Lin Y P, Wharton S A, Martín J, Skehel J J, Wiley D C, Steinhauer D A
National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom.
Virology. 1997 Jul 7;233(2):402-10. doi: 10.1006/viro.1997.8626.
A series of eight transfectant influenza viruses was generated by reverse genetics for studies of the palmitylated cysteine residues in the cytoplasmic tail of the hemagglutinin glycoprotein (HA). Following amplification of these viruses in MDCK cells we found that all had developed an elevated pH of membrane fusion--an unexpected result since previous mutant HA expression studies had shown that substitutions of the cysteine residues had no effect on fusion properties. Sequence analyses revealed that each of the viruses had at least one additional mutation in the ectodomain of HA which was responsible for the increase in fusion pH. Similarly, when we passaged egg-grown wild-type X-31 virus in three different lines of MDCK cells or in MDBK cells, high pH fusion mutants were selected within a few passages in every case. The locations of the substitutions in the HA structure are in or near the "fusion peptide" or at subunit interfaces throughout the length of the trimer--reminiscent of the changes selected in earlier studies on amantadine resistance. The observation that passage of certain viruses in mammalian cells can result in the selection of mutants with elevated fusion pH has potential implications both for reverse genetic experiments and, perhaps more importantly, for the choice of substrates for propagation of vaccine viruses.
通过反向遗传学方法构建了一系列八种转染流感病毒，用于研究血凝素糖蛋白（HA）细胞质尾部的棕榈酰化半胱氨酸残基。在MDCK细胞中扩增这些病毒后，我们发现所有病毒的膜融合pH值均升高——这是一个意外的结果，因为之前的突变HA表达研究表明，半胱氨酸残基的取代对融合特性没有影响。序列分析显示，每种病毒在HA的胞外结构域至少还有一个额外突变，这导致了融合pH值的升高。同样，当我们在三种不同的MDCK细胞系或MDBK细胞中传代鸡胚培养的野生型X-31病毒时，每种情况下在几代内都选择到了高pH值融合突变体。HA结构中取代位点位于“融合肽”内或附近，或在整个三聚体长度的亚基界面处——这让人想起早期关于金刚烷胺抗性研究中选择的变化。某些病毒在哺乳动物细胞中传代可导致选择出融合pH值升高的突变体，这一观察结果对反向遗传实验可能具有潜在影响，也许更重要的是，对疫苗病毒增殖底物的选择也有潜在影响。
