Probing blocks to infectious HIV release in mouse cells

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。大多数治疗针对病毒逆转录酶或蛋白酶的抑制。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完全允许HIV复制，可能是由于病毒复制的额外阻断。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。我们已经分离出几个单染色体小鼠-人杂交细胞系，允许感染性病毒的释放。这些细胞系不是完全允许的，因为与人细胞融合后病毒释放进一步增加。我们现在寻求进一步探索小鼠细胞中复制阻滞的本质。在第一个目标中，我们将采用相对宽容的小鼠-人细胞与各种啮齿动物-人杂交细胞系进行融合实验，包括一组单染色体小鼠-人细胞系，小鼠-人微细胞杂交细胞系，和一组良好表征的仓鼠-人辐射杂交细胞克隆。我们还将测试特定的候选基因，特别是那些参与Rev/RRE功能的基因。如果分离出允许增加HIV产生的额外嵌合细胞克隆，则这些将在特异性和病毒mRNA和蛋白质产生方面进一步表征。第二个目标是集中在已经分离的小鼠-人杂交细胞系的染色体工程上，这些细胞系允许HIV释放，并且只有一条人类染色体。这将首先通过用包括LoxP位点和转座子睡美人的逆转录病毒载体转导细胞并鉴定具有整合到人染色体中的载体的细胞克隆来实现。转座酶的瞬时表达应该允许转座子顺式“跳跃”到另一个位置，从而产生一组具有在人类染色体上分开的LoxP位点的细胞克隆。通过加入Cre重组酶删除间质染色体片段，并测试所得细胞克隆的HIV释放。一旦感兴趣的染色体区域被减少到合理的区间，将使用更传统的方法来识别允许HIV释放的因素。在这些研究完成后，希望能够更好地了解艾滋病毒释放所涉及的宿主要求，为建立艾滋病毒小鼠模型奠定更坚实的基础。叙述 尽管在了解和治疗艾滋病毒方面取得了进展，但仍然没有这种病毒的小动物模型。在小鼠中，病毒复制存在多个阻断，这可以通过提供细胞进入和转录所需的宿主因子来部分克服。仍然很少有艾滋病毒从小鼠细胞中释放出来。通过遗传学手段，我们已经分离出几种小鼠-人杂交细胞系，这些细胞系释放了合理数量的病毒。这些细胞系只有一条人类染色体，不像人类细胞那样宽容。本申请的目的是i）通过与多种其他杂交细胞系的细胞融合进一步评价分离的细胞系，以鉴定可能参与HIV释放的其他人类染色体，和ii）使用染色体工程的新方法来鉴定相对允许的细胞系中的负责基因。在这项研究的最后，希望能够鉴定出参与病毒生命周期的新的人类基因，这些基因可能作为药物靶点，也有助于建立艾滋病毒的小动物模型。

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 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, likely due to additional blocks to viral replication. Mouse-human cell fusions produce infectious virus, suggesting that mouse cells lack one or more factors required for HIV replication. We have isolated several monochromosomal mouse-human hybrid cell lines that allow infectious virus release. These cell lines are not fully permissive in that there is a further increase in virus release after fusion with human cells. We now seek to further explore the nature of the replicative block in mouse cells. In the first aim we will employ the relatively permissive mouse-human cells to perform fusion experiments with a variety of rodent-human hybrid cell lines, including a panel of monochromosomal mouse-human cell lines, mouse-human microcell hybrid cell lines, and a set of well-characterized hamster-human radiation hybrid cell clones. We will also test specific candidate genes, especially those involved in Rev/RRE function. If additional chimeric cell clones are isolated that allow increased HIV production, these will be further characterized in terms of specificity and viral mRNA and protein production. The second aim is focused on chromosome engineering of the already isolated mouse-human hybrid cell lines that are permissive for HIV release and only have a single human chromosome. This will be accomplished first by transducing the cells with a retroviral vector that includes LoxP sites and the transposon Sleeping Beauty and identifying cell clones that have vector integrated into the human chromosome. Transient expression of the transposase should allow the transposon to `hop' to another location in cis, thus creating a panel of cell clones with the LoxP sites variably separated on the human chromosome. Interstitial chromosomal segments will be deleted by addition of Cre recombinase, and the resulting cell clones tested for HIV release. Once the chromosomal region of interest has been reduced to a reasonable interval, more conventional approaches will be used to identify factors allowing HIV release. At the completion of these studies it is hoped that a better understanding of the host requirements involved in HIV release will be achieved, with firmer footing towards a mouse model of HIV.Narrative Despite the progress made in understanding and treatment of HIV, there is still no small animal model for the virus. In mouse, there are multiple blocks to virus replication, which can be partially overcome by provision of host factors required for cell entry and transcription. Still little HIV is released from mouse cells. By genetic means we have isolated several mouse-human hybrid cell lines that release a reasonable amount of virus. These cell lines, which have only a single human chromosome, are not as permissive as human cells. The goals of this application are to i)further evaluate the isolated cell lines by cell fusions with a variety of other hybrid cell lines to identify other human chromosomes that might be involved in HIV release, and ii) to use a new method of chromosome engineering to identify the responsible genes in the relatively permissive cell lines. At the end of this study it is hoped that new human genes involved in the virus life cycle will be identified that may serve as drug targets and also help establish a small animal model of HIV.