Conformational dynamics of henipavirus fusion proteins

Henipaviruses are a genus of emerging pathogens which includes the deadly Nipah and Hendra viruses. Within recent years, the number of viral species has proliferated and expanded into new animal reservoirs, underscoring the risk for zoonotic transmission to humans and therefore the need for further research into the antigenicity and conformational dynamics of henipavirus glycoproteins. Like with many enveloped viruses, henipaviruses use a fusion protein to facilitate entry into host cells, but distinct from many other well-studied viruses, such as coronaviruses or influenzaviruses, the fusion protein has no receptor binding capability, instead remaining in a metastable pre-fusion state before receiving a “triggering” signal from a separate attachment protein. The structural basis for this triggering signal is not yet understood. Recent structural studies with Langya virus, a novel member of the genus, have discovered that the fusion peptide domain of the fusion protein is held in a “spring-loaded” state while in the pre-fusion conformation, offering a starting point to begin exploring metastability. By combining electron microscopy, differential scanning fluorimetry, and binding assays that use antigenicity to probe conformation, we assess the intrinsic stability of henipavirus fusion proteins, and the effect of mutations on the pre-to-post fusion conformation conversion. Specifically, we have developed a protocol for using heat incubation to force conversion to the post-fusion state, clarified how the intrinsic fluorescence during heat-ramp experiments can reveal the conformational state of unknown species or constructs, and used structure-based design to create henipavirus mutants that stabilize either the pre- or the post-fusion conformation. These findings add to our understanding of the source of metastability in henipavirus fusion proteins and will enable the development of pre-fusion stabilizing mutations to aid with vaccine development.