Murphy C I, Lennick M, Lehar S M, Beltz G A, Young E
Cambridge Bioscience Corporation, Worcester, MA 01605.
Genet Anal Tech Appl. 1990 Oct;7(6):160-71. doi: 10.1016/0735-0651(90)90030-j.
Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.
三种不同的I型人类免疫缺陷病毒（HIV-1）包膜衍生重组蛋白和全长人类CD4多肽在草地贪夜蛾（Sf9）细胞中表达。编码CD4、gp120、gp160和gp160 delta（全长gp160减去gp41的跨膜和胞质区域）的DNA构建体被克隆到杆状病毒表达载体pVL941或其衍生物中，并用于在与苜蓿银纹夜蛾核型多角体病毒（AcMNPV）的DNA共转染中产生重组病毒。重组HIV-1蛋白细胞提取物的蛋白质印迹分析表明，对于每个构建体，有两条主要条带与抗HIV-1包膜抗血清特异性反应。通过对感染细胞进行3H-甘露糖标记和衣霉素处理确定，这些条带对应于HIV蛋白的糖基化和非糖基化形式。HIV包膜表达的时间进程显示，在感染后早期（24小时），蛋白质完全糖基化且可溶于非离子去污剂。然而，在感染后后期（48小时），重组蛋白的表达水平达到最大值，但大部分增加是由于非糖基化物种水平的上升，其在很大程度上不溶于非离子去污剂。因此，似乎Sf9细胞不能有效地处理大量糖基化重组蛋白。作为生物活性的一种衡量，在共免疫沉淀试验中测试了糖基化和非糖基化重组HIV包膜蛋白的CD4结合能力。结果表明，在该分析中，CD4与重组gp120或gp160 delta的糖基化形式特异性相互作用。来自衣霉素处理培养物的非糖基化gp120或gp160 delta蛋白确实能与抗HIV-1抗血清进行免疫沉淀，但不与CD4相互作用。我们得出结论，通过添加碳水化合物侧链和结合CD4的能力来衡量，天然HIV包膜蛋白的产生在杆状病毒感染的昆虫细胞中感染后早期达到峰值。
