title: Bimolecular complementation of paramyxovirus fusion and hemagglutinin-neuraminidase proteins enhances fusion: implications for the mechanism of fusion triggering.
abstract: For paramyxoviruses, entry requires a receptor-binding protein (hemagglutinin-neuraminidase [HN], H, or G) and a fusion protein (F). Like other class I viral fusion proteins, F is expressed as a prefusion metastable protein that undergoes a refolding event to induce fusion. HN binding to its receptor triggers F refolding by an unknown mechanism. HN may serve as a clamp that stabilizes F in its prefusion state until HN binds the target cell (the "clamp model"). Alternatively, HN itself may undergo a conformational change after receptor binding that destabilizes F and causes F to trigger (the "provocateur model"). To examine F-HN interactions by bimolecular fluorescence complementation (BiFC), the cytoplasmic tails of parainfluenza virus 5 (PIV5) F and HN were fused to complementary fragments of yellow fluorescent protein (YFP). Coexpression of the BiFC constructs resulted in fluorescence; however, coexpression with unrelated BiFC constructs also produced fluorescence. The affinity of the two halves of YFP presumably superseded the F-HN interaction. Unexpectedly, coexpression of the BiFC F and HN constructs greatly enhanced fusion in multiple cell types. We hypothesize that the increase in fusion occurs because the BiFC tags bring F and HN together more frequently than occurs in a wild-type (wt) scenario. This implies that normally much of wt F is not associated with wt HN, in conflict with the clamp model for activation. Correspondingly, we show that wt PIV5 fusion occurs in an HN concentration-dependent manner. Also inconsistent with the clamp model are the findings that BiFC F does not adopt a postfusion conformation when expressed in the absence of HN and that HN coexpression does not provide resistance to the heat-induced triggering of F. In support of a provocateur model of F activation, we demonstrate by analysis of the morphology of soluble F trimers that the hyperfusogenic mutation S443P has a destabilizing effect on F.
authors.affiliation: Howard Hughes Medical Institute.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Connolly
authors.forename: George P
authors.initials: GP
authors.lastname: Leser
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Jardetzky
authors.forename: Robert A
authors.initials: RA
authors.lastname: Lamb
mesh_terms: D000818:Animals; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D015707:HN Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D009439:Neuraminidase / Q000378:metabolism; D018098:Paramyxovirinae / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D053585:Virus Attachment; D053586:Virus Internalization
chemical_list: D015707:HN Protein; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D009439:Neuraminidase