FUNCTIONAL STUDIES WITH HIV-1 VPR

HIV-1 VPR 的功能研究

• 批准号: 2330352
• 负责人: Alagarsamy Srinivasan
• 金额: $ 26.97万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-11-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330352
• 关键词: chimeric proteins; density gradient ultracentrifugation; gene mutation; human immunodeficiency virus 1; human immunodeficiency virus 2; human tissue; molecular cloning; protein structure function; tissue /cell culture; virus assembly; virus infection mechanism; virus protein; virus replication

DESCRIPTION: (Adapted from investigator's abstract) Human immunodeficiency virus, the etiologic agent of AIDS, is a complex retrovirus of the lentivirus subfamily. The HIV-1 genome contains six accessory gene known as vif, vpr, tat, rev, vpu and nef. The functions of tat and rev are essential for HIV-1 replication, but the functions of the other genes vary depending on the target cells employed for analysis. Contradictory data has been reported for some genes. In the initial funding period the applicant noted in structure-function studies of HIV-1 using a macrophage-tropic molecular clone designated 89.6, that Vpr plays a significant role in the productive infection of primary macrophages as observed for HIV-2 and chimeric HIV-1 containing partial sequences from macrophage-tropic virus. The characteristic features of Vpr noted by several studies including the applicant's are: (i) Vpr is stable in cells, (ii) Vpr has the ability to oligomerize, (iii) Vpr is transported to the nucleus, (iv) Vpr is incorporated into the virus particle, (v) Vpr has a positive effect on HIV-I infection in macrophages, (vi) Vpr prevents establishment of infected cells that chronically produce virus, and (vii) Vpr induces differentiation and arrest cell progression at G2 phase of the cell cycle. Despite the demonstration of these characteristics there is limited information available regarding the essential domains of Vpr for function. The hypothesis to be tested in this proposal is that Vpr has either distinct functional domains contributing to specific features of Vpr or a specific domain contributes to multiple features. It is likely that a combination of these may also be operative. An understanding of the structure-function relationship of Vpr will yield useful information regarding the interrelationship between oligomerization, nuclear localization, virion incorporation and the effect of Vpr at the level of virus infection. Based on preliminary studies and data published by others on vpr, the applicants propose to investigate in detail the following: (I) The requirement of Vpr for incorporation into the virus particle will be determined. The Vpr coding sequences will be altered utilizing several strategies based on secondary structure prediction and molecular modeling studies and characterized using different biochemical biological assays. As Vpr and Vpx incorporated into HIV-1 and -2 Gag directed virus particles respectively, chimeragenesis of Vpr and the related Vpx will be undertaken to define the sequences underlying the specificity of Vpr incorporation into the virus particles; (II) Structural protein Gag p6 domain will be analyzed in detail to localize the residues that participate in the incorporation of Vpr into the virus particle; (III) The molecular mechanism(s) of Vpr incorporation into the virus particle will be elucidated by analyzing the protein-protein interactions involving Gag and Vpr; (IV) The role of Vpr in HIV- I replication will be studied utilizing several Vpr mutants; and (V) The structural constraints associated with Vpr incorporation into the virus particle will be addressed. Vpr- fusion proteins and Vpr linked dimers will be generated and incorporation into virus particles will be addressed. This information will then be used to generate Vpr-fusion proteins that might interfere with HIV-1 replication. The structure- function studies of HIV-1 Vpr in terms of incorporation into the virus particle and its role in viral replication will be useful for understanding AIDS pathogenesis and for development of therapeutic agents.

描述：（改编自研究者的摘要）人类 免疫缺陷病毒是艾滋病的病原体，是一种复杂的病毒 慢病毒亚科的逆转录病毒。 HIV-1基因组包含六个 辅助基因称为 vif、vpr、tat、rev、vpu 和 nef。功能 tat 和 rev 对 HIV-1 复制至关重要，但是 其他基因根据用于分析的靶细胞而变化。 对于某些基因，已经报道了相互矛盾的数据。 在最初的 申请人在 HIV-1 结构功能研究中指出的资助期限 使用称为 89.6 的巨噬细胞嗜性分子克隆，Vpr 发挥作用 在原代巨噬细胞的生产性感染中发挥重要作用 观察到含有部分序列的 HIV-2 和嵌合 HIV-1 嗜巨噬细胞病毒。 Vpr 的特征由 包括申请人在内的几项研究是： (i) Vpr 在 细胞，(ii) Vpr 具有寡聚化能力，(iii) Vpr 被运输 进入细胞核，(iv) Vpr 并入病毒颗粒，(v) Vpr 对巨噬细胞中的 HIV-1 感染具有积极作用，(vi) Vpr 防止长期产生病毒的受感染细胞的建立， (vii) Vpr 在 G2 诱导分化并阻止细胞进展 细胞周期的阶段。尽管展示了这些 特征 有关的可用信息有限 Vpr 功能的基本域。要检验的假设 该提议是 Vpr 具有不同的功能域 对 Vpr 的特定功能或特定领域的贡献 到多个特征。这些的组合也可能 可以操作。理解结构与功能的关系 Vpr 将产生有关之间相互关系的有用信息 寡聚化、核定位、病毒体掺入和 Vpr 在病毒感染水平上的影响。 根据初步 其他人在 vpr 上发表的研究和数据，申请人建议 详细考察以下内容： （一）Vpr的要求 将确定掺入病毒颗粒中的情况。 Vpr 编码 序列将利用基于二级的几种策略进行改变 结构预测和分子建模研究和表征 使用不同的生化生物学测定。 作为 Vpr 和 Vpx 分别掺入HIV-1和-2 Gag定向病毒颗粒中， 将进行 Vpr 和相关 Vpx 的嵌合来定义 Vpr 掺入特异性的序列 病毒颗粒； (二) 结构蛋白Gag p6结构域分析 详细定位参与掺入的残基 Vpr进入病毒颗粒； （三）Vpr的分子机制 通过分析病毒颗粒的掺入将被阐明 涉及 Gag 和 Vpr 的蛋白质-蛋白质相互作用； （四）Vpr的作用 将利用几种Vpr突变体来研究HIV-1复制中的作用；和 (V) 与 Vpr 纳入相关的结构性限制 病毒颗粒将得到解决。 Vpr-融合蛋白与Vpr相连 将产生二聚体并掺入病毒颗粒中 已解决。然后，该信息将用于生成 Vpr-fusion 可能干扰 HIV-1 复制的蛋白质。 结构- HIV-1 Vpr 掺入病毒的功能研究 颗粒及其在病毒复制中的作用将有助于 了解艾滋病发病机制并开发治疗方法 代理。