FUNCTIONAL ANALYSIS OF SIGNAL SEQUENCES OF INFLUENZA VIRUS HEMAGGLUTININ

流感病毒信号肽的氨基酸需求 使用流感血凝素（HA）cDNA-SV 40进行了研究 在非洲绿色猴肾（AGMK）细胞中的表达系统。 当地 进行位点特异性诱变以产生一系列 HA-SV 40的重组体，其在HA-SV 40的区域中含有点突变， 编码信号肽的流感病毒血凝素（HA）基因 序列的 利用这些突变HA-SV 40重组体对AGMK进行了转染 细胞以实现突变血凝素的表达。 功能 通过细胞表面免疫荧光表征这种HA产物 测定、红细胞吸附和糖基化分析表明，大多数 的突变对HA的功能特性没有影响。 然而，在这方面， 一个分离株（突变体28）持续了几个突变，包括一个氨基 在信号切割位点的酸取代是有缺陷的， 细胞表面表达。 NH 2末端的氨基酸序列分析 结果表明，细胞内积累的HA不能 进行信号切割。 此外，缺陷突变HA仅含有 在所述组合物中加入的内切糖苷酶H敏感性碳水化合物组分 内质网 这些发现表明，含有 未切割的疏水信号序列跨微粒体易位 膜，但未能进行到高尔基体，其中内切糖苷酶 H抗性碳水化合物被并入。 点突变，使用 已经尝试了确定的寡核苷酸引物，目的是 分离出一种特异的切割突变体，这将使我们能够确认 突变体28中存在信号切割缺陷确实是导致其 HA易位和细胞表面表达缺陷。

The amino acid requirements of a functional influenza virus signal peptide were investigated using the influenza hemagglutinin (HA) cDNA-SV40 expression system in African green monkey kidney (AGMK) cells. Local site-specific mutagenesis was carried out to generate a series of recombinants of HA-SV40 containing point mutations in the region of the influenza virus hemagglutinin (HA) gene that codes for the signal peptide sequences. These mutant HA-SV40 recombinants were used to transfect AGMK cells in order to achieve expression of mutant hemagglutinins. Functional characterization of such HA products by cell surface immunofluorescence assay, hemadsorption and analysis of glycosylation showed that a majority of the mutations had no effect on functional properties of HA. However, one isolate (mutant 28) that sustained several mutations including an amino acid substitution at the signal cleavage site was defective with regard to cell surface expression. Amino acid sequence analysis of the NH2-terminus of mutant HA showed that the intracellularly accumulated HA failed to undergo signal cleavage. Also, the defective mutant HA contained only endoglycosidase H sensitive carbohydrate components that are added in the endoplasmic reticulum. These findings suggest that HA containing an uncleaved hydrophobic signal sequence translocates across the microsomal membrane but fails to proceed to the Golgi apparatus where endoglycosidase H resistant carbohydrates are incorporated. Point mutagenesis using a defined oligonucleotide primer has been attempted with the intention of isolating a specific cleavage mutant that will allow us to confirm that the signal cleavage defect present in mutant 28 is indeed responsible for its defect in HA translocation and cell surface expression.