Mutational evidence for an internal fusion peptide in flavivirus envelope protein E

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) viru s promotes cell entry by inducing fusion of the viral membrane with an intr acellular membrane after uptake by endocytosis, This protein differs from o ther H ell-studied viral and cellular fusion proteins because of its distin ct molecular architecture and apparent lack of involvement of coiled coils in the low-pH-induced structural transitions that lead to fusion. A highly conserved loop (the ed loop), which resides at the distal tip of each subun it and is mostly buried in the subunit interface of the native E homodimer at neutral pH, has been hypothesized to function as an internal fusion pept ide at low pH, but this has not pet been shown experimentally. It was predi cted by examination of the X-ray crystal structure of the TEE virus E prote in (F, A. Rey et al,, Nature 375:291-298, 1995) that mutations at a specifi c residue within this loop (Leu 107) would not cause the native structure t o be disrupted. We therefore introduced amino acid substitutions at this po sition and, using recombinant subviral particles, investigated the effects of these changes on fusion and related properties. Replacement of Leu with hydrophilic amino acids strongly impaired (Thr) or abolished (Asp) fusion a ctivity, whereas a Phe mutant still retained a significant degree of fusion activity. Liposome coflotation experiments showed that the fusion-negative Asp mutant did not form a stable interaction with membranes at low pH, alt hough it was still capable of undergoing the structural rearrangements requ ired for fusion, These data support the hypothesis that the ed loop may be directly involved in interactions with target membranes during fusion.