Study on functions of HEV-1 accessory gene Vpr

HIV-1Vpr是一种长度为96个氨基酸的病毒相关蛋白，具有多种生物学功能，包括其核定位活性、细胞周期G2/M期阻滞、细胞凋亡的正调控和负调控。Vpr对巨噬细胞中预整合复合体（pre - integration complex， PIC）的核输入至关重要，尽管Vpr在PIC进入机制中的作用尚不清楚。我们首先证明了Vpr是靶向核膜的，然后通过单独输入a以不依赖于输入13的方式转运到细胞核中。接下来，我们证明了加入巨噬细胞的胞浆提取物可以强烈促进Vpr的核输入，而不是单核细胞，并且胞浆提取物中输入蛋白q的免疫消耗会使核输入活性丧失。通过免疫印迹和实时PCR分析，未成熟单核细胞低水平表达输入蛋白a，而三种主要输入蛋白a亚型的表达在向巨噬细胞分化时显著增加，表明Vpr的核输入需要输入蛋白a的表达。此外，输入a和Vpr之间的相互作用不仅对于Vpr的核输入，而且对于巨噬细胞中的HIV-1复制都是必不可少的。最后，我们证明了Vpr与一个新的核输入过程之前的输入a的结合是治疗干预的潜在目标。另一方面，我们发现Vpr在体内和体外选择性抑制细胞前mrna剪接中的新作用。Vpr不仅通过与必需剪接因子SAP145的相互作用，还通过与功能性剪接体复合物的相互作用发挥这种作用。我们还发现Vpr的表达选择性地抑制HIV-1 mRNA的剪接，导致4kb形式的病毒mRNA的积累和2kb形式的减少，从而改变了相应蛋白的表达。此外，Vpr增强了病毒粒子相关蛋白的从头合成，增加了病毒的感染性。这是第一个证明Vpr通过选择性抑制病毒前mrna剪接来增强病毒传染性的报道。少

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