Bos T J, Davis A R, Nayak D P
Proc Natl Acad Sci U S A. 1984 Apr;81(8):2327-31. doi: 10.1073/pnas.81.8.2327.
Influenza virus neuraminidase (NA), unlike the majority of integral membrane proteins, does not contain a cleavable signal sequence. It contains an NH2-terminal hydrophobic domain that functions as an anchor. We have investigated the signal function for translocation of this NH2-terminal hydrophobic domain of NA by constructing chimeric cDNA clones in which the DNA coding for the first 40 NH2-terminal hydrophobic amino acids of NA was joined to the DNA coding for the signal-minus hemagglutinin (HA) of influenza virus. The chimeric HA (N4OH) containing the NH2 terminus of NA was expressed in CV1 cells by using a simian virus 40 late-expression vector. The chimeric HA is synthesized, translocated into the rough endoplasmic reticulum, and glycosylated, whereas HA lacking the signal sequence is present only in small amounts and is unglycosylated. These results clearly show that the NH2 terminus of NA, in addition to its anchor function, also provides the signal function in translocation. However, the acquisition of complex oligosaccharides and the transport of N4OH to the cell surface are greatly retarded. To determine if the presence of two anchor sequences, one provided by NA at the NH2 terminus and the other provided by HA at the COOH terminus of N4OH, was responsible for the slow transport, the NH2 terminus of NA was fused to an "anchorless" HA. The resulting chimeric HA (N4OH482) contains the hydrophobic domain of NA at the NH2 terminus but lacks the HA anchor at the COOH terminus. N4OH482 was synthesized and glycosylated; however, as with N4OH, the acquisition of complex oligosaccharides and the migration to the cell surface are greatly retarded. Immunofluorescence data also support that, compared to the native HA, only a small amount of chimeric HA proteins is transported to the cell surface. Thus, the hydrophobic NH2 terminus of NA, although capable of providing the signal function in translocation across the rough endoplasmic reticulum, interferes with the transport of the chimeric HA to the cell surface.
与大多数整合膜蛋白不同，流感病毒神经氨酸酶（NA）不含可裂解的信号序列。它含有一个作为锚定功能的NH2末端疏水结构域。我们通过构建嵌合cDNA克隆来研究NA的这个NH2末端疏水结构域的转运信号功能，在这些克隆中，编码NA前40个NH2末端疏水氨基酸的DNA与编码流感病毒信号缺失血凝素（HA）的DNA连接。通过使用猿猴病毒40晚期表达载体，在CV1细胞中表达含有NA的NH2末端的嵌合HA（N4OH）。嵌合HA被合成、转运到糙面内质网并进行糖基化，而缺乏信号序列的HA仅以少量存在且未糖基化。这些结果清楚地表明，NA的NH2末端除了其锚定功能外，还在转运中提供信号功能。然而，复杂寡糖的获得以及N4OH向细胞表面的转运被大大延迟。为了确定两个锚定序列的存在是否导致了缓慢转运，一个由NA在NH2末端提供，另一个由HA在N4OH的COOH末端提供，将NA的NH2末端与一个“无锚定”的HA融合。所得的嵌合HA（N4OH482）在NH2末端含有NA的疏水结构域，但在COOH末端缺乏HA锚定。N4OH482被合成并糖基化；然而，与N4OH一样，复杂寡糖的获得和向细胞表面的迁移被大大延迟。免疫荧光数据也支持，与天然HA相比，只有少量的嵌合HA蛋白被转运到细胞表面。因此，NA的疏水NH2末端虽然能够在跨糙面内质网的转运中提供信号功能，但会干扰嵌合HA向细胞表面的转运。
