In vitro reconstitution reveals key intermediate states of trimer formation by the dengue virus membrane fusion protein

J Virol. 2010 Jun;84(11):5730-40. doi: 10.1128/JVI.00170-10. Epub 2010 Mar 24.

Abstract

The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Membrane / metabolism
  • Dengue Virus / chemistry*
  • Hydrogen-Ion Concentration
  • Protein Folding
  • Protein Multimerization*
  • Viral Envelope Proteins
  • Viral Fusion Proteins / chemistry*
  • Virus Internalization

Substances

  • Viral Envelope Proteins
  • Viral Fusion Proteins