Garten W, Will C, Buckard K, Kuroda K, Ortmann D, Munk K, Scholtissek C, Schnittler H, Drenckhahn D, Klenk H D
Institut für Virologie, Philipps-Universität, Marburg, Germany.
J Virol. 1992 Mar;66(3):1495-505. doi: 10.1128/JVI.66.3.1495-1505.1992.
Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.
对甲型流感病毒A/FPV/Rostock/34（H7N1）的五个温度敏感突变体ts206、ts293、ts478、ts482和ts651进行了研究，这些突变体在允许温度下能将正确的血凝素（HA）插入MDCK细胞的顶端质膜，但在限制温度下向细胞表面的转运存在缺陷。对这些突变体及其回复体的HA基因进行核苷酸序列分析表明，每个突变体中单个氨基酸的变化是导致转运受阻的原因。将这些氨基酸替换与之前分析过的突变体ts1和ts227的替换进行了比较（W. Schuy、C. Will、K. Kuroda、C. Scholtissek、W. Garten和H.-D. Klenk，《欧洲分子生物学组织杂志》5:2831 - 2836，1986年）。除ts206外，所有突变体和回复体中发生变化的氨基酸集中在三维HA模型的三个不同区域：（i）在80埃（8纳米）长的α螺旋末端，（ii）在球状区域与茎部的连接处，以及（iii）在茎部的基部结构域。这些区域对HA组装以及因此对转运至关重要这一概念得到了以下发现的支持：即在非允许温度下无法离开内质网的突变体不能正确三聚化。通过密度梯度离心、交联以及用胰蛋白酶和内切葡糖胺酶H消化分析，这些突变体可分为两组：ts1、ts227和ts478的HA形成大的不可逆聚集体，而ts206和ts293的HA在内质网中以单体形式保留。对于第三组，包括进入高尔基体的突变体ts482和ts651，三聚化未受损害。
