Hemming A, Bolmstedt A, Flodby P, Lundberg L, Gidlund M, Wigzell H, Olofsson S
Department of Clinical Virology, University of Göteborg, Sweden.
Arch Virol. 1989;109(3-4):269-76. doi: 10.1007/BF01311087.
A DNA fragment encoding the CD4-binding region of human immunodeficiency virus type 1 (HIV) gp 120 was excised from an SV40-based expression vector containing gp 160, and subcloned into phage M13 for site-directed mutagenesis. Mutant vectors were constructed and CV-1 cells were transfected with constructs, where Cys402 was substituted for a serine, and metabolically labelled with [3H]-N-acetylglucosamine (GlcN). Radioimmunoprecipitation with an hyperimmunserum, specific for gp 120/gp 160, and subsequent SDS-polyacrylamide gel electrophoresis demonstrated presence of gp 160, whereas gp 120 was replaced by [3H]-GlcN-labelled material, migrating as a diffuse band corresponding to 80-105k, suggesting increased sensitivity of mutant env gene products to proteolysis after cleavage to gp 120. Wild type gp 120 and gp 160 bound to CD4, whereas neither gp 160 nor gp 120 from mutant-transfected cell lysates did bind to CD4. Altogether the results indicated that Cys402, probably by participating in a disulfide bridge, is essential for (i) the CD4-binding ability of env gene products and for (ii) the physical stability of gp 120.
从一个含有gp160的基于SV40的表达载体中切下一段编码人免疫缺陷病毒1型（HIV）gp120的CD4结合区的DNA片段，并亚克隆到噬菌体M13中进行定点诱变。构建了突变载体，并用构建体转染CV-1细胞，其中将半胱氨酸402替换为丝氨酸，并用[3H]-N-乙酰葡糖胺（GlcN）进行代谢标记。用对gp120/gp160特异的超免疫血清进行放射免疫沉淀，随后进行SDS-聚丙烯酰胺凝胶电泳，结果显示存在gp160，而gp120被[3H]-GlcN标记的物质所取代，迁移为一条对应于80-105k的弥散带，表明突变的env基因产物在裂解为gp120后对蛋白水解的敏感性增加。野生型gp120和gp160与CD4结合，而来自突变转染细胞裂解物的gp160和gp120均不与CD4结合。总之，结果表明半胱氨酸402可能通过参与二硫键，对（i）env基因产物的CD4结合能力和（ii）gp120的物理稳定性至关重要。
