STRUCTURAL, BIOCHEMICAL, AND BIOLOGICAL CHARACTERIZATION OF HIV NEF AND VPU

HIV NEF 和 VPU 的结构、生化和生物学特征

• 批准号: 3874624
• 负责人: J A LAUTENBERGER
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3874624
• 关键词: AIDS; Escherichia; HIV infections; Xenopus; affinity labeling; antibody formation; bacterial genetics; calcium binding protein; calcium channel; cell free system; enzyme linked immunosorbent assay; enzyme substrate; gene expression; genome; human immunodeficiency virus 1; human immunodeficiency virus 2; human tissue; immunochemistry; insect virus; ion exchange chromatography; laboratory rabbit; molecular cloning; monoclonal antibody; mutant; oncogenes; oncogenic virus; oncoproteins; peripheral blood vessel; protein biosynthesis; protein kinase C; protein purification; protooncogene; radioimmunoassay; radiotracer; transfection; transposon /insertion element; virus genetics; virus protein

Relatively high levels of the HIV-1 and HIV-2-negative effector factor (Nef) proteins were expressed in the insect cell line, Sf-9, with a baculovirus vector. Two forms of the baculovirus produced HIV-1 Nef proteins were purified to greater than 95% homogeneity by affinity chromatography on a Nef monoclonal antibody (S-897-55S) column. A bacterially-produced HIV-2 Nef protein was also produced at relatively high levels and purified to near homogeneity by gel filtration and ion exchange chromatography. A high-titered rabbit antiserum was developed against the full-length HIV-2 Nef protein, and was used to identify a 23kD Nef protein in HIV-2 (NIHZ)-infected H9 cells by radioimmunoprecipitation (RIP) assay. Both the HIV-1 Nef monoclonal antibody S-897-55S and the HIV-2 Nef rabbit anti-serum were used to study the in vitro oligomerization of Nef proteins. In addition, both the HIV-1 baculovirus and HIV-2 bacterial Nef proteins demonstrate autophosphorylation activities and are efficient substrates in vitro for protein kinase C activity. We have also shown that the HTLV-IIIB-infected H9 cell line expresses two distinct species of Nef proteins (p25 and p27), which are coded by two different nef genes expressed in different genetic variants of HIV-1. In addition, we are using a bacterially-expressed HIV-1 vpu protein to study its potential for binding calcium and/or binding calcium channel activator or inhibitor agents.

HIV-1和HIV-2阴性效应因子水平相对较高 (Nef)蛋白质在昆虫细胞系Sf-9中表达， 杆状病毒载体两种形式的杆状病毒产生HIV-1 Nef 通过亲和层析将蛋白质纯化至大于95%的均一性 通过在Nef单克隆抗体（S-897- 55 S）柱上的层析纯化。一 细菌产生的HIV-2 Nef蛋白也以相对较高的水平产生， 水平，并通过凝胶过滤和离子交换纯化至接近均匀 层析制备了高滴度的兔抗血清， 全长HIV-2 Nef蛋白，并用于鉴定23 kD Nef蛋白 在HIV-2（NIHZ）感染的H9细胞中通过放射免疫沉淀（RIP）测定。 HIV-1 Nef单克隆抗体S-897- 55 S和HIV-2 Nef兔 抗血清用于研究Nef蛋白的体外寡聚化。 此外，HIV-1杆状病毒和HIV-2细菌Nef蛋白均 证明了自身磷酸化活性，并且是 体外测定蛋白激酶C活性。 我们还表明，HTLV-IIIB感染的H9细胞系表达两个 不同种类的Nef蛋白（p25和p27），它们由两个不同的基因编码。 不同的nef基因在HIV-1的不同遗传变异体中表达。在 此外，我们正在使用细菌表达的HIV-1 vpu蛋白来研究 其结合钙和/或结合钙通道激活剂的潜力 或抑制剂。