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Journal of Virology, November 2009, p. 10857-10868, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01191-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Bimolecular Complementation of Paramyxovirus Fusion and Hemagglutinin-Neuraminidase Proteins Enhances Fusion: Implications for the Mechanism of Fusion Triggering ^,

Sarah A. Connolly,^1,2 George P. Leser,² Theodore S. Jardetzky,³ and Robert A. Lamb^1,2*

Howard Hughes Medical Institute,¹ Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500,² Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305³

Received 10 June 2009/ Accepted 14 August 2009

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 The affinity of the two halves of YFP presumably superseded the F-HN interaction.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.

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* Corresponding author. Mailing address: BMBCB, Northwestern University, 2205 Tech Drive, Hogan 2-100, Evanston, IL 60208-3500. Phone: (847) 491-5433. Fax: (847) 491-2467. E-mail: ralamb{at}northwestern.edu

Published ahead of print on 26 August 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, November 2009, p. 10857-10868, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01191-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.