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

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. Membrane fusion implies coordinated movements of protein domains through a series of sequential steps. Structures for the initial and final conformations are available for several fusogens, but folding intermediates remain largely unresolved, and the 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 followed the experimental evolution of CHIKV under the selective pressure of a novel entry inhibitor. Mutations arising from selection mapped to two residues located in the distal domains of the E2 and E1 heterodimer and spikes. Here, we demonstrate that the antiviral mechanism involves inhibition of membrane 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. In addition, molecular dynamics simulations suggest that these two residues modulate the conformational rearrangement of the E1-E2 heterodimer. In this line and supporting a functional link between residues, the double-mutant virus displayed a higher pH threshold for fusion than single-mutant viruses. Finally, mutations resulted in distinct replication and spreading outcomes in mice and infection rates in mosquitoes, underscoring the fine-tuning of envelope proteins' function as a determinant for the establishment of infection. Altogether, our approach captured an otherwise unresolved interdomain interaction.IMPORTANCEChikungunya virus (CHIKV) is a reemergent pathogen that has caused large outbreaks in the last 20 years. There are no available antiviral therapies, and a vaccine has only recently been approved. We describe the mode of action of an inhibitor designed to target CHIKV envelope proteins, blocking entry at the stage of fusion between the virus envelope and host membranes. Fusion is common to the entry of enveloped viruses. Virus envelope proteins drive fusion, undergoing a series of transitions from an initial metastable conformational state to a more stable post-fusion state. Intermediate conformations are transient and have mostly remained inaccessible to structure determination. Here, a selection of viruses that are resistant to antiviral inhibition of fusion uncovered a functional interaction between two residues residing in domains that are apart in both the pre-fusion and post-fusion states. Thus, we provide new insight into the molecular detail of the inner working of virus fusion machinery.