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，剖析大分子核质运输的机制，并研究VPR作为转录激活因子和细胞周期调节因子的作用机制。 我们研究了几种HIV-1蛋白的功能，包括Tat、Rev和Vpr。我们发现，啮齿动物细胞中REV的缺陷是由于REV在细胞核中延迟输入所致。造成这一效果的因素正在调查中，可能会导致针对基本REV因素的新型干预措施。 已经确定并表征了一种新的CRM1定向核出口的抑制剂。这种抑制物对REV和许多通过CRM1途径输出的细胞蛋白的研究是有用的。 我们利用突变的HIV-1DNA前病毒克隆作为一种新型的分子陷阱，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。与这一元素有关的因素正在调查中。这一结果表明，基因组中存在能够直接进行核输出的额外的RNA元件，并可能定义一条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。虽然已有多种细胞蛋白与VPR相互作用，但其作用机制尚未完全阐明。我们已经检测到VPR与其他细胞蛋白的直接结合，并研究了这种结合对细胞周期和VPR反式激活效应的影响。我们已经证明，VPR直接与糖皮质激素和其他核受体相互作用，并在激活转录过程中发现与这些因子的复合体。我们还发现VPR直接与p300结合，p300是许多细胞启动子的重要共激活因子。这种结合导致这些启动子的激活增加，并解释了VPR的一些反式激活效应。 对HIV基因表达调控机制的了解已被用于开发改进的DNA疫苗接种方法。我们开发了一种通用的方法来增加不稳定的mRNAs的表达，方法是在其编码区引入多个点突变。这导致了有效的运输，并增加了几个mRNAs的稳定性和翻译。基于我们对HIV的Gag和env序列的研究结果，我们构建了高效的DNA免疫表达载体。我们发现，更好的抗原表达可以提高免疫原性。这导致了DNA疫苗的高效表达，并在小鼠体内实现了抗体、CTL和辅助反应的产生。相比之下，灵长类动物没有达到同样有效的免疫反应，这表明了物种之间的差异。我们开发了额外的DNA疫苗载体，产生修饰的抗原，以便在灵长类动物中诱导更有效的免疫反应。我们测试了几个这样的载体，表达分泌或细胞内降解的抗原，并显示一些组合似乎提高了免疫原性。结果以小鼠为实验对象，设计猕猴疫苗接种实验。我们致力于进一步优化疫苗载体，以改进DNA疫苗接种的方法学，旨在利用多种载体的组合对多种HIV抗原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. 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.