anionic polymers and anionic antiviral agents (viprolaxikine) for DENV. These amphipathic α-helices could enable design of molecules for inhibiting α-helix mediated protein-protein interactions. Finally, comparison of these secondary structures in proteins from related families illuminate subtle changes in the proteins that might render them ineffective to previously successful drugs and vaccines, which are difficult to identify by a simple sequence or structural alignment. Finally, conflicting results about residues that are involved in neutralizing a DENV-E protein by the potent antibody 5J7 (PDB ID:3J6U) are reported.

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Structure of a dengue virus envelope protein late-stage fusion intermediate.

Publications

Structure of a dengue virus envelope protein late-stage fusion intermediate.

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