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Data from: A fusion protein’s weak link: functional constraints revealed by inhibitory peptide interaction with the parainfluenza fusion protein

Crosby, Maximilian1; Kuhn, Ariel2; Marcink, Tara1; Gellman, Samuel2; Moscona, Anne1

Published Mar 17, 2026 on Dryad. https://doi.org/10.5061/dryad.wh70rxx1d

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Abstract

Human parainfluenza viruses (HPIVs) cause significant respiratory illnesses including croup and pneumonia. HPIV infection begins with fusion of viral and host cell membranes, driven by the coordinated actions of the attachment (HN) and fusion (F) glycoproteins, which together form the fusion/entry complex. We have described fusion inhibitory peptides derived from the heptad repeat (HRC) domain of the F glycoprotein with potent antiviral activity against HPIV3 and other paramyxoviruses. These peptides inhibit fusion by binding to the transiently exposed N-terminal heptad repeat (HRN) segments of the F prehairpin intermediate and preventing the six-helix bundle (6HB) formation that is required for the membrane fusion process. We report viral variants that escape inhibition by an α/β-substituted HRC peptide of HPIV3 through an HN mutation that enhances HN’s activation of F, and a mutation in F’s HRN domain that destabilizes the 6HB. The F HRN domain bearing the alteration shows reduced α-helicity relative to the wildtype HRN and forms an assembly with the HRC domain that is destabilized relative to the wildtype 6HB. This viral variant is not resistant to an HRC-derived α-peptide inhibitor that forms a more stable 6HB relative to the HRC-derived α/β-peptide. The emergence of this variant suggests that improved inhibitor potency against HPIV3 could be achieved by increasing the stability of the α/β-peptide/HRN 6HB. The unique mutation in F that reduces sensitivity to inhibitor also compromises viral fitness in a human airway model and impairs viral infection, presumably as a result of diminished post-fusion state stability.