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: 7996613
• 负责人: Richard Sutton
• 金额: $ 40.55万
• 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/7996613
• 关键词: Affect; Animal Model; Candidate Disease Gene; Cell Line; Cell fusion; Cells; Chromosomes; Clone Cells; Data; Drug Delivery Systems; Engineering; Future; Genes; Genetic; Genetic Recombination; 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; Mediating; Messenger RNA; Methods; Molecular Genetics; Mus; Nature; Peptide Hydrolases; Population; Production; Proteins; RNA; RNA-Directed DNA Polymerase; Radiation Hybrid; Retroviral Vector; Rodent; Site; Sleeping Beauty; Small Interfering RNA; Specificity; Testing; Transposase; Viral; Virus; Virus Assembly; Virus Replication; World Health Organization; autosome; cellular transduction; chemokine; cyclin T1; foot; interest; interstitial; mouse model; murine retroviral vector; novel; receptor; recombinase; research study; therapeutic target; vector; viral RNA

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.

世界卫生组织估计，到今年年底， 世界人口将对人类免疫缺陷病毒（艾滋病毒）呈血清反应阳性。大多数治疗都是 涉及抑制病毒逆转录酶或蛋白酶。虽然在过去的两年里 几十年来，人们对艾滋病毒的复制周期和所涉及的细胞因子已经了解了很多， 在我们的知识中仍然存在空白，这些空白可能代表未来的治疗目标。例如在 通过表达人源化的抗HIV抗体，小鼠进入和进入后阻断HIV复制已经被规避， CD 4，一种趋化因子共受体，和细胞周期蛋白T1。然而，这些小鼠细胞仍然没有完全 允许HIV复制，可能是由于病毒复制的额外阻断。鼠-人细胞 融合产生感染性病毒，表明小鼠细胞缺乏HIV所需的一种或多种因子， 复制的我们已经分离出几个单染色体小鼠-人杂交细胞系， 传染性病毒释放这些细胞系不是完全允许的，因为存在进一步的增加。 与人体细胞融合后释放病毒。我们现在试图进一步探索复制的本质 在小鼠细胞中阻断。 在第一个目标中，我们将使用相对允许的小鼠-人细胞进行融合 用各种啮齿动物-人杂交细胞系进行的实验，包括一组单染色体的 小鼠-人细胞系、小鼠-人微细胞杂交细胞系和一组充分表征的 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 参与Rev/RRE功能的人员。如果分离出额外的嵌合细胞克隆， 艾滋病毒的生产，这些将进一步表征方面的特异性和病毒的mRNA和蛋白质 生产 第二个目标是集中在已经分离的小鼠-人的染色体工程 允许HIV释放且仅具有单个人类染色体的杂交细胞系。这 将首先通过用包括LoxP位点的逆转录病毒载体转导细胞来完成， 转座子睡美人和鉴定具有整合到人类中的载体的细胞克隆 染色体转座酶的瞬时表达应该允许转座子"跳“到另一个转座子上， 顺式定位，从而产生一组具有在人类上间隔开的LoxP位点的细胞克隆。 染色体间质染色体区段将通过添加Cre重组酶而缺失，并且 测试所得细胞克隆的HIV释放。一旦感兴趣的染色体区域已经被 减少到一个合理的区间，将使用更传统的方法来确定因素， 艾滋病毒释放在完成这些研究后，希望能对主持人有更好的了解 将实现艾滋病毒释放所涉及的要求，为建立小鼠模型奠定更坚实的基础。 艾滋病。