Evolution of antiviral resistance captures a transient interdomain functional interaction between chikungunya virus...

Envelope proteins drive virus and host-cell membrane fusion to achieve virus entry. Fusogenic proteins are classified into structural classes that function with remarkable mechanistic similarities. Fusion proceeds through coordinated movements of protein domains in a sequence of orchestrated steps. Structures for the initial and final conformations are available for several fusogens, but folding intermediates have largely remained unresolved and interdependency between regions that drive conformational rearrangements is not well understood. Chikungunya virus (CHIKV) particles display heterodimers of envelope proteins E1 and E2 associated as trimeric spikes that respond to acidic pH to trigger fusion. We have followed experimental evolution of CHIKV under the selective pressure of a novel small-molecule entry inhibitor. Mutations arising from selection mapped to two residues located in distal domains of E2 and E1 heterodimer and spikes. Here, we pinpointed the antiviral mode of action to inhibition of fusion. Phenotypic characterization of recombinant viruses indicated that the selected mutations confer a fitness advantage under antiviral pressure, and that the double-mutant virus overcame antiviral inhibition of fusion while single-mutants were sensitive. Further supporting a functional connection between residues, the double-mutant virus displayed a higher pH-threshold for fusion than single-mutant viruses. Finally, mutations implied distinct outcomes of replication and spreading in mice, and infection rates in mosquitoes underscoring the fine-tuning of envelope protein function as a determinant for establishment of infection. Together with molecular dynamics simulations that indicate a link between these two residues in the modulation of the heterodimer conformational rearrangement, our approach captured an otherwise unresolved interaction.