Identification of Host Factor(s) Involved in HIV Release

描述（由申请人提供）：到今年年底，世界卫生组织估计，大约0.7%的世界人口将对人类免疫缺陷病毒（HIV）呈血清阳性。这些人大多生活在发展中国家，无法获得高效抗逆转录病毒疗法。一种安全有效的疫苗可能需要十年的时间。大多数治疗都是针对抑制病毒逆转录酶或蛋白酶。 虽然在过去的二十年里，我们已经了解了很多关于艾滋病毒的复制周期和所涉及的细胞因子，但我们的知识仍然存在差距，可能代表未来的治疗目标。例如，在小鼠中，通过表达人CD 4（一种趋化因子辅助受体）和细胞周期蛋白T1，可以规避对HIV复制的进入和进入后阻断。然而，这些小鼠细胞仍然不能完全允许HIV复制，这可能是由于Gag处理过程中的缺陷。小鼠-人细胞融合产生感染性病毒，表明小鼠细胞缺乏HIV复制所需的一种或多种因子。该提案寻求通过遗传手段来识别新的宿主 细胞因子，可能促进艾滋病毒的释放，使用不良的许可啮齿动物细胞作为模型。 在第一个目标中，将使用遗传筛选来识别这样的缺失因子。已基于HIV开发了cDNA文库表达载体，其具有高滴度并具有选择标记。将其引入表达细胞周期蛋白T1（cycT 1）的小鼠细胞中。通过提供反式功能回收感染性病毒，滴定并用于重新培养小鼠cycT 1细胞。然后重复这个循环，希望扩增和富集促进小鼠中HIV复制的cDNA。在第二个目标中，将常规的基于质粒的cDNA文库引入小鼠细胞（已经表达cycT 1并且用编码荧光素酶和选择标记的HIV载体转导）。将细胞分成池，并通过与目的1相似的方法从每个池中回收感染性病毒。支持最高程度的HIV释放的细胞克隆将通过同胞选择分离。为了这一目的和前述目的，将通过PCR回收来自单个克隆的cDNA，通过DNA测序表征，并重新引入小鼠细胞中以确定小鼠细胞随后是否完全允许HIV复制。在第三个目标中，斯坦福大学小组的辐射杂交（RH）细胞系将单独转导的复制缺陷型HIV载体编码cycT 1和标记。通过以下方式从每个RH中回收感染性病毒： 方法类似于目标1。最有希望的RH将被测试其拯救HIV Gag加工和支持野生型M嗜性HIV复制的能力，类似于前两个目标。该RH面板应允许功能遗传图谱小于1 Mb。从这一目标，它是希望，一个合理的近似的数量（和染色体位置）的缺失的主机因素将获得，这将与前两个目标的结果。如果时间允许，将测试来自最感兴趣区域的候选基因介导小鼠细胞释放HIV的能力。在完成这些研究后，希望能够更好地了解艾滋病毒释放所涉及的宿主因素。

DESCRIPTION (provided by applicant): By the end of this year, the World Health Organization has estimated that approximately 0.7% of the world's population will be seropositive for human immunodeficiency virus (HIV). Most of these individuals live in the developing world and do not have access to highly active anti-retroviral therapy (HAART). A safe and efficacious vaccine may be a decade away. Most therapy is directed towards inhibition of viral reverse transcriptase or protease. Although over the last two decades much has been learned regarding the replicative cycle of HIV and the cellular factors involved, there are still gaps in our knowledge that could represent future therapeutic targets. For example, in the mouse entry and post-entry blocks to HIV replication have been circumvented by expressing human CD4, a chemokine co-receptor, and cyclin T1. These mouse cells, however, are still not fully permissive for HIV replication, perhaps due to a defect in Gag processing. Mouse-human cell fusions produce infectious virus, suggesting that mouse cells lack one or more factors required for HIV replication. This proposal seeks by genetic means to identify novel host cellular factors that may facilitate HIV release, using poorly permissive rodent cells as a model. In the first aim, a genetic screen will be used to identify such a missing factor(s). A cDNA library expression vector has been developed based upon HIV which is of high titer and has a selectable marker. This will be introduced into mouse cells expressing cyclin T1 (cycT1). Infectious virus will be recovered by providing trans functions, titered, and used to re-transduce na'fve mouse.cycT1 cells. This cycle will then be repeated in the hopes of amplifying and enriching for cDNAs that facilitate HIV replication in the mouse. In the second aim, a conventional plasmid-based cDNA library will be introduced into mouse cells (already expressing cycT1 and transduced with an HIV vector encoding both luciferase and a selectable marker). Cells will be divided into pools, and infectious virus recovered from each pool by methods similar to aim 1. Cell clones supporting the highest degree of HIV release will be isolated by sib selection. For both this and the preceding aim, cDNAs from individual clones will be recovered by PCR, characterized by DNA sequencing, and re-introduced into mouse cells to determine if the mouse cells are then rendered fully permissive for HIV replication. In the third aim, the Stanford panel of radiation hybrid (RH) cell lines will be individually transduced with a replication-defective HIV vector encoding cycT1 and a marker. Infectious virus will be recovered from each RH by methods similar to aim 1. The most promising RHs will be tested for their ability to rescue HIV Gag processing and support wild-type M-tropic HIV replication similar to the first two aims. This RH panel should allow functional genetic mapping to less than 1 Mb. From this aim it is hoped that a reasonable approximation of the number (and chromosomal location) of missing host factors will be obtained and this will be correlated with the results from the first two aims. Time permitting, candidate genes from the region of greatest interest will be tested for their ability to mediate HIV release from mouse cells. At the completion of these studies it is hoped that a better understanding of the host factors involved in HIV release will be achieved.