Guo H G, Veronese F M, Tschachler E, Pal R, Kalyanaraman V S, Gallo R C, Reitz M S
Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20892.
Virology. 1990 Jan;174(1):217-24. doi: 10.1016/0042-6822(90)90070-8.
The envelope proteins of retroviruses are derived from a polypeptide precursor protein by cleavage adjacent to a cluster of basic amino acids. Site-specific mutagenesis was used to construct a mutant of the human immunodeficiency virus type 1 (HIV-1) in which the arginine residue at the carboxy-terminus of the gp120 was changed to a threonine residue. This single substitution was sufficient to abolish all detectable cleavage of the gp160 envelope precursor polypeptide as well as virus infectivity. The gp160 was produced in normal quantities from a biologically active clone of the mutant virus after transfection into cos-1 cells. The mutant gp160 contained N-linked oligosaccharide chains with mannose-rich cores similar to those of the gp160 produced by the wild-type clone. Immunofluorescence assays showed that gp160 was transported to the surface of transfected CD4+ HeLa cells. No envelope proteins of known size could be detected in the media of cells transfected with the mutant virus, suggesting that functional virions were not formed. Binding of the mutant gp160 to the CD4 receptor molecule was unimpaired. Despite this and the presence of gp160 on the cell surface, neither growth of mutant-transfected CD4+ HeLa cells nor cocultivation of transfected cos-1 cells with H9 cells resulted in significant syncytium formation. The data indicate that the carboxy-terminal arginine residue of HIV-1 gp120 is necessary for envelope protein cleavage and suggest cleavage is important in the virus life cycle in both functional virus release and membrane fusion.
逆转录病毒的包膜蛋白是通过在一簇碱性氨基酸附近进行切割，从多肽前体蛋白衍生而来的。位点特异性诱变被用于构建1型人类免疫缺陷病毒（HIV-1）的突变体，其中gp120羧基末端的精氨酸残基被改变为苏氨酸残基。这一单一位点的替换足以消除gp160包膜前体多肽的所有可检测到的切割以及病毒的感染性。将突变病毒转染到cos-1细胞后，从该突变病毒的生物活性克隆中正常量地产生了gp160。突变的gp160含有与野生型克隆产生的gp160相似的富含甘露糖核心的N-连接寡糖链。免疫荧光分析表明，gp160被转运到转染的CD4+ HeLa细胞表面。在用突变病毒转染的细胞培养基中未检测到已知大小的包膜蛋白，这表明未形成功能性病毒颗粒。突变的gp160与CD4受体分子的结合未受损害。尽管如此，并且细胞表面存在gp160，但转染了突变体的CD4+ HeLa细胞的生长以及转染的cos-1细胞与H9细胞的共培养均未导致明显的合胞体形成。数据表明，HIV-1 gp120的羧基末端精氨酸残基对于包膜蛋白的切割是必需的，并表明切割在病毒生命周期中对于功能性病毒释放和膜融合都很重要。
