Research for determinants of fusion activity of viral glycoprotein

流感病毒的血凝素(HA)通过膜融合过程介导病毒基因组进入宿主细胞。目前尚不清楚HA的胞浆结构域是否参与了这一融合过程。我们发现，胞质结构域的延长会导致融合活性的急剧下降。这一效应依赖于添加的氨基酸的数量，但与氨基酸的种类无关。加入5个氨基酸后，细胞融合活性完全消失。另一方面，尽管细胞质结构域的活性显著降低，但细胞融合活性仍保持不变。为了研究影响融合活性的因素(S)，我们比较了野生型和修饰的细胞质结构域中HA的表达水平、寡糖加工、裂解、酰化、细胞表面分布和细胞膜的横向移动。用荧光素标记的抗HA抗体Fab在Video-FRAP(荧光漂白后恢复)中检测侧向运动。两种HAS之间在任何因素上均未检测到差异。我们还发现HA的受体结合亲和力是决定融合活性的一个因素，即缺失受体结合部位附近的寡糖会按比例提高结合亲和力和降低细胞融合活性，而降低结合亲和力的突变则恢复了融合活性。因此，观察到了两项活动之间的互惠关系。具有高受体结合亲和力的HA形成融合孔，钙黄绿素等小分子可以通过这些孔。然而，血红蛋白几乎没有穿过融合孔。可能会干扰融合孔的扩大。由于病毒的核衣壳蛋白比血红蛋白大，这一问题对病毒感染至关重要。因此，受体结合亲和力的控制对病毒来说是一个至关重要的问题。

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.