Probing blocks to infectious HIV release in mouse cells

描述(申请人提供)：世界卫生组织估计，到今年年底，大约0.7%的世界人口将是人类免疫缺陷病毒(HIV)血清阳性。大多数治疗都是针对抑制病毒逆转录酶或蛋白酶。尽管在过去的二十年里，关于艾滋病毒的复制周期和所涉及的细胞因素已经学到了很多，但在我们的知识中仍然存在空白，可以代表未来的治疗目标。例如，在小鼠的进入和进入后，通过表达人类CD4、趋化因子共受体和细胞周期蛋白T1，可以绕过HIV复制的障碍。然而，这些小鼠细胞仍然不能完全允许艾滋病毒复制，这可能是由于病毒复制的额外障碍。鼠与人的细胞融合产生了传染性病毒，这表明老鼠细胞缺乏复制艾滋病毒所需的一个或多个因素。我们已经分离了几个单染色体的鼠-人杂交细胞系，这些细胞系可以释放传染性病毒。这些细胞系并不是完全允许的，因为在与人类细胞融合后，病毒释放会进一步增加。我们现在寻求进一步探索小鼠细胞中复制块的性质。在第一个目标中，我们将利用相对宽松的鼠-人细胞与各种啮齿动物-人杂交细胞系进行融合实验，包括一组单染色体鼠-人细胞系，鼠-人微细胞杂交细胞系，以及一组特征良好的仓鼠-人辐射杂交细胞克隆。我们还将测试特定的候选基因，特别是那些与REV/RRE功能有关的基因。如果分离出更多的嵌合细胞克隆，使艾滋病毒产量增加，这些克隆将在特异性以及病毒mRNA和蛋白质产量方面得到进一步表征。第二个目标是对已经分离的人-鼠杂交细胞系进行染色体工程，这些细胞系允许释放艾滋病毒，并且只有一个人类染色体。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来完成，并识别具有整合到人类染色体中的载体的细胞克隆。转座酶的瞬时表达应该允许转座子‘跳’到顺式结构中的另一个位置，从而创建一组细胞克隆，其中loxP位点在人类染色体上可变地分离。通过添加Cre重组酶，间质染色体片段将被删除，并对产生的细胞克隆进行HIV释放测试。一旦感兴趣的染色体区域被减少到一个合理的区间，将使用更传统的方法来确定允许艾滋病毒释放的因素。随着这些研究的完成，希望能够更好地理解艾滋病毒释放所涉及的宿主要求，并为建立艾滋病毒的小鼠模型奠定更坚实的基础。 尽管在了解和治疗艾滋病毒方面取得了进展，但仍然没有针对该病毒的小动物模型。在小鼠体内，病毒复制有多个障碍，这些障碍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。然而，从小鼠细胞中释放出的艾滋病毒仍然很少。通过基因手段，我们已经分离出了几个可以释放相当数量病毒的人鼠杂交细胞系。这些只有一条人类染色体的细胞系不像人类细胞那样具有容许性。这项应用的目标是i)通过与各种其他杂交细胞系的细胞融合来进一步评估分离的细胞系，以确定可能参与HIV释放的其他人类染色体，以及ii)使用一种新的染色体工程方法来确定相对允许的细胞系中的负责基因。在这项研究结束时，希望能识别出参与病毒生命周期的新的人类基因，这些基因可能成为药物靶标，并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.