Fenouillet E, Jones I, Powell B, Schmitt D, Kieny M P, Gluckman J C
Laboratoire de Biologie et Génétique des Pathologies Immunitaires, CNRS URA 1463, France.
J Virol. 1993 Jan;67(1):150-60. doi: 10.1128/JVI.67.1.150-160.1993.
To examine the role of the glycans of human immunodeficiency virus type 1 transmembrane glycoprotein gp41, conserved glycosylation sites within the env sequence (Asn-621, Asn-630, and Asn-642) were mutated to Gln. The mutated and control wild-type env genes were introduced into recombinant vaccinia virus and used to infect BHK-21 or CD4+ CEM cells. Mutated gp41 appeared as a 35-kDa band in a Western blot (immunoblot), and it comigrated with the deglycosylated form of wild-type gp41. Proteolytic cleavage of the recombinant wild-type and mutant forms of the gp160 envelope glycoprotein precursor was analyzed by pulse-chase experiments and enzyme-linked immunosorbent assay: gp160 synthesis was similar whether cells were infected with control or mutated env-expressing recombinant vaccinia virus, but about 10-fold less cleaved gp120 and gp41 was produced by the mutated construct than the control construct. The rates of gp120-gp41 cleavage at each of the two potential sites appeared to be comparable in the two constructs. By using a panel of antibodies specific for gp41 and gp120 epitopes, it was shown that the overall immunoreactivities of control and mutated gp41 proteins were similar but that reactivity to epitopes at the C and N termini of gp120, as present on gp160 produced by the mutated construct, was enhanced. This was no longer observed for cleaved gp120 in supernatants. Both gp120 proteins, from control and mutated env, were expressed on the cell surface under a cleaved form and could bind to membrane CD4, as determined by quantitative immunofluorescence assay. In contrast, and despite sufficient expression of env products at the cell membrane, gp41 produced by the mutated construct was unable to induce membrane fusion. Therefore, while contradictory results reported in the literature suggest that gp41 individual glycosylation sites are dispensable for the bioactivity and conformation of env products, it appears that such is not the case when the whole gp41 glycan cluster is removed.
为研究人类免疫缺陷病毒1型跨膜糖蛋白gp41聚糖的作用，将env序列中的保守糖基化位点（天冬酰胺-621、天冬酰胺-630和天冬酰胺-642）突变为谷氨酰胺。将突变的和对照野生型env基因导入重组痘苗病毒，并用于感染BHK-21或CD4+CEM细胞。突变的gp41在蛋白质印迹法（免疫印迹法）中呈现为一条35 kDa的条带，并且它与野生型gp41的去糖基化形式迁移位置相同。通过脉冲追踪实验和酶联免疫吸附测定分析重组野生型和突变型gp160包膜糖蛋白前体的蛋白水解切割：无论细胞是感染对照还是表达突变env的重组痘苗病毒，gp160的合成相似，但与对照构建体相比，突变构建体产生的切割后的gp120和gp41减少了约10倍。在两个构建体中，两个潜在位点处的gp120-gp41切割速率似乎相当。通过使用一组对gp41和gp120表位具有特异性的抗体，结果表明对照和突变的gp41蛋白的总体免疫反应性相似，但对突变构建体产生的gp160上存在的gp120 C端和N端表位的反应性增强。对于上清液中切割后的gp120，不再观察到这种情况。通过定量免疫荧光测定确定，来自对照和突变env的两种gp120蛋白均以切割形式表达在细胞表面，并且可以结合膜CD4。相比之下，尽管env产物在细胞膜上有足够的表达，但突变构建体产生的gp41无法诱导膜融合。因此，虽然文献中报道的相互矛盾的结果表明gp41单个糖基化位点对于env产物的生物活性和构象是可有可无的，但当整个gp41聚糖簇被去除时，情况似乎并非如此。
