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 The affinity of the two halves of YFP presumably superseded the F-HN interaction.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

journal_name

J Virol

journal_title

Journal of virology

authors

Connolly SA,Leser GP,Jardetzky TS,Lamb RA

doi

10.1128/JVI.01191-09

subject

Has Abstract

pub_date

2009-11-01 00:00:00

pages

10857-68

issue

eissn

0022-538X

issn

1098-5514

pii

JVI.01191-09

journal_volume

pub_type

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Bimolecular complementation of paramyxovirus fusion and hemagglutinin-neuraminidase proteins enhances fusion: implications for the mechanism of fusion triggering.