Synthetic Fusion Peptides of Tick-Borne Encephalitis Virus as Models for Membrane Fusion

Abstract

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.

The Structural Dynamics of the Flavivirus Fusion Peptide–Membrane Interaction

“…It has recently been proposed that the flavivirus histidines function as molecular switches, and they therefore control the fusion process by acting as pH sensors in the Flavivirus membrane fusion process [27]. However, a recent work publication showed that His104 protonation does not inhibit the ability of TBEV FP to bind to membranes [28]. Indeed, the data presented here, as well as in other studies, suggest that this is not the case for His104 in TBEV FP.…”

HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation, membrane binding, and electrostatics, and implications for HIV-host cell fusion

“…Fusion of some enveloped viruses other than HIV (such as influenza) occurs in the acidified endosome at pH between 5 and 6 (Pan et al 2010; White et al 2008). This has motivated earlier studies that examined the pH dependence of vesicle fusion induced by influenza fusion protein constructs including the full ectodomain with fusion peptide (Curtis-Fisk et al 2007; Epand et al 1999).…”

The Interaction of Alphavirus E1 Protein with Exogenous Domain III Defines Stages in Virus-Membrane Fusion

“…Indeed, in the absence of the linker, the core trimer is unstable. Studies of a synthetic flavivirus fusion peptide suggested that residues in the fusion loop can affect trimer stability (35). Although G91D E1 inserts into target membranes and forms the extended trimer, intratrimer interactions of the fusion loops may be disrupted.…”