文章基本信息

标题：Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting
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作者：Taylor A. Poor ; Lisa M. Jones ; Amika Sood 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：25
页码：E2596-E2605
DOI：10.1073/pnas.1408983111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe activities of the fusion proteins that mediate virus-cell fusion are an absolute requirement for virus entry and infectivity of enveloped viruses such as HIV, influenza virus, measles virus, and respiratory syncytia virus, among others. Viral fusion proteins are translated initially in a metastable prefusion state and, upon triggering, undergo an extensive and irreversible refolding process. Membrane fusion is coupled to the energy released by the fusion proteins adopting a stable, low-energy postfusion state. Here we use oxidative footprinting of the parainfluenza virus 5 fusion protein to reveal new details of this critical event in the viral lifecycle. A greater understanding of the dynamic nature of these metastable proteins may reveal novel opportunities for the development of targeted therapeutics. To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.
关键词：protein refolding ; viral fusion protein ; mass spectroscopy