Chikungunya virus E1 fusion loop-membrane interaction: Conserved residues and shallow insertion

Fusion of viral and cellular membranes, essential for entry of enveloped viruses, is mediated by viral fusion proteins (FPs). These proteins insert hydrophobic elements into target membranes to destabilize bilayers and drive fusion. For class I FPs, sequence and structural requirements of the fusion peptide are well characterized. In contrast, class II FPs, such as the alphavirus E1 glycoprotein, employ compact fusion loops, whose conserved sequence and structural features remain less understood. Here, we examined the chikungunya virus (CHIKV) E1 fusion loop using molecular dynamics (MD) simulations, mutational analysis, depth-dependent fluorescence quenching, liposome fusion assays, and Langmuir monolayer experiments. Comparative analyses of alphavirus and flavivirus fusion loops identified a conserved glycine at the N terminus and a tryptophan at the loop tip. Atomistic and coarse-grained MD simulations revealed that the loop inserts shallow into the lipid headgroup region, adopting a tilted interfacial orientation stabilized by hydrogen bonding. Depth probing by tryptophan fluorescence quenching in the parallax method confirmed placement of Trp89 at the headgroup-acyl chain boundary. Functional assays showed that substitution of the conserved glycine (G83A) abolished both membrane insertion and fusion, whereas W89A retained near-wild-type activity. Langmuir monolayer measurements further demonstrated that the E1 protein decreased compressibility and altered lipid packing, whereas with the W89A mutant, the effect is reduced, implying that both G83 and W89 in the CHIKV E1 fusion loop contribute to membrane destabilization. These findings establish that class II fusion loops employ shallow, interfacial anchoring mediated by conserved residues. This mode of binding perturbs lipid organization sufficiently to promote fusion, defining a conserved mechanistic principle that distinguishes class II fusion loops from the deeper-penetrating peptides of class I proteins.