Study on functions of HEV-1 accessory gene Vpr

HEV-1辅助基因Vpr的功能研究

基本信息

批准号：
16017304
负责人：
AIDA Yoko
金额：
$ 9.6万
依托单位：
RIKEN
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16017304/
关键词：
HIV-1 Vpr nuclear entry importin a macrophages SAP145 splicing inhibitor ceuular pre-mRNA HIV-1 mRNA HIV-1Vpr 核移行核輸送担体imporitn α 結合阻害 ELISA 低分子化合物 X線構造解析蛍光イメージング技術 Vpr変異体ウイルスintegration complex (PIC)in vitro nucleor im

项目摘要

HIV-1Vpr, a virion-associated viral protein 96 amino-acids in length, has multiple biological functions including its nuclear localization activity, arrest at the G2/M phase of the cell cycle, positive and negative regulation of apoptosis.Vpr is essential for the nuclear import of preintegration complex (PIC) in Macrophages, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We firstly demonstrated that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin a alone in an importin 13-independent manner. Next, we demonstrated that the nuclear import of Vpr is strongly promoted by the addition of the cytoplasmic extract from macrophages but not from monocyte, and the nuclear import activity is lost by immunodepletion of importin q from the cytoplasmic extract. Immature monocytes express importin a at low level by immunoblot analysis and real-time PCR, while the expression of three major importin a isoforms markedly increases … More upon differentiation to macrophages, indicating requirement the expression of importin a for a nuclear import of Vpr. Furthermore, interaction between importin a and Vpr is indispensable not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. Finally, we demonstrated that the binding of Vpr to importin a that precedes a novel nuclear import process is potential target for therapeutic intervention.On the other hand, we discovered a novel role for Vpr in the selective inhibition of cellular pre-mRNA splicing both in vivo and in vitro. Vpr exerted this effect via interactions not only with the essential splicing factor SAP145 but also with functional spliceosomal complexes. We also found that expression of Vpr selectively inhibit splicing of HIV-1 mRNA, resulting in an accumulation of the 4kb-form of viral mRNA and a decrease in the 2kb-form, and consequently altered the expression of corresponding proteins. Moreover, Vpr enhanced de novo synthesis of virion-associated proteins and increases the virus infectivity. This is the first report to demonstrate that Vpr enhances the virus infectivity via selective inhibition of viral pre-mRNA splicing. Less

HIV-1VPR是一种长度与病毒体相关的病毒蛋白96氨基酸，具有多种生物学功能，包括其核定位活性，在细胞周期的G2/m期停滞，凋亡的阳性和负调控。VPR对摩托噬细胞的核导入（PIC）在摩托噬细胞中的核导入至关重要，尽管该作用在摩托车中仍然是vpr的作用。我们首先证明了VPR是针对核包络的，然后以13个独立的方式单独将Importin A运送到核中。接下来，我们证明，通过从巨噬细胞中添加细胞质提取物，而不是从单核细胞中添加细胞质提取物，从而强烈促进了VPR的核进口，并且通过从细胞质提取物中的ImportIn Q进行免疫原理而丧失了核进口活性。未成熟的单核细胞通过免疫印迹分析和实时PCR表达低水平的导入蛋白A，而三种主要importin a同工型的表达显着增加……更多地增加了与巨噬细胞的区分，表明需要对vpr的核导入表达importin a。此外，Importin A与VPR之间的相互作用不仅对于VPR的核进口是必不可少的，而且对于巨噬细胞中的HIV-1复制也是必不可少的。最后，我们证明了VPR与进口蛋白A的结合是在新的核进口过程之前进行治疗干预的潜在靶标。另一方面，我们发现了VPR在选择性抑制细胞前MRNA中既固定体内和体内和体内剪接的新作用。 VPR不仅通过与必需的剪接因子SAP145而且与功能剪接体复合物相互作用发挥了这种影响。我们还发现，VPR的表达有选择地抑制HIV-1 mRNA的剪接，从而导致4KB形式的病毒mRNA的积累和2KB形式的降低，从而改变了相应的蛋白质的表达。此外，VPR增强了病毒相关蛋白的从头合成，并增加了病毒感染。这是第一个证明VPR通过选择性抑制病毒前MRNA剪接增强病毒感染的报告。较少的