The central proline of an internal viral fusion peptide serves two important roles

The fusion peptide of the avian sarcoma/leukosis virus (ASLV) envelope prot ein (Env) Is internal, near the N terminus of its transmembrane (TM) subuni t. As for most internal viral fusion peptides, there is a proline near the center of this sequence. Robson-Garnier structure predictions of the ASLV f usion peptide and immediate surrounding sequences indicate a region of orde r (B-sheet), a tight reverse rum containing the proline, and a second regio n of order (alpha-helix). Similar motifs (order, turn or loop, order) are p redicted for other internal fusion peptides, In this study, we made and ana lyzed 12 Env proteins with substitutions for the central proline of the fus ion peptide. Env proteins were expressed in 293T cells and in murine leukem ia virus pseudotyped virions. We found the following. (i) All mutant Envs f orm trimers, but when the bulky hydrophobic residues phenylalanine or leuci ne are substituted for proline, trimerization is weakened. (ii) Surprisingl y, the proline is required for maximal processing of the Env precursor into its surface and TM subunits; the amount of processing correlates linearly with the propensity of the substituted residue to be found in a reverse tur n, (iii) Nonetheless, proteolytically processed forms of all Envs are prefe rentially incorporated into pseudotyped virions. (iv) All Envs bind recepto r with affinity greater than or equal to wild-type affinity: (v) Residues t hat support high infectivity cluster with proline at intermediate hydrophob icity. Infectivity is not supported by mutant Envs in which charged residue s are substituted for proline, nor is it supported by: the trimerization-de fective phenylalanine and leucine mutants, Our findings suggest that the ce ntral proline in the ASLV fusion peptide is important for the formation of the native (metastable) Env structure as well as for membrane interactions that lead to fusion.