Mutational evidence for an internal fusion peptide in flavivirus envelope protein E.
The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus promotes cell entry by inducing fusion of the viral membrane with an intracellular membrane after uptake by endocytosis. This protein differs from other well-studied viral and cellular fusion proteins because of its distinct 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 cd loop), which resides at the distal tip of each subunit 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 peptide at low pH, but this has not yet been shown experimentally. It was predicted by examination of the X-ray crystal structure of the TBE virus E protein (F. A. Rey et al., Nature 375:291-298, 1995) that mutations at a specific residue within this loop (Leu 107) would not cause the native structure to be disrupted. We therefore introduced amino acid substitutions at this position 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 activity, 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, although it was still capable of undergoing the structural rearrangements required for fusion. These data support the hypothesis that the cd loop may be directly involved in interactions with target membranes during fusion.
Allison
S L
SL
Institute of Virology, University of Vienna, A-1095 Vienna, Austria. steve.allison@univie.ac.au
Schalich
J
J
Stiasny
K
K
Mandl
C W
CW
Heinz
F X
FX
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004669:Encephalitis Viruses, Tick-Borne / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000235:genetics; D008051:Lipid Bilayers / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020836:Protein Structure, Quaternary; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D008051:Lipid Bilayers; D010455:Peptides; D014759:Viral Envelope Proteins; C065696:glycoprotein E, Flavivirus; D007930:Leucine