Research for determinants of fusion activity of viral glycoprotein

流感病毒血凝素（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的细胞质结构域是否参与了这种融合过程。我们发现细胞质结构域的伸长导致融合活性急剧下降。这种效应与添加的氨基酸数量有关，但与氨基酸种类无关。添加5种氨基酸完全破坏了细胞融合活性。另一方面，即使在细胞质结构域缺失后，细胞融合活性仍然存在，尽管活性大大降低。为了研究影响融合活性的因素，我们比较了野生型和细胞质域修饰型HA的表达水平、低聚糖加工、可切割性、酰化、细胞表面分布和细胞膜内HA的横向运动。荧光素标记的抗ha抗体Fab在Video-FRAP（光漂白后荧光恢复）中检测横向运动。两组间的任何因素均未发现差异。其他参数需要搜索。我们还发现，HA的受体结合亲和力是融合活性的一个决定因素，即在受体结合位点附近缺失寡糖，会成比例地增强结合亲和力，降低细胞融合活性，而降低结合亲和力的突变会恢复融合活性。因此，观察到两种活动之间的相互关系。具有高受体结合亲和力的HA形成融合孔，钙黄蛋白等小分子可以通过。然而，血红蛋白很难通过融合孔。熔合孔的扩大可能受到干扰。由于病毒核衣壳比血红蛋白大，这个问题对病毒感染至关重要。因此，对病毒的受体结合亲和力的控制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.