FUNCTIONAL STUDIES WITH HIV-1 VPR

描述：（改编自研究者摘要）人 免疫缺陷病毒是艾滋病的病原体，是一种复杂的 慢病毒亚科的逆转录病毒。 HIV-1基因组包含六个 辅助基因被称为vif、vpr、达特、rev、vpu和nef。的功能 达特和rev对HIV-1复制至关重要，但tat和rev的功能 其它基因根据用于分析的靶细胞而变化。 一些基因的数据相互矛盾。 在初始 申请人在HIV-1的结构-功能研究中指出的资助期 使用命名为89.6的嗜巨噬细胞分子克隆，Vpr发挥 在初级巨噬细胞的生产性感染中起重要作用， 观察到HIV-2和嵌合HIV-1含有来自 嗜巨噬细胞病毒Vpr的特征由 包括申请人的几项研究是：（i）Vpr在 细胞，（ii）Vpr具有寡聚化的能力，（iii）Vpr被转运 （iv）Vpr掺入病毒颗粒，（v）Vpr 对巨噬细胞中的HIV-I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， 和（vii）Vpr诱导分化并将细胞进展阻滞在G2 细胞周期的阶段。尽管这些示威活动 关于这些特征，现有信息有限。 Vpr函数的基本域。待检验的假设 这一建议是Vpr具有不同的功能域， 有助于Vpr的特定特征或特定域有助于 到多个功能。很可能这些因素的结合也可能 要有行动力。对结构-功能关系的理解 Vpr将产生关于以下方面的相互关系的有用信息： 寡聚化，核定位，病毒体掺入和 Vpr在病毒感染水平的作用。 根据初步 根据其他人发表的关于vpr的研究和数据，申请人建议 详细研究了以下几个方面：（一）Vpr对 将确定病毒颗粒中的掺入。 Vpr编码 序列将利用基于次级的几种策略来改变， 结构预测和分子模拟研究，并表征 使用不同的生化生物测定。 当Vpr和Vpx 分别掺入HIV-1和-2 Gag定向病毒颗粒， Vpr和相关Vpx的嵌合体形成将被用来定义 Vpr掺入细胞的特异性的基础序列 病毒颗粒;（II）分析结构蛋白Gag p6结构域 详细定位参与掺入的残基 Vpr进入病毒颗粒的分子机制 将通过分析结合到病毒颗粒中的蛋白质来阐明结合到病毒颗粒中的方法。 涉及Gag和Vpr的蛋白质-蛋白质相互作用;（IV）Vpr的作用 将利用几种Vpr突变体研究HIV-I复制; (V)与Vpr结合到 病毒颗粒将被处理。 Vpr-融合蛋白和Vpr连接的 将产生二聚体，并将掺入病毒颗粒中。 处理。然后，该信息将用于生成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 了解艾滋病的发病机制和发展治疗 剂.

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.