Otteken A, Earl P L, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0455, USA.
J Virol. 1996 Jun;70(6):3407-15. doi: 10.1128/JVI.70.6.3407-3415.1996.
Monoclonal antibodies (MAbs) that bind linear or conformational epitopes on monomeric or oligomeric human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were screened for their recognition of maturational intermediates. On the basis of reactivities with gp160 at different times after pulse-labeling, the MAbs were sorted into groups that exhibited binding which was immediate and constant, immediate but transient, delayed, late, or very late. This grouping was consistent with the selectivity of the MAbs for structural features of gp160. Thus, a MAb to the V3 loop reacted with envelope proteins at all times, in accord with the relative conformational independence and accessibility of the epitope. Several MAbs that preferentially react with monomeric gp160 exhibited diminished binding after the pulse. A 10-min tag occurred before gp160 reacted with conformational MAbs that inhibited CD4 binding. The availability of epitopes for other conformational MAbs, including some that react equally with monomeric and oligomeric gp160 and some that react better with oligomeric forms, was half-maximal in 30 min and closely followed the kinetics of gp160 oligomerization. Remarkably, there was a 1- to 2-h delay before gp160 reacted with stringent oligomer-specific MAbs. After 4 h, approximately 20% of the gp160 was recognized by these MAbs. Epitopes recognized by monomerspecific or CD4-blocking MAbs but not by oligomer-dependent MAbs were present on gp160 molecules associated with the molecular chaperone BiP/GRP78. MAbs with a preference for monomers reacted with recombinant or HIV-1 envelope proteins in the endoplasmic reticulum, whereas the oligomer-specific MAbs recognized them in the Golgi complex. Additional information regarding gp160 maturation and intracellular trafficking was obtained by using brefeldin A, dithiothreitol, and a low temperature.
筛选了与单体或寡聚体人类免疫缺陷病毒1型（HIV-1）包膜糖蛋白上的线性或构象表位结合的单克隆抗体（MAb），以检测它们对成熟中间体的识别情况。根据脉冲标记后不同时间与gp160的反应性，将这些单克隆抗体分为几组，它们表现出立即且持续的结合、立即但短暂的结合、延迟的结合、晚期的结合或非常晚期的结合。这种分组与单克隆抗体对gp160结构特征的选择性一致。因此，针对V3环的单克隆抗体在所有时间都与包膜蛋白反应，这与表位相对的构象独立性和可及性相符。一些优先与单体gp160反应的单克隆抗体在脉冲后结合能力减弱。在gp160与抑制CD4结合的构象单克隆抗体反应之前有10分钟的标记时间。其他构象单克隆抗体的表位可及性，包括一些与单体和寡聚体gp160反应相同的以及一些与寡聚体形式反应更好的，在30分钟时达到最大值的一半，并紧密跟随gp160寡聚化的动力学。值得注意的是，在gp160与严格的寡聚体特异性单克隆抗体反应之前有1至2小时的延迟。4小时后，约20%的gp160被这些单克隆抗体识别。与分子伴侣BiP/GRP78相关的gp160分子上存在单体特异性或CD4阻断单克隆抗体识别但寡聚体依赖性单克隆抗体不识别的表位。偏好单体的单克隆抗体与内质网中的重组或HIV-1包膜蛋白反应，而寡聚体特异性单克隆抗体在高尔基体复合物中识别它们。通过使用布雷菲德菌素A、二硫苏糖醇和低温获得了关于gp160成熟和细胞内运输的更多信息。
