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Research for determinants of fusion activity of viral glycoprotein

病毒糖蛋白融合活性决定因素的研究

基本信息

批准号：
09670321
负责人：
OHUCHI Masanobu
金额：
$ 2.05万
依托单位：
Kawasaki Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Hemagglutinin (HA) of influenza virus mediates the entry of viral genome into the host cell though the membrane fusion process. It was not clear whether the cytoplasmic domain of HA should involve this fusion process or not. We have found that elongation the cytoplasmic domain causes drastic decrease in the fusion activity. This effect depended on the number of amino acid added but was independent of the species of amino acid. Addition of 5 amino acids abolished the cell fusion activity entirely. On the other hand, the cell fusion activity remained even after deletion of the cytoplasmic domain, although the activity was considerably reduced.To investigate what factor(s) is for the fusion activity, we compared the expression level, oligosaccharide processing, cleavability, acylation, cell surface distribution, and lateral movement of HA in the cellular membrane between the wild type and cytoplasmic domainmodified Has. The lateral movement was assayed with Fab of fluorescein-labeled anti-HA antibody in Video-FRAP (fluorescence recovery after photobleaching). No difference was detected in any factors between both Has. Other parameter must be searched.We also found that receptor binding affinity of HA is a determinant for the fusion activity, that is, deletion of oligosaccharides near the receptor binding site enhanced the binding affinity and reduced the cell fusion activity proportionally, and the mutation which reduced the binding affinity restored the fusion activity. Thus, the reciprocal relationship was observed between both activities. The HA with high receptor binding affinity formed fusion pores, through which small molecules such as calcein could pass. However, hemoglobin hardly passed through the fusion pore. Enlargement of the fusion pore may be interfered with. Since the viral nucleocapside is larger than hemoglobin, this problem is critical for the viral infection. Accordingly, the control of receptor binding affinity is a vital problem for the virus.

流感病毒的血凝素（HA）介导病毒基因组通过膜融合过程进入宿主细胞。目前尚不清楚HA的胞质结构域是否应参与该融合过程。我们发现胞质结构域的延长导致融合活性的急剧下降。这种效应依赖于添加的氨基酸数量，但与氨基酸种类无关。添加5个氨基酸完全消除了细胞融合活性。另一方面，细胞融合活性即使在胞质结构域缺失后仍然保持，尽管活性显著降低。为了研究融合活性的因素，我们比较了野生型和胞质结构域修饰的Has之间的表达水平、寡糖加工、切割能力、酰化、细胞表面分布和HA在细胞膜中的横向运动。在Video-FRAP（光漂白后的荧光恢复）中用荧光素标记的抗HA抗体的Fab测定侧向运动。两种Has之间的任何因子均未检测到差异。我们还发现HA的受体结合亲和力是融合活性的决定性因素，即受体结合位点附近的寡糖的缺失增加了结合亲和力，降低了细胞融合活性，而降低结合亲和力的突变恢复了融合活性。因此，在这两种活动之间观察到了相互关系。具有高受体结合亲和力的HA形成融合孔，小分子如钙黄绿素可以通过该融合孔。然而，血红蛋白几乎不通过融合孔。融合孔的包裹可能受到干扰。由于病毒核衣壳比血红蛋白大，这个问题对于病毒感染来说是关键的。因此，受体结合亲和力的控制是病毒的重要问题。