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

被引:80
作者
Connolly, Sarah A. [2 ]
Leser, George P. [2 ]
Jardetzky, Theodore S. [3 ]
Lamb, Robert A. [1 ,2 ]
机构
[1] Northwestern Univ, BMBCB, Howard Hughes Med Inst, Evanston, IL 60208 USA
[2] Northwestern Univ, Dept Biochem Mol Biol & Cell Biol, Evanston, IL 60208 USA
[3] Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA
基金
美国国家卫生研究院;
关键词
NEWCASTLE-DISEASE VIRUS; VIRAL MEMBRANE-FUSION; F-SPECIFIC DOMAIN; CELL-FUSION; MEASLES-VIRUS; HN PROTEIN; ATTACHMENT GLYCOPROTEIN; ENVELOPE GLYCOPROTEINS; CRYSTAL-STRUCTURE; RECEPTOR-BINDING;
D O I
10.1128/JVI.01191-09
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 The affinity of the two halves of YFP presumably superseded the F-HN interaction.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.
引用
收藏
页码:10857 / 10868
页数:12
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