FUNCTIONAL STUDIES WITH HIV-1 VPR

HIV-1 VPR 的功能研究

• 批准号: 2653810
• 负责人: Alagarsamy Srinivasan
• 金额: $ 28.05万
• 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/2653810
• 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：（根据调查员的摘要改编）人类 免疫缺陷病毒是艾滋病的病因学药物，是一个复杂的 慢病毒亚科逆转录病毒。 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的复制至关重要，但是 其他基因根据用于分析的目标细胞而变化。 已经报道了某些基因的矛盾数据。 在最初 资金期HIV-1的结构 - 功能研究中指出的申请人 使用指定的巨噬细胞 - 循环分子克隆89.6，VPR播放 在原发性巨噬细胞的生产感染中起着重要作用 观察到HIV-2和嵌合HIV-1包含部分序列 巨噬细胞 - 递归病毒。 VPR的特征 包括申请人在内的几项研究是：（i）VPR稳定 细胞，（ii）VPR具有寡聚的能力，（iii）VPR运输 在细胞核中，（iv）vpr被纳入病毒颗粒中，（v）vpr 对巨噬细胞中的HIV-I感染有积极作用，（VI）VPR 防止建立感染的细胞，这些细胞长期产生病毒， （vii）VPR在G2处诱导分化和阻滞细胞进展 细胞周期的阶段。尽管证明了这些 特征有限的信息可用 VPR的基本域用于功能。要检验的假设 该建议是VPR具有不同的功能域 有助于VPR或特定领域的特定特征有助于 具有多个功能。这些结合可能也可能 要手术。了解结构功能关系的关系 VPR将产生有关相互关系的有用信息 寡聚，核定位，病毒体掺入和 VPR在病毒感染水平上的影响。 基于初步 其他人在VPR上发表的研究和数据，申请人建议 详细调查以下内容：（i）VPR对 将确定掺入病毒颗粒中。 VPR编码 序列将通过基于次级的几种策略来改变序列 结构预测和分子建模研究并进行了表征 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指向病毒颗粒中， VPR和相关VPX的嵌合作用将被定义 VPR融合到该特异性的基础的序列 病毒颗粒； （ii）将分析结构蛋白GAG P6结构域 详细介绍参加合并的残留物 VPR进入病毒颗粒； （iii）VPR的分子机制 将掺入病毒颗粒的掺入将通过分析来阐明 涉及GAG和VPR的蛋白质蛋白质相互作用； （iv）VPR的作用 在HIV中，将使用几个VPR突变体研究复制。和 （v）与vpr融合有关的结构约束 病毒颗粒将被解决。 VPR融合蛋白和VPR连接 将生成二聚体，并掺入病毒颗粒中 解决。然后，此信息将用于生成VPR融合 可能干扰HIV-1复制的蛋白质。 结构 - HIV-1 VPR的功能研究在掺入病毒方面 粒子及其在病毒复制中的作用将有用 了解有助于发病机理和治疗的发展 代理商。