The membrane-proximal region of murine leukemia virus envelope (Env) is a critical modulator of its functionality. We have previously shown that the insertion of one amino acid (+1 leucine) within the membrane-spanning domain (MSD) abolished protein functionality in infectivity assays. However, functionality could be restored to this+1 leucine mutant by either inserting two additional amino acids (+3 leucine) or by deleting the cytoplasmic tail domain (CTD) in the +1 leucine background. We inferred that the ectodomain and CTD have protein interfaces that have to be in alignment for Env to be functional. Here, we made single residue deletions to the Env mutant with the+1 leucine insertion to restore the interface alignment (gain of functionality) and therefore define the boundaries of the two interfaces. We identified the glycine-proline pairs near the N terminus (positions 147 and 148) and the C terminus (positions 159 and 160) of the MSD as being the boundaries of the two interfaces. Deletions between these pairs restored function, but deletions outside of them did not. In addition, the vast majority of the single residue deletions regained function if the CTD was deleted. The exceptions were four hydroxyl-containing amino acid residues (T139, T140, S143, and T144) that reside in the ectodomain interface and the proline at position 148, which were all indispensable for functionality. We hypothesize that the hydroxyl-containing residues at positions T139 and S143 could be a driving force for stabilizing the ectodomain interface through formation of a hydrogen-bonding network.