THE ATTACHMENT FUNCTION OF THE NEWCASTLE-DISEASE VIRUS HEMAGGLUTININ-NEURAMINIDASE PROTEIN CAN BE SEPARATED FROM FUSION PROMOTION BY MUTATION
THE ATTACHMENT FUNCTION OF THE NEWCASTLE-DISEASE VIRUS HEMAGGLUTININ-NEURAMINIDASE PROTEIN CAN BE SEPARATED FROM FUSION PROMOTION BY MUTATION
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DOI：
10.1006/viro.1993.1180
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发表时间：
1993-04-01
期刊：
影响因子：
3.7
通讯作者：
MORRISON, TG
中科院分区：
文献类型：
--
作者：
SERGEL, T;MCGINNES, LW;MORRISON, TG
利用Cos-7细胞和以SV40为基础的载体，建立了新城疫病毒血凝素神经氨酸酶(HN)与融合糖蛋白融合的定量检测方法。这些检测数据显示，HN蛋白和融合蛋白DNA共转染后，合胞体的大小逐渐增加，而单独转染HN蛋白或F蛋白DNA后，合胞体的大小没有改变。利用免疫荧光技术，评估糖蛋白引导的合胞体形成的效率。事实上，所有单独表达融合蛋白或HN蛋白的细胞都以单核细胞的形式存在。而共表达这两种基因的细胞均存在于4~50个核的合胞体中。利用这些方法对HN蛋白的两个缺失突变体的融合促进活性进行了定量检测。一个突变缺失了4到26个氨基酸，有效地去除了蛋白质的细胞质区域。这种突变蛋白是在细胞表面发现的，尽管数量非常少。突变蛋白可以结合红细胞并促进融合，尽管形成的合胞体比野生型蛋白促进的合胞体小(4~9个核)。第二个突变缺失了9个氨基酸(91-99)，位于胞外区跨膜区的37个氨基酸。该突变体在细胞表面检测到的水平是野生型的33%，并保持了接近正常的结合红细胞的能力。然而，该突变完全消除了HN蛋白的融合促进活性。该突变有效地将HN蛋白的附着功能从融合促进中分离出来。
Using Cos-7 cells and an SV40-based vector, quantitative assays for fusion directed by the Newcastle disease virus hemagglutinin-neuraminidase (HN) and fusion glycoproteins were developed. Data from these assays showed that after cotransfection of the HN protein and the fusion protein DNAs, there was a progressive increase in syncytia size over background levels while transfection with HN protein or F protein DNA alone resulted in no changes over background. Using immunofluorescence, the efficiency of syncytia formation directed by the glycoproteins was assessed. Virtually all cells expressing the fusion protein alone or the HN protein alone existed as single nucleated cells. However, all cells coexpressing the two genes existed in syncytia with 4 to 50 nuclei. Using these assays, the fusion promotion activity of two deletion mutants of the HN protein was quantitated. One mutation deleted amino acids 4 through 26, effectively removing the cytoplasmic domain of the protein. This mutant protein was found at the cell surface, although in very reduced amounts. The mutant protein could bind red blood cells and could promote fusion, although the syncytia formed were smaller (4 to 9 nuclei) than those promoted by the wild-type protein. A second mutation deleted nine amino acids (91-99) located 37 amino acids from the transmembrane region in the ectodomain. This mutant was detected at the cell surface at 33% the level of wild type and it retained near normal ability to bind red blood cells. However, this mutation completely eliminated the fusion promotion activity of the HN protein. This mutation effectively separates the attachment function of the HN protein from fusion promotion.