Contrasting effects of two arginine residues on the stability of the trimer-of-hairpins Based on homology modelling, we previously suggested that Arg403 of the LHR projects out from one side of the a-helix and is repelled electrostatically by N-helix residue Arg345 [17] . This prediction is substantiated by the crystal structure presented here (Fig. 1C) . Moreover, the R403A substitution yields an envelope that produces extensive syncytia and is significantly more fusogenic than wild-type envelope (Fig. 2C and Fig. 4C) . Thus, the substitutions R403A and R413A have very different effects on the fusogenicity of BLV envelope, increasing activity by over two-fold and almost completely abolishing activity respectively (Fig. 2C  and 4C ). Using a modified thermal aggregation assay [33] , we assessed the relative effects of these two substitutions on the thermostability of the BLV trimer-of-hairpins. Control experiments revealed that after a five minute incubation at 40uC, on average 53% of the wild-type MBP-BLV-hairpin protein was present in high-order aggregates (Fig. 4A, B) . However, when the same heat treatment was applied to the protein bearing the R403A mutation, we found that only 37% of protein aggregated. The difference to wild-type hairpin was significant (P#0.005, t-test) (Fig. 4A, B) . By contrast, the R413A substitution resulted in aggregation of over 70% of total protein, again a significant difference to wild type (P#0.005, t-test) ( Fig. 4A and B) . Notably, even without heat treatment over 50% of the R413A protein was aggregated (Fig. 4A) . Furthermore, these relative differences in the propensity of the recombinant proteins to aggregate were maintained following heat treatment at 50 and 60uC (Fig. 4B) . Hence, the contrasting effects of the arginine substitutions on envelope fusogenicity are directly due to changes in stability of the post-fusion trimer-of-hairpins structure.