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

在大肠杆菌中合成了重组HIV-2（NIH-Z）Nef蛋白。杆菌和 通过去污剂/离液剂提取技术纯化并制备 SDS-PAGE：在Western印迹上具有免疫反应性，抗Nef 抗体;一个很好的底物在体外磷酸化， 纯化的蛋白激酶C（PKC）和成熟促进因子（MPF） 从非洲爪蟾卵母细胞中纯化的激酶;表现出内在的低水平 自身激酶活性，并形成稳定的同源二聚体和同源四聚体 复合物在体外，这是显着增加的情况下， 巯基还原剂。 体内磷酸化的初步结果 连接和寡聚化实验表明，天然的25 kD HIV-2（NIH-Z）Nef蛋白在培养的感染T淋巴细胞中成为 用钙离子载体刺激后高度磷酸化，和 该蛋白质的二聚体（50 kD）和四聚体（100 kD）形式可以是 特异性抗HIV-2 Nef单克隆抗体放射免疫沉淀 抗体的 此外，一种假定的Nef细胞蛋白的证据 也得到了配合物。 结构研究还显示， 存在亮氨酸拉链样重复结构在保守的 Nef蛋白的中心“核心”区域，具有特征性的4，3 重复类似于bZIP因子的七肽亮氨酸重复基序。 此外，在Nef蛋白的C-末端是高度酸性的， 序列（净电荷为-5至-8）延伸超过40个氨基酸， 包含两个预测的α-螺旋，由预测的β-转角分开 结构，与已知的酸性激活结构域同源， 转录激活因子 的生物化学表征 仅表达p27 nef的单个生物Nef克隆（克隆3B-3）或 p25 nef（克隆3B-5），通过有限稀释HTLV-IIIB感染的 H9细胞仍在继续。 此外，杆状病毒载体表达的 HIV-1 Vpu蛋白，其中含有潜在的钙结合或钙 通道样序列类似于S-IV序列之一， 二氢吡啶（DHP）受体，正在纯化用于钙 通道药物结合研究。

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.