Dengue virus (DEN) is currently the most important flavivirus causing human disease in the tropics and subtropics. The mosquito-borne DEN is the causative agent of dengue fever and its associated complications of dengue hemorrhagic fever and dengue shock syndrome. The envelope protein (E) of DEN mediates entry into host cells through the dual functions of receptor binding and membrane fusion. During virion assembly and maturation, E associates with a second protein prM, the precursor of membrane protein M, in a heterodimeric complex that protects E from premature inactivation. Maturation of the virion involves cleavage of prM and reorientation of the E proteins as homodimers. Fusion occurs between the viral membrane and an intracellular membrane following a conformational change in E leading to the formation of homotrimers, a process triggered by the mildly acidic pH of the endosome. Three regions of the E protein previously identified as playing a
role in fusion activity, a putative hinge region between domains I and II, the fusion peptide itself and the stem-anchor region in the C-terminal end of prM/E were the focus of this study. We first characterised the fusion phenotype of two DEN2 strains, NGC and PR159 using both wildtype virus and expression of recombinant prM/E proteins using a SFV expression system. Here we provide evidence that two residues located within this domain, E-126 and E- 202, are responsible for determining the threshold pH for fusion activity. These residues are located at the base of domain II of E, a region that is believed to serve as a hinge during the low pH-mediated conformational change and is also the site of numerous, defined molecular virulence determinants for flaviviruses. The actual membrane fusion event is promoted via an internal fusion peptide sequence located at the distal tip of E that is believed to insert into the host membrane during the initial stages of entry. To better understand
the nature of the fusion peptide, we introduced amino acid substitutions within this sequence and investigated the effects of these changes on prM/E heterodimerisation, intracellular trafficking and fusion. The fusion peptide is thought to present a hydrophobic face on one side of a disulphide-bonded loop that interacts with the target membrane. It was our intention to either perturb or enhance this interaction by substituting conservative or non-conservative residues at specific locations. The residues targeted for mutagenesis included the hydrophobic residue Trp101, which forms part of the hydrophobic face, Cys74 and Cys145 that form the stabilising disulphide bond and Gly104, the only amino acid demonstrating variability within the fusion peptide. Other fusion peptide mutations targeted the highly conserved GLFG motif with substitutions being made at Gly106 and
Phe108. Two of the fusion peptide mutants, W101A and C74/105SS, were unable to form a stable prM/E heterodimer and these E proteins were not detected on the cell surface. Of the other mutants that did form heterodimers, surface E protein was detected for three mutants, G103H, G106F and G108A, while mutants W101F and G106A had impaired intracellular E transport. For the most part substitutions within the fusion peptide were not tolerated, however, G106F had a fusion phenotype similar to parental E, while a G103H change dramatically increased the pH threshold for fusion activation. The stem-anchor region of E contains sequences that are important for membrane anchoring, prM interactions and low-pH-induced conformational changes associated with fusion. To
explore the function of specific elements within this region, a series of prM/E C-terminal deletion constructs and alanine substitution mutants were generated and the biological properties of the resulting mutant prM/E proteins examined. The membrane-spanning regions of both prM and E were found to be unimportant for the stability of the prM/E heterodimer and the regions responsible for prM/E association were localised to prM106-146 and E406-450. However, while full-length prM and E proteins expressed both in cis and in trans induced syncytial formation in insect cells the removal of one or more transmembrane stretches abrogated fusion potential. Further analysis revealed that the region of E responsible for prM/E association contained five highly conserved phenylalanine and tyrosine residues. Alanine substitution demonstrated that these residues were not critical to prM/E heterodimerisation however multiple substitutions did abrogate syncytial
formation suggesting a possible role for these residues in membrane fusion. It can therefore be concluded from this study that amino acid residues within the hinge domain, the fusion peptide and the stem/anchor of DEN E influence both prM/E heterodimerisation and membrane fusion.