A panel of mutants in BLV envelope are processed and expressed identically in vivo Guided by the crystal structure, a panel of mutants in BLV envelope were designed to perturb key features of the trimer-ofhairpins structure. A common difficulty with mutagenesis of viral envelopes, particularly the TM, is that at particular positions even conservative substitutions can dramatically impair the proteolytic processing and cell surface expression of envelope [27, 28] . Therefore, we compared each of the mutants with the parental ''wild-type'' envelope and confirmed appropriate expression, processing and cell surface presentation of the TM mutants. No significant differences in either the expression level of gp51 or the cleavage of the precursor protein, gp72, were observed ( Fig. 2A) . Moreover, flow cytometry analysis confirmed that in each case the mutant envelope was displayed on the cell surface at levels equivalent to wild type (Fig. 2B) . Subsequently, the envelopeexpressing cells were used as effector cells in syncytium formation assays, and the efficiency of each mutant envelope relative to wildtype in catalysing membrane fusion was calculated as the relative fusogenic index. The effect of each mutation on fusogenicity (Fig. 2C) , and the mutant phenotypes are interpreted with reference to the crystal structure below. Solvent molecules are critical for stable assembly of the core coiled-coil The trimerisation of the N-helices appears to be facilitated by presentation of aliphatic residues to the interacting faces of the TM monomers, thereby forming a hydrophobic core down the axis of the central coiled coil. Notably, in the BLV trimer-of-hairpins there are two positions along this interface that harbour polar residues, Thr353 and Asn367 (Fig. 3A, B ). Thr353 is located approximately half way up the N-helix, and it is oriented such that the methyl group of the side chain points toward the centre of the coiled coil and the hydroxyl group faces toward the neighbouring N-helix. In this position, Thr353 participates in a complex network of hydrogen bonding via several buried ordered water molecules. In particular, Thr353 makes a contact through one water molecule with the main-chain of Glu397 and through a separate water molecule to the side-chain of His354, one of the residues which forms the wall of the groove into which the LHR