A Fusion Protein Weak Link Disrupting the Post-Fusion Conformation of the Parainfluenza Virus Fusion Protein

Human parainfluenza viruses (HPIVs) cause respiratory illnesses including croup, bronchiolitis, and pneumonia. Infection begins with the 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. HPIV3 HN binds to a host cell surface receptor, triggering the activation of the F protein. The homotrimeric F protein, initially anchored to the viral envelope in a metastable prefusion state by its transmembrane segments and adjacent C-terminal heptad repeat (HRC) domains, then undergoes a structural transition. The N-terminal heptad repeat (HRN) domains of F extend, inserting the fusion peptide segments into the host cell membrane and forming a transient prehairpin intermediate, followed by refolding of the F protein resulting in the assembly of a stable six-helix bundle (6HB). This 6HB assembly step is tightly coupled to the membrane fusion process. Peptides corresponding to the HRC domain can inhibit fusion by binding to the transiently exposed HRN segments of the prehairpin intermediate and preventing 6HB formation. We have described fusion inhibitory peptides derived from the HPIV3 F HRC domain with potent antiviral activity against HPIV3 and other paramyxoviruses, and we have enhanced their antiviral activity and resistance to protease degradation by lipid conjugation and beta-amino acid incorporation. Resistant variant viruses that escape the inhibitory effect of a prototype alpha/beta-substituted HRC peptide bear an alteration in HN that enhances HN activation of F conformational transition, and an alteration in F HRN domain that destabilizes the 6HB. The F HRN domain bearing the alteration shows reduced alpha-helicity as well as reduced affinity for the HRC peptide. The emergence of this variant suggests that improved potency vs. HPIV-3 could be achieved by increasing the stability of the alpha/beta-peptide/HRN 6HB. The unique mutation in F that disrupts the stability of the 6HB highlights the critical nature of the proper formation of the 6HB in the fusion process. The destabilized F reduces viral fitness in a human airway model, impairing viral infection.