Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal hydrophobic region
Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal hydrophobic region
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流感病毒神经氨酸酶的糖基化和表面表达需要 N 端疏水区
DOI：
10.1128/mcb.4.1.8-16.1984
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发表时间：
1984
影响因子：
5.3
通讯作者：
C. Lai
中科院分区：
文献类型：
--
作者：
L. Markoff;B. Lin;M. Sveda;C. Lai
将甲型H1N1流感病毒神经氨酸酶(NA)基因的全长双链DNA拷贝克隆到pSV2330的晚期区域，构建了含有pBR322质粒DNA序列和猴病毒40早期区和40后期区启动子、剪接序列和转录终止位点的杂交表达载体pSV2330。克隆的野生型NA基因编码的蛋白经N_2单抗间接免疫荧光证实存在于固定细胞的细胞质和“活”或未固定细胞的表面。免疫沉淀法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法对野生型载体感染细胞的[35S]蛋氨酸标记蛋白的分析表明，克隆的NA DNA与流感病毒感染细胞的糖基化NA共游走，与细胞内膜结合，形成膜和胞质组分，并可形成可免疫共沉淀的二聚体。载体感染细胞经猴病毒40裂解后可检测到NA酶活性。将克隆的野生型基因产物的这些性质与同样克隆到pSV2330载体末端的3个缺失突变的NA DNA所产生的多肽的性质进行了比较。这些突变体缺少7(DLK)、21(DLI)或所有23个氨基酸(DLZ)的氨基(N)末端可变疏水区，该区域锚定感染细胞或流感病毒膜中成熟的野生型NA四聚体结构。对这些突变体表型的比较表明，NA分子中的这一区域还包括控制新生多肽转移到膜细胞器中进行糖基化的序列。
A full-length double-stranded DNA copy of an influenza A virus N2 neuraminidase (NA) gene was cloned into the late region of pSV2330, a hybrid expression vector that includes pBR322 plasmid DNA sequences and the simian virus 40 early region and simian virus 40 late region promoters, splice sequences, and transcription termination sites. The protein encoded by the cloned wild-type NA gene was shown to be present in the cytoplasm of fixed cells and at the surface of "live" or unfixed cells by indirect immunofluorescence with N2 monoclonal antibodies. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of [35S]methionine-labeled proteins from wild-type vector-infected cells with heterospecific N2 antibody showed that the product of the cloned NA DNA comigrated with glycosylated NA from influenza virus-infected cells, remained associated with internal membranes of cells fractionated into membrane and cytoplasmic fractions, and could form an immunoprecipitable dimer. NA enzymatic activity was detectable after simian virus 40 lysis of vector-infected cells. These properties of the product of the cloned wild-type gene were compared with those of the polypeptides produced by three deletion mutant NA DNAs that were also cloned into the late region of the pSV2330 vector. These mutants lacked 7 (dlk), 21 (dlI), or all 23 amino acids (dlZ) of the amino (N)-terminal variable hydrophobic region that anchors the mature wild-type NA tetrameric structure in the infected cell or influenza viral membrane. Comparison of the phenotypes of these mutants showed that this region in the NA molecule also includes sequences that control translocation of the nascent polypeptide into membrane organelles for glycosylation.
使用猿猴病毒 40 载体在猴细胞中表达人流感病毒血凝素。
DOI：
10.1073/pnas.79.2.233
发表时间：
1982
影响因子：
11.1
作者：
Hartman,JR;Nayak,DP;Fareed,GC
通讯作者：
Fareed,GC
用单克隆抗体选择和分析 N2 流感病毒神经氨酸酶抗原变异体。
影响因子：
3.7
作者：
Webster,RG;Hinshaw,VS;Laver,WG
通讯作者：
Laver,WG