Simpson D A, Lamb R A
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
J Virol. 1992 Feb;66(2):790-803. doi: 10.1128/JVI.66.2.790-803.1992.
The influenza virus hemagglutinin (HA) contains a cytoplasmic domain that consists of 10 to 11 amino acids, of which five residues have sequence identity for 10 of 13 HA subtypes. To investigate properties of these conserved residues, oligonucleotide-directed mutagenesis was performed, using an HA cDNA of influenza virus A/Udorn/72 (H3N2) to substitute the conserved cysteine residues with other residues, to delete the three C-terminal conserved residues, or to remove the entire cytoplasmic domain. The altered HAs were expressed in eukaryotic cells, and the rates of intracellular transport were examined. It was found that substitution of either conserved cysteine residue within the cytoplasmic domain did not affect the rate of intracellular transport, whereas deletion of residues within the C-terminal domain resulted in delayed cell surface expression. All the altered HAs were biologically active in hemadsorption and fusion assays. To investigate whether the wild-type HA and HAs with altered cytoplasmic tails could complement the influenza virus temperature-sensitive transport-defective HA mutant A/WSN/33 ts61S, the HA cDNAs were expressed by using a transient expression system and released virus was assayed by plaque analysis. The wild-type HA expression resulted in a release of approximately 10(3) PFU of virus per ml. Antibody neutralization of complemented virus indicated that the infectivity was due to incorporation of wild-type H3 HA into ts61S virions. Sucrose density gradient analysis of released virions showed that each of the HA cytoplasmic domain mutants was incorporated into virus particles. Virions containing HAs with substitution of the cysteine residues in the cytoplasmic domain were found to be infectious. However, no infectivity could be detected from virions containing HAs that had deletions in their cytoplasmic domains. Possible roles of the HA cytoplasmic domain in forming protein-protein interactions in virions and their involvement in the initiation of the infection process in cells are discussed.
流感病毒血凝素（HA）含有一个由10至11个氨基酸组成的胞质结构域，其中五个残基在13种HA亚型中的10种具有序列同一性。为了研究这些保守残基的特性，利用A型流感病毒A/Udorn/72（H3N2）的HA cDNA进行寡核苷酸定向诱变，用其他残基替代保守的半胱氨酸残基，删除三个C末端保守残基，或去除整个胞质结构域。将改变后的HA在真核细胞中表达，并检测细胞内运输速率。结果发现，胞质结构域内任一保守半胱氨酸残基的替代均不影响细胞内运输速率，而C末端结构域内残基的缺失导致细胞表面表达延迟。所有改变后的HA在血细胞吸附和融合试验中均具有生物学活性。为了研究野生型HA和胞质尾巴改变的HA是否能互补流感病毒温度敏感型运输缺陷型HA突变体A/WSN/33 ts61S，通过瞬时表达系统表达HA cDNA，并通过噬斑分析检测释放的病毒。野生型HA的表达导致每毫升释放约10³个病毒空斑形成单位（PFU）。对互补病毒的抗体中和表明，感染性是由于野生型H3 HA掺入ts61S病毒粒子中。对释放的病毒粒子进行蔗糖密度梯度分析表明，每个HA胞质结构域突变体均被掺入病毒颗粒中。发现含有胞质结构域中半胱氨酸残基被替代的HA的病毒粒子具有感染性。然而，从含有胞质结构域有缺失的HA的病毒粒子中未检测到感染性。讨论了HA胞质结构域在病毒粒子中形成蛋白质-蛋白质相互作用及其参与细胞感染过程起始中的可能作用。
