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：(改编自调查人员摘要)人类 免疫缺陷病毒，艾滋病的病原体，是一种复杂的 慢病毒亚家族的逆转录病毒。HIV-1基因组包含六个 辅助基因包括vif、vpr、tat、rev、vPU和nef。这些功能 Tat和rev的功能对于HIV-1复制是必不可少的，但 其他基因因用于分析的目标细胞而异。 关于某些基因的数据也有相互矛盾的报道。在最初 申请者在HIV-1结构-功能研究中注明的资助期 使用命名为89.6的巨噬细胞亲和性分子克隆，VPR扮演 在原代巨噬细胞生产性感染中的重要作用 观察HIV-2和包含部分序列的嵌合HIV-1 嗜巨噬细胞病毒。VPR的特征由 包括申请者在内的几项研究是：(I)VPR在 细胞，(Ii)vpr具有寡聚的能力，(Iii)vpr被运输 到细胞核，(Iv)vpr被结合到病毒颗粒中，(V)vpr 对巨噬细胞中HIV-I感染有积极作用，(Vi)vpr 防止长期产生病毒的受感染细胞的建立， 和(Vii)vpr诱导分化并阻止G2期细胞进展 细胞周期的阶段。尽管有这些演示 特征有关的信息有限 函数VPR的本质域。待检验的假说 这一建议是VPR具有不同的功能域 对VPR的特定功能或特定领域的贡献 到多个功能。很可能，这些因素的组合也可能 行动起来。对结构与功能关系的认识 VPR将提供关于两国之间相互关系的有用信息 寡聚、核定位、病毒粒子掺入和 VPR在病毒感染水平的作用。基于初步的 其他人在VPR上发布的研究和数据，申请者建议 详细调查以下内容：(I)VPR对 病毒颗粒中的掺入情况将被确定。VPR编码 序列将使用基于次要信息的几种策略进行更改 结构预测和分子模拟研究及表征 使用不同的生化生物学检测方法。AS 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的嵌合发生，以确定 VPR掺入的特异性背后的序列 病毒颗粒；(Ii)结构蛋白Gag p6结构域将被分析 详细地定位参与掺入的残基 VPR进入病毒颗粒；(III)VPR的分子机制(S) 病毒颗粒中的结合将通过分析 涉及GAG和VPR的蛋白质相互作用；(IV)VPR的作用 将利用几个vpr突变体研究艾滋病毒-I型复制；以及 (V)将越南船级社纳入 病毒粒子将得到解决。VPR-融合蛋白与VPR连锁 将产生二聚体，并将其整合到病毒颗粒中 地址。然后，该信息将被用于生成VPR融合 可能干扰HIV-1复制的蛋白质。这个结构-- HIV-1vpr与病毒整合的功能研究 粒子及其在病毒复制中的作用将有助于 了解艾滋病的发病机制和治疗方法的发展 探员们。