Ohuchi M, Fischer C, Ohuchi R, Herwig A, Klenk H D
Institut für Virologie, Philipps-Universität Marburg, Germany.
J Virol. 1998 May;72(5):3554-9. doi: 10.1128/JVI.72.5.3554-3559.1998.
The hemagglutinin (HA) of fowl plague virus was lengthened and shortened by site-specific mutagenesis at the cytoplasmic tail, and the effects of these modifications on HA functions were analyzed after expression from a simian virus 40 vector. Elongation of the tail by the addition of one to six histidine (His) residues did not interfere with intracellular transport, glycosylation, proteolytic cleavage, acylation, cell surface expression, and hemadsorption. However, the ability to induce syncytia at a low pH decreased dramatically depending on the number of His residues added. Partial fusion (hemifusion), assayed by fluorescence transfer from octadecylrhodamine-labeled erythrocyte membranes, was also reduced, but even with the mutant carrying six His residues, significant transfer was observed. However, when the formation of fusion pores was examined with hydrophilic fluorescent calcein, transfer from erythrocytes to HA-expressing cells was not observed with the mutant carrying six histidine residues. The addition of different amino acids to the cytoplasmic tail of HA caused an inhibitory effect similar to that caused by the addition of His. On the other hand, a mutant lacking the cytoplasmic tail was still able to fuse at a reduced level. These results demonstrate that elongation of the cytoplasmic tail interferes with the formation and enlargement of fusion pores. Thus, the length of the cytoplasmic tail plays a critical role in the fusion process.
通过对禽瘟病毒血凝素（HA）胞质尾进行位点特异性诱变来延长和缩短其长度，并在从猿猴病毒40载体表达后分析这些修饰对HA功能的影响。通过添加一至六个组氨酸（His）残基来延长尾端，这并不干扰细胞内运输、糖基化、蛋白水解切割、酰化、细胞表面表达和血细胞吸附。然而，在低pH下诱导细胞融合的能力会根据添加的His残基数量而显著下降。通过从十八烷基罗丹明标记的红细胞膜进行荧光转移测定的部分融合（半融合）也有所降低，但即使是携带六个His残基的突变体，仍观察到显著的转移。然而，当用亲水性荧光钙黄绿素检测融合孔的形成时，携带六个组氨酸残基的突变体未观察到从红细胞到表达HA的细胞的转移。向HA的胞质尾添加不同氨基酸会产生与添加His类似的抑制作用。另一方面，缺乏胞质尾的突变体仍能在较低水平上融合。这些结果表明，胞质尾的延长会干扰融合孔的形成和扩大。因此，胞质尾的长度在融合过程中起关键作用。
