There is greater sequence divergence between BLV and HTLV-1 than, for example, between SIV and HIV-1 [34, 35] , nonetheless the crystal structures of the BLV and HTLV-1 trimerof-hairpins are similar. The incorporation of ordered water molecules and ions to facilitate trimerisation of the coiled coil and assembly of the trimer-of-hairpins is seen in several viral fusion proteins [20, 29, 30, 36] . The crystal structure of SIV gp41 implicates multiple water molecules in the folding of the trimerof-hairpins [37] . However, the number of water molecules concentrated in one region of the BLV coiled coil is unusual. The interaction of Thr353 with an array of water molecules appears to play a critical role in assembly of the coiled coil, as substitution of T353 eliminates both in vitro trimerisation and in vivo fusogenicity. Notably, for HTLV-1 the threonine residue is replaced by an asparagine, which by virtue of its larger side chain directly hydrogen bonds with the adjacent N-helix. By contrast, Thr353 of BLV makes this contact through water-mediated hydrogen bonds. However, the contribution of polar interactions to coiled coil assembly is maintained at this location in HTLV-1 by interaction of a chloride ion on the symmetry axis of the coiled coil. Moreover, for both viruses three conserved asparagine Fusogenicity of HTLV-1 C-terminal LHR arginine mutants. The fusogenic index was calculated as described (mean 6SD from triplicate assays). (D) A ''hotspot'' for binding of HTLV-1 fusion inhibitors based on the previously published HTLV-1 structure and modelled incorporating the data from this study. Four key residues (Leu413, Arg416, Leu419 and Arg422, shown as yellow sticks) interact with the HTLV-1 coiled coil (grey space-filling model, region interacting with LHR residues shown in red) within a compact binding site. The contact made by Arg422 is critical to the binding and functional activity of HTLV-1 peptide inhibitors. doi:10.1371/journal.ppat.1001268.g005 residues located towards the membrane distal end of the coiled coil interact with a chloride ion. The properties of the N367L substitution and the failure of N367D to rescue envelope function indicate that the observed chloride-mediated interactions are essential for stable assembly of the coiled coil and for TMmediated membrane fusion. Significantly, the asparagine (Asn367) and adjacent arginine (Arg368) residues of BLV are conserved between BLV, HTLV-1 and a number of disparate viral fusion proteins, and the ability of the asparagines to interact with a chloride ion is maintained. Critically, the chloride ion is present within the post-fusion conformation of Ebola GP2 (30), but is conspicuously absent in a pre-fusion structure resolved by Lee et al [38] wherein the conserved asparagines adopt a more open arrangement and the coiled coil is only partially trimerised. We suggest that Asn367, as a direct consequence of conformational changes in SU resultant from receptor binding, and in concert with the equivalent asparagines from neighbouring monomers, mediates electrostatic interactions with a chloride ion, which brings the N-helices together, thus driving the trimerisation of the coiled coil to yield the pre-hairpin intermediate in an event pivotal to successful fusion. In addition, our results indicate that trimerisation of the N-terminus of TM is not a requirement for Env maturation and surface display of trimeric pre-fusogenic envelope, offering a novel insight into the conformation of retroviral Env spikes prior to activation. Taken together, the data suggest that while the ''knobs-into-holes'' interactions of aliphatic residues at the centre of the coiled-coil regions of class 1 viral fusion proteins are important for trimerisation of the fusion-active coiled coil the polar layers among these hydrophobic interactions are critical. This information should be considered when designing trimeric immunogens based on the coiled coil of TM. Moreover, the structure in and around the chloride-interacting region has been conserved in diverse viral fusion proteins and may be susceptible to coiled coil destabilising drugs.