USE OF ACCESSORY GENE MUTANTS FOR THE DEVELOPMENT OF ATTENUATED HIV VACCINES

本项目的长期目标是：1)评估可行性 生产HIV-1和HIV-2的减毒活病毒疫苗 由于辅助基因突变而变得非致病的 单独或结合在一起；以及2)探索 辅助基因蛋白可以作为抗病毒治疗的靶点。 最近使用SIV系统的研究表明，病毒在 Nef的产生对猴子是非致病的，并提供保护 对那些来自随后携带野生型SIV的猴子的挑战。突变 在其他辅助基因中，HIV-1的vif、vpr、vPU和vif、vpr、vPU HIV-2的VPX尚未在动物模型系统中进行探索。AS 两种候选减毒活病毒研制的先决条件 针对艾滋病的疫苗和抗HIV药物的开发 辅助基因产物，我们一直在进行研究，以确定 这些蛋白在HIV-1和HIV-2体外生命周期中的作用 因为对它们如何运作的了解对这两个目标都至关重要。我们的 早期的研究已经证明了HIV-1 Vif对病毒的关键作用 在初级T细胞(外周血单核细胞， PBMC)，尽管该蛋白不是HIV-1在体内复制所必需的 大多数CD4阳性细胞系。我们继续表明，Vif也 在原代单核细胞来源的HIV-1复制中起关键作用 巨噬细胞(MDM)。在Nef的案例中，一直存在争议，因为 这种蛋白质对病毒的生长是积极的还是消极的影响。 我们已经证明了Nef对病毒是否有可测量的影响 复制取决于使用的特定病毒主机系统。而Nef 几种HIV-1毒株的突变体的复制能力都略低于 在PBMC中为野生型，在嗜巨噬细胞HIV-1的情况下，在MDM中， 病毒复制可能没有影响，也可能大幅减少 在CD4阳性细胞中检测HIV-1和HIV-2的Nef突变 台词。对于VPR，我们已经表明， 在HIV-1和HIV-2中突变该基因以在CD4阳性中复制 细胞系或PBMC，尽管这种蛋白似乎是一个积极的因素 以促进MDM的增长。我们已展开研究，以调查 Nef和Vpr的分子机制。到目前为止的结果表明， 这些蛋白质在几种哺乳动物细胞中的表达导致 停止细胞生长(细胞停滞)，我们目前正在研究 导致这一现象的细胞通路。

The long-term goals of this project are: 1) to evaluate the feasibility of generating live attenuated virus vaccines of HIV-1 and HIV-2 that are rendered non-pathogenic by mutation of accessory genes, either individually or in combination; and 2) to explore the possibility that the accessory gene proteins can be targets for anti-viral therapy. Recent studies using the SIV system have shown that viruses defective in the production of Nef are non-pathogenic in monkeys and confer protection to those monkeys from subsequent challange with wild-type SIV. Mutations in the other accessory genes, vif, vpr and vpu of HIV-1 and vif, vpr and vpx of HIV-2, have yet to be explored in animal model systems. As prerequisites to both the development of candidate live attenuated virus vaccines and the development of anti-HIV drugs directed against the accessory gene products, we have been engaged on studies to determine the role of these proteins in the life cycle of HIV-1 and HIV-2 in vitro, since a knowledge of how they function is critical to both goals. Our earlier work had demonstrated the critical role of HIV-1 Vif to virus replication in primary T cells (peripheral blood mononuclear cells, PBMC), although this protein is not obligatory for HIV-1 replication in most CD4-positive cell lines. We have gone on to show that Vif also plays a critical role for HIV-1 replication in primary monocyte-derived macrophages (MDM). In the case of Nef, there has been a controversy as to whether this protein positively or negatively influences virus growth. We have shown that whether or not Nef has a measurable effect on virus replication depends on the particular virus-host system used. While Nef mutants of several HIV-1 strains all replicate slightly less well than wild type in PBMC and, in the case of a macrophage-tropic HIV-1, in MDM, there can be either no effect or dramatic reductions in virus replication when Nef mutants of HIV-1 and HIV-2 are assayed in CD4-positive cell lines. For Vpr, we have shown that there is little consequence of mutating this gene in HIV-1 and HIV-2 for the replication in CD4-positive cell lines or PBMC, although this protein appears to be a positive factor for growth in MDM. We have commenced studies to investigate the molecular mechanism of Nef and Vpr. Results so far have indicated that expression of these proteins in several mammalian cells results in the cessation of cell growth (cytostasis), and we are currently investigating the cellular pathways that bring this about.