Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

Abstract

Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin–neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design.

Nitazoxanide inhibits paramyxovirus replication by targeting the Fusion protein folding: role of glycoprotein-specific thiol oxidoreductase ERp57

“…Because of its critical role in cell entry, the fusion-protein represents an attractive target for novel antivirals 34 . Most of the current attempts to block F-protein function are aimed at halting the cascade of conformational changes driving membrane fusion through stabilization of the pre-fusion conformation of virion F-trimers by designing specific peptide inhibitors, or developing selective or cross-reactive neutralizing antibodies 35 – 37 . We now propose a novel approach to target paramyxovirus fusion proteins by drug-directed misfolding of newly synthesized F-protein.…”

Conserved amino acids around the DIII-DI linker region of the Newcastle disease virus fusion protein are critical for protein folding and fusion activity

“…Thus, conservative amino acid mutant introduction may destroy the relative activities of these two fragments, affecting the fusion process F proteins. Given that the DIII-DI connection region appears to be a conserved structure in paramyxovirus F proteins, this region presents an alternative way to investigate structure-based rational vaccine or drug designs (30,31).…”