PATHOGENIC MECHANISMS OF HIV

实验室的一个中心项目是了解艾滋病毒表达和致病性的机制，并将这些信息应用于开发新的治疗和疫苗方法。我们研究了原代细胞中细胞因子、趋化因子和HIV受体的复杂相互作用；HIV辅助蛋白VPR作为病毒共激活因子的作用；以及CCR5基因的调节，CCR5基因是人类感染HIV必不可少的受体。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。我们已经证明了HIV-1VPR蛋白是核受体的转录辅助激活因子。这一发现解释了VPR对几个病毒和细胞基因表达的混杂激活效应。我们发现VPR直接与p300/CBP结合，p300/CBP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。我们在趋化因子受体CCR5的基因中发现了CCAAT增强子结合蛋白(C/EBPB)的多个结合位点，提示C/EBPB可能参与了CCR5的调控。RNA分析显示，与健康献血者相比，HIV-1感染者循环T淋巴细胞中C/EBPB的表达增加了5倍。同样，对PBMCs中CCR5表达的分析表明，与健康献血者相比，HIV-1感染者的CCR5+T淋巴细胞数量增加。CCR5+T淋巴细胞在疾病的各个阶段均有升高，并与CD_4细胞下降有关。我们的结果表明，C/EBPB是CCR5表达的重要调节因子，可能在HIV疾病的发病机制中发挥相关作用。我们研究了核糖核酸核出口的替代机制。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，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。我们还启动了对新的HIV重组体的研究，以了解与HIV亚型重组相关的复杂的流行病学问题。改进的艾滋病DNA疫苗我们已经开发出用于艾滋病DNA疫苗接种的改进疫苗载体。结果表明，DNA载体的免疫原性增强，肌肉注射DNA后，Gag DNA具有很强的免疫原性。我们还表明，GAG抗原朝向分泌途径的方向会导致免疫反应的增强。我们继续改进DNA疫苗接种的方法，旨在利用针对多种HIV抗原的载体组合进行有效的疫苗接种。动物模型我们已经开发出一种系统，可以通过Cre-loxP重组系统将任何感兴趣的基因引入到预先确定的基因组位置，在那里基因表达受到严格调控。在胚胎干细胞中发现了允许严格控制基因表达的染色体位点，并通过从胚胎干细胞中培育出小鼠并与含有四环素控制的反式激活剂(TTA)的小鼠杂交，对其在许多组织中的表达进行了广泛的分析。只有少数基因座表现出较低的基础活性和高达1000倍的诱导性。一般来说，诱导性与TTA在不同组织中的表达水平相关。为了进一步提高基因调控的严密性，将鸡b-珠蛋白基因的绝缘子序列引入到同一基因组中。绝缘体的存在进一步将荧光素酶指示剂的基本活性降低到每个细胞不到一个荧光素酶分子。该系统允许对基因转移后的表达进行严格的调控，是研究转基因动物基因功能的宝贵工具。

A central project in the laboratory is the understanding of the mechanisms of HIV expression and pathogenicity and the application of this information to the development of new therapeutic and vaccine approaches. We have studied the complex interactions of cytokines, chemokines, and HIV receptors in primary cells; the role of the HIV accessory protein Vpr as a virus coactivator; and the regulation of the CCR5 gene, a receptor essential for HIV infection in humans. 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. We have demonstrated that the HIV-1 Vpr protein is a transcriptional coactivator of nuclear receptors. This finding explains the promiscuous activating effects of Vpr on the expression of several viral and cellular genes. We found that Vpr binds directly to p300/CBP, an important coactivator of many cellular promoters. This binding leads to increased activation of such promoters. We identified multiple binding sites for CCAAT-enhancer-binding protein (C/EBPb) within the gene for chemokine receptor CCR5, suggesting that C/EBPb may be involved in CCR5 regulation. RNA analysis demonstrated a 5-fold increase in C/EBPb expression in circulating T-lymphocytes from HIV-1-infected individuals compared with healthy blood donors. Similarly, analysis of CCR5 expression in PBMCs demonstrated increased numbers of CCR5+ T lymphocytes in HIV-1-infected individuals as compared with healthy blood donors. The increase in CCR5+ T lymphocytes was found in all stages of the disease and correlated with the decline in CD4 counts. Our results suggest that C/EBPb is an important regulator of CCR5 expression and may play a relevant role in the pathogenesis of HIV disease. We have studied alternative mechanisms for the nuclear export of mRNAs. We identified a new RNA element 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. We have also initiated the study of new HIV recombinants to understand the complex epidemiologic questions associated with HIV subtype recombination. Improved DNA vaccines against AIDS We have developed improved vaccine vectors for DNA vaccination against AIDS. We showed that improved expression leads to better immunogenicity of DNA vectors and also that gag DNA is very immunogenic after intramuscular DNA injection. We also showed that direction of the gag antigen towards the secretory pathway results in increased immune response. We continue to improve the methodology of DNA vaccination, aiming at efficient vaccination using combinations of vectors against multiple HIV antigens. Animal models We have developed a system to introduce any gene of interest by the Cre-loxP recombination system into predetermined genomic loci where gene expression is tightly regulated. Chromosomal loci that permit tight control of gene expression were identified in embryonic stem (ES) cells and analyzed extensively for expression in many tissues by creating mice from ES cells and breeding them with mice containing tetracycline-controlled transactivator (tTA). Only a small number of loci showed low basal activity and up to a 1000-fold inducibility. In general, inducibility correlated with tTA expression levels in different tissues. To further improve the stringency of gene regulation, the insulator sequence of chicken b-globin locus was introduced into the same genomic loci. The presence of insulator further reduced the basal activity of the luciferase indicator to less than one luciferase molecule per cell. This system allows the tight regulation of expression after gene transfer and is a valuable tool for gene function studies in transgenic animals.