Probing blocks to infectious HIV release in mouse cells

探究小鼠细胞中感染性艾滋病毒释放的阻断

• 批准号: 7750034
• 负责人: Richard Sutton
• 金额: $ 40.96万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7750034
• 关键词: Animal Model; Budgets; Candidate Disease Gene; Cell Line; Cell fusion; Cells; Chromosome Deletion; Chromosomes; Clone Cells; Data; Drug Delivery Systems; Engineering; Eye; Frequencies; Future; Genes; Genetic; Genetic Transcription; Goals; HIV; Hamsters; Human; Human Cell Line; Human Chromosomes; Human immunodeficiency virus test; Humulus; Hybrid Cells; Integration Host Factors; Knowledge; Learning; Life Cycle Stages; Location; Maps; Messenger RNA; Methods; Mus; Nature; Partner in relationship; Peptide Hydrolases; Phenotype; Population; Production; Proteins; Published Comment; RNA-Directed DNA Polymerase; Radiation Hybrid; Research Personnel; Retroviral Vector; Retroviridae; Rodent; Site; Sleeping Beauty; Specificity; System; Testing; Text; Time; Transposase; Viral; Virus; Virus Replication; World Health Organization; cellular transduction; chemokine; cyclin T1; design; embryonic stem cell; experience; foot; high reward; high risk; interest; interstitial; mouse model; murine retroviral vector; receptor; recombinase; research study; therapeutic target; vector

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.

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。大多数治疗是针对病毒逆转录酶或蛋白酶的抑制。尽管在过去的二十年里，人们对HIV的复制周期和所涉及的细胞因素有了很多了解，但我们对未来治疗目标的了解仍然存在差距。例如，在小鼠体内，通过表达人类CD4（一种趋化因子共受体）和细胞周期蛋白T1，可以规避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-人类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。我们已经分离出几种允许传染性病毒释放的单染色体小鼠-人类杂交细胞系。这些细胞系不是完全允许的，因为在与人类细胞融合后，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，我们将使用相对宽松的小鼠-人类细胞与各种啮齿动物-人类杂交细胞系进行融合实验，包括一组单染色体小鼠-人类细胞系，小鼠-人类微细胞杂交细胞系，以及一组具有良好特征的仓鼠-人类辐射杂交细胞克隆。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。如果进一步分离出允许增加HIV产生的嵌合细胞克隆，这些将在特异性和病毒mRNA和蛋白质产生方面进一步表征。第二个目标集中在已经分离的小鼠-人类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在内的逆转录病毒载体对细胞进行转导，并识别将载体整合到人类染色体中的细胞克隆来实现。转座酶的瞬时表达应该允许转座子“跳跃”到顺式中的另一个位置，从而创建一个具有人类染色体上可变分离的LoxP位点的细胞克隆面板。通过添加Cre重组酶来删除间质染色体片段，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减少到一个合理的间隔，将使用更传统的方法来确定允许HIV释放的因素。在完成这些研究后，希望能够更好地了解HIV释放过程中涉及的宿主需求，为HIV小鼠模型的建立奠定坚实的基础。叙述