A spherical density feature was observed on the central axis of the molecule; comparison of refined temperature factors with those of the surrounding protein atoms suggests an entity more electron-dense than water. Based on the chemical environment, the shape of the density and B factor comparisons we have modeled this feature as a chloride ion. This chloride ion is situated towards the chain reversal end of the coiled coil between a group of three asparagines, one from each monomer of TM (Asn367). These asparagine residues establish electrostatic interactions with the chloride ion by creating a slightly positively charged microenvironment between the N-helices. An alignment of envelope sequences from diverse viral groups (Fig. 3C ) reveals that this asparagine is conserved. Moreover, comparison of the crystal structures for the fusion protein ectodomains of HTLV-1 [20] , MoMLV [29] , Ebola [24, 30] , and Syncytin-1 [31] , a protein derived from a human endogenous retrovirus that plays a critical role in the implantation of the trophoblast into the wall of the uterus [32] , show that the ability to coordinate chloride is retained (Fig. 3D) . To test the importance of the chloride interacting asparagines, we introduced amino acid substitutions of similar size and geometry. Introducing a leucine residue at position 367 of BLV TM renders envelope entirely non-fusogenic. The N367L substitution does not completely prevent trimerisation of the BLV coiled coil in vitro but it significantly destabilises the trimer and a large broad peak corresponding to higher order oligomers and aggregated material is observed by gel filtration chromatography (Fig. 3E ). Interestingly, a substitution whereby Asn367 is replaced by an aspartic acid residue produces a decrease in fusogenicity of almost 90% (Fig. 2C ). The N367D substitution replaces an amide with a carboxylate group in the centre of the coiled coil, which inverts the surface charge of the binding pocket, and therefore would not support interaction with a chloride. In contrast to N367L, the N367D mutation should maintain the hydrogen bonding in and around the chain-reversal region mediated by the carbonyl group that is common to both asparagine and aspartic acid, and therefore the disruption of fusion should be due primarily to the absence of the chloride ion, albeit we cannot exclude the possibility that the close proximity of negatively charged aspartate side chains impairs trimerisation. However, our biological assays demonstrate that the N367D substitution, unlike N367L, retains some membrane fusion activity. In vitro, rather than producing a range of higher order species, the introduction of the N367D mutation into pMBP-BLVhairpin produces a recombinant protein with a major peak at an elution volume consistent with the molecular weight of a tetramer rather than a trimer (Fig. 3E ). Tetramerisation is unlikely to be the reason for the compromised fusogenic activity of the N367D-envelope; nonetheless, the results demonstrate the importance of the chloride-interacting asparagines to the stability of the trimeric coiled coil.