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: 3853416
• 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/3853416
• 关键词: DNA virus; HIV infections; SDS polyacrylamide gel electrophoresis; antiviral antibody; calcium binding protein; calcium channel; cell line; enzyme substrate; human immunodeficiency virus 1; human immunodeficiency virus 2; human tissue; immunoprecipitation; molecular cloning; molecular oncology; phosphoproteins; phosphorylation; protein kinase C; protein purification; protein sequence; protein structure; tissue /cell culture; transfection; transposon /insertion element; viral carcinogenesis; virus protein; western blottings

A recombinant HIV-2 (NIH-Z) Nef protein synthesized in E. coli, and purified by the detergent/chaotrope extraction technique and preparative SDS-PAGE is: immunologically reactive on Western blots with anti-Nef antibodies; an excellent substrate in vitro for phosphorylation both by purified protein kinase C (PKC) and the maturation promoting factor (MPF) kinase purified from Xenopus oocytes; exhibits an intrinsic low-level autokinase activity, and forms stable homodimers and homotetrameric complexes in vitro, which are significantly increased in the absence of sulfhydryl-reducing agents. Preliminary results of in vivo phosphory- lation and oligomerization experiments suggest that the native 25 kD HIV-2 (NIH-Z) Nef protein in infected T-lymphocytes in culture becomes highly phosphorylated following stimulation with calcium ionophores, and the dimeric (50 kD) and tetrameric (100 kD) forms of this protein can be radioimmunoprecipitated with specific anti-HIV-2 Nef monoclonal antibodies. In addition, evidence of a putative Nef cellular protein complex was also obtained. Structural studies have also revealed the presence of a leucine zipper-like repeat structure at the conserved central "core" region of the Nef proteins, with a characteristic 4,3 repeat similar to the heptad leucine repeat motif of the bZIP factors. Moreover, at the C-terminus of the Nef proteins is a highly acidic sequence (net charge of -5 to -8) stretched over 40 amino acids, and it contains two predicted alpha-helices separated by a predicted beta-turn structure, with homology to known acidic activation domains of transcriptional activation factors. Biochemical characterization of individual biological Nef clones expressing only p27nef (clone 3B-3) or p25nef (clone 3B-5), isolated by limiting dilution of HTLV-IIIB-infected H9 cells, are continuing. In addition, a baculovirus vector-expressed HIV-1 Vpu protein, which contains a potential calcium binding or calcium channel-like sequence similar to one of the S-IV sequences of the dihydropyridine (DHP)-receptor, is being purified for use in calcium channel drug-binding studies.

在大肠杆菌中合成的重组HIV-2(NIH-Z)Nef蛋白，以及 洗涤剂/杂多酸提取技术提纯及制备 抗Nef蛋白免疫印迹的SDS-PAGE分析 抗体；一种良好的体外磷酸化底物， 蛋白激酶C(PKC)与成熟促进因子(MPF) 从非洲爪哇卵母细胞中纯化的激酶；表现出一种固有的低水平 自身激酶活性，并形成稳定的同源二聚体和同源四聚体 在体外，在没有药物的情况下，这些化合物会显著增加 巯基还原剂。体内磷光的初步结果-- 裂解和齐聚实验表明，天然的25kD 培养中感染的T淋巴细胞中的HIV-2(NIH-Z)Nef蛋白成为 在钙离子载体刺激后高度磷酸化，以及 这种蛋白质的二聚体(50kD)和四聚体(100kD)形式可以是 抗HIV-2Nef特异性单抗的放射免疫沉淀 抗体。此外，有证据表明可能存在Nef细胞蛋白 还得到了络合物。结构研究也揭示了 在保守区存在亮氨酸拉链样重复结构 Nef蛋白的中心“核心”区域，具有4，3 重复序列类似于bZIP因子的七肽亮氨酸重复序列。 此外，在Nef蛋白的C-末端是一个高度酸性的 序列(净电荷为-5到-8)延伸了40多个氨基酸，它 包含两个预测的阿尔法螺旋，由预测的贝塔转向隔开 结构，与已知的酸性激活结构域同源 转录激活因子。生物化学特性研究 仅表达p27nef的单个生物学Nef克隆(克隆3B-3)或 P25nef(克隆3B-5)，用HTLV-IIIB感染的有限稀释法分离 H9细胞，仍在继续。此外，杆状病毒载体--表达 HIV-1 VPU蛋白，含有潜在的钙结合或钙 与S-IV序列之一相似的通道状序列 二氢吡啶(DHP)-受体，正在提纯用于钙 渠道药物结合研究。