Synthetic fusion peptides of tick-borne encephalitis virus as models for membrane fusion.

The fusion peptide of TBEV is a short segment of the envelope protein that mediates viral and host cell membrane fusion at acidic pH. Previous studies on the E protein have shown that mutations at L107 have an effect on fusogenic activity. Structural studies have also suggested that during the fusion process the E protein rearranges to form a trimer. In the present study, a number of short peptides were synthesized, and their structure/activity was examined: (1) monomers consisting of residues 93-113 of the wild-type E protein with Leu at position 107 (WT) and two mutants, namely, L107F and L107T; (2) a monomer consisting of residues 93-113 of the E protein with a C105A mutation (TFPmn); (3) a trimer consisting of three monomers described in (2), linked at the C-terminus via 1 Lys (TFPtr); (4) a monomer consisting of residues 93-113 of the E protein plus six additional Lys at the C-terminus; and (5) a trimer consisting of three monomers described in (3), linked via the side chain of the sixth lysine. The secondary structure content of all peptides was investigated using circular dichroism (CD). Approximately seven of the residues were in beta-strand conformation, in the presence of POPC/POPE/cholesterol. The structures did not depend on pH significantly. The fusogenicity of the peptides was measured by FRET and photon correlation spectroscopy. The data suggest that TFPtr is the most fusogenic at acidic pH and that the mutation from L107 to T reduces activity. Molecular dynamics simulations of WT, L107T, and L107F suggest that this reduction in activity may be related to the fact that the mutations disrupt trimer stability. Finally, tryptophan fluorescence experiments were used to localize the peptides in the membrane. It was found that WT, L107F, TFPmn, and TFPtr could penetrate better into the acyl chain region of the lipids than the other peptides tested. The implications of these results on the fusion mechanism of TBEV E protein will be presented.

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