Engineered Intermonomeric Disulfide Bonds in the Globular Domain of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein: Implications for the Mechanism of Fusion Promotion
Engineered Intermonomeric Disulfide Bonds in the Globular Domain of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein: Implications for the Mechanism of Fusion Promotion
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DOI：
10.1128/jvi.00581-08
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发表时间：
2008-11-01
影响因子：
5.4
通讯作者：
Iorio, Ronald M.
中科院分区：
文献类型：
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作者：
Mahon, Paul J.;Mirza, Anne M.;Iorio, Ronald M.
新城疫病毒（NDV）促进膜融合需要病毒血凝素-神经氨酸酶（HN）和融合(F)蛋白之间的相互作用，尽管这种相互作用调节融合的机制尚不清楚。NDV HN蛋白是由一对二聚体组成的四聚体。基于NDV HN球状畴的x射线晶体学研究（S. Crennell et al., Nat. Struct.）。Biol. 7:1068-1074, 2000)，有人提出蛋白质经历了一个显著的构象变化，从一个具有最小单体间接触的初始结构到具有更广泛的二聚体界面的结构。这种构象变化被预测为融合促进不可或缺的一部分，其最小的界面形式需要维持F在其预融合状态直到HN结合受体。然而，没有证据表明这种构象变化存在于任何其他副粘病毒附着蛋白。为了测试NDV模型，我们通过引入半胱氨酸取代残基T216和D230，在非二硫化物连接的NDV HN蛋白的球状结构域的二聚体界面上设计了一对单体间二硫键。二硫化物连接二聚体在细胞内和没有受体结合的情况下形成，并在细胞表面有效表达。二硫键阻止了蛋白质最小界面形式的形成，但增强了37°C下受体结合活性和融合促进。这些结果证实，HN的最小界面形式和提出的蛋白质的剧烈构象变化都不是融合所必需的。
The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S. Crennell et al., Nat. Struct. Biol. 7:1068-1074, 2000), it was proposed that the protein undergoes a significant conformational change from an initial structure having minimal intermonomeric contacts to a structure with a much more extensive dimer interface. This conformational change was predicted to be integral to fusion promotion with the minimal interface form required to maintain F in its prefusion state until HN binds receptors. However, no evidence for such a conformational change exists for any other paramyxovirus attachment protein. To test the NDV model, we have engineered a pair of intermonomeric disulfide bonds across the dimer interface in the globular domain of an otherwise non-disulfide-linked NDV HN protein by the introduction of cysteine substitutions for residues T216 and D230. The disulfide-linked dimer is formed both intracellularly and in the absence of receptor binding and is efficiently expressed at the cell surface. The disulfide bonds preclude formation of the minimal interface form of the protein and yet enhance both receptor-binding activity at 37°C and fusion promotion. These results confirm that neither the minimal interface form of HN nor the proposed drastic conformational change in the protein is required for fusion.