Berman P W, Riddle L, Nakamura G, Haffar O K, Nunes W M, Skehel P, Byrn R, Groopman J, Matthews T, Gregory T
Department of Developmental Biology, Genentech, Inc., South San Francisco, California 94080.
J Virol. 1989 Aug;63(8):3489-98. doi: 10.1128/JVI.63.8.3489-3498.1989.
The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.
1型人类免疫缺陷病毒的包膜糖蛋白以前体gp160的形式合成，随后被切割产生成熟的gp120和gp41。在这些研究中，编码gp160的基因被诱变，以指导合成一种由gp120和gp41的细胞外结构域组成的截短蛋白。这种变异蛋白称为sgp160，由458个gp120氨基酸和172个gp41氨基酸组成。为便于蛋白质纯化，通过定点诱变删除了gp120和gp41之间的正常多聚糖蛋白加工位点。这使得能够合成一种分子，该分子可通过亲和色谱法纯化，采用酸洗脱，而无需将gp120多肽与gp41多肽解离。sgp160变异体的构象似乎具有功能相关性，这体现在它能够以与变异体rgp120相当的亲和力结合CD4。含sgp160的多肽结构与rgp120不同，因为它倾向于形成高分子量聚集体，在还原剂存在下可解离为单体和二聚体。针对sgp160蛋白的抗体与真正的病毒衍生gp160、gp120和gp41发生反应；中和病毒感染性；并抑制rgp120与CD4的结合。针对sgp160蛋白的兔抗体与针对rgp120产生的抗体不同，因为它们富含阻断CD4结合但不阻止1型人类免疫缺陷病毒诱导的合胞体形成的群体。
