HIV Molecular Biology and DNA Vaccine Approaches Against

本项目旨在了解HIV调节蛋白和辅助蛋白在病毒生命周期和疾病发展中的作用和机制;剖析大分子的核质运输机制;并研究Vpr作为转录激活剂和细胞周期调节剂的作用机制。这项工作的结论适用于设计更好的艾滋病疫苗的方法。 我们研究了几种HIV-1蛋白的功能，包括达特，Rev和Vpr。我们发现，在啮齿类动物细胞中的缺陷Rev是由于延迟输入Rev的核。负责这种影响的因素正在调查中，并可能导致针对基本Rev因素的新型干预措施。 一种新的CRM 1定向核输出抑制剂已被确定和表征。这种抑制剂将有助于研究Rev和许多通过CRM 1途径输出的细胞蛋白。 我们从小鼠基因组中鉴定出一种新的RNA元件（命名为RTE），它能够取代HIV-1的Rev和RRE，使用突变的HIV-1 DNA前病毒克隆作为新型分子陷阱。与这一要素有关的因素正在调查之中。这一结果表明，在基因组中存在额外的RNA元件，能够指导核输出，并可能定义一个独特的核质运输途径。 Vpr在病毒生命周期中的作用是复杂的，需要更详细地研究。虽然有几种细胞蛋白质与Vpr相互作用，但其功能机制尚未完全阐明。我们已经检测到Vpr的直接结合到其他细胞蛋白质，并研究了这种结合对细胞周期和Vpr的反式激活作用的影响。我们已经表明，Vpr直接与糖皮质激素和其他核受体相互作用，并发现在活性转录过程中与这些因子的复合物。我们还发现，Vpr直接结合到p300，许多细胞启动子的一个重要的辅激活因子。这种结合导致这些启动子的激活增加，并解释了Vpr的一些反式激活作用。 对HIV基因表达调控机制的理解已被应用于开发改进的DNA疫苗接种方法。我们已经开发了一种通用的方法，通过在其编码区引入多个点突变来增加不稳定mRNA的表达。这导致有效的运输，并增加几种mRNA的稳定性和翻译。基于我们对HIV的gag和env序列的研究结果，我们开发了用于基于DNA的免疫的有效表达载体。我们表明，更好的抗原表达导致免疫原性增加。这导致DNA疫苗在小鼠中有效表达并实现抗体、CTL和辅助应答的产生。相比之下，灵长类动物没有达到同样有效的免疫反应，表明物种之间的差异。我们开发了额外的DNA疫苗载体，产生修饰的抗原，以引发灵长类动物更有效的免疫应答。我们测试了几种表达分泌或细胞内降解抗原的此类载体，并显示某些组合似乎增强免疫原性。小鼠中的结果用于设计猴疫苗接种实验。我们致力于进一步优化疫苗载体以改进DNA疫苗接种的方法，旨在使用针对多种HIV抗原的载体组合进行有效的疫苗接种。

This project aims to understand the role and mechanism of function of HIV regulatory and accessory proteins in the virus life cycle and disease development; to dissect the mechanisms of nucleocytoplasmic trafficking of macromolecules; and to examine the mechanism of Vpr function as transcription activator and as cell cycle modulator. The conclusions of this work are applied towards the design of better vaccine approaches against AIDS. We study the function of several HIV-1 proteins, including Tat, Rev, and Vpr. We showed that the defect of Rev in rodent cells is caused by delayed import of Rev in the nucleus. The factors responsible for this effect are under investigation and may lead to new types of interventions targeted against the essential Rev factor. A new inhibitor of CRM1-directed nuclear export has been identified and characterized. This inhibitor will be useful for studies of Rev and many cellular proteins exported through the CRM1 pathway. We identified a new RNA element (named RTE) from the mouse genome that is able to replace the Rev and RRE of HIV-1, using a mutated HIV-1 DNA proviral clone as a new type of molecular trap. The factors binding to this element are under investigation. This result showed the presence of additional RNA elements in the genome that are able to direct nuclear export and may define a distinct pathway for nucleocytoplasmic transport. The role of Vpr on virus life cycle is complex and needs to be examined in more detail. Although several cellular proteins were shown to interact with Vpr, the mechanisms of its function are not fully elucidated. We have detected direct binding of Vpr to additional cellular proteins and have studied the effect of this binding on the cell cycle and on transactivator effects of Vpr. We have shown that Vpr interacts directly with the glucocorticoid and other nuclear receptors, and is found in complexes with these factors during active transcription. We also found that Vpr binds directly to p300, an important coactivator of many cellular promoters. This binding leads to increased activation of such promoters, and explains some of the transactivation effects of Vpr. The understanding of the regulatory mechanisms of HIV gene expression has been applied for the development of improved DNA vaccination approaches. We have developed a general method to increase the expression of unstable mRNAs by introducing multiple point mutations in their coding regions. This results in efficient transport, and increased stability and translation of several mRNAs. Based on our results with gag and env sequences of HIV, we developed efficient expression vectors for DNA-based immunization. We showed that better antigen expression results in increased immunogenicity. This led to DNA vaccines that are efficiently expressed and achieve the production of antibodies, CTL, and helper responses in mouse. In contrast, primates do not achieve an equally effective immune response, indicating differences among the species. We developed additional DNA vaccine vectors producing modified antigens in order to elicit more potent immune responses in primates. We tested several such vectors expressing either secreted or intracellularly degraded antigens and showed that some combinations appear to boost immunogenicity. Results in mice were used to design monkey vaccination experiments. We work on further optimization of the vaccine vectors to improve the methodology of DNA vaccination, aiming at efficient vaccination using combinations of vectors against multiple HIV antigens.