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）：世界卫生组织估计，到今年年底，世界上大约 0.7% 的人口将出现人类免疫缺陷病毒 (HIV) 血清反应阳性。大多数疗法针对抑制病毒逆转录酶或蛋白酶。尽管在过去的二十年里，我们对艾滋病毒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，在小鼠中，通过表达人CD4（一种趋化因子辅助受体）和细胞周期蛋白T1，可以避免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艾滋病毒复制，这可能是由于对病毒复制的额外阻碍。小鼠与人类细胞融合产生感染性病毒，表明小鼠细胞缺乏艾滋病毒复制所需的一种或多种因子。我们分离出了几种可以释放传染性病毒的单染色体小鼠-人杂交细胞系。这些细胞系并不完全允许，因为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块的性质。在第一个目标中，我们将利用相对宽松的小鼠-人细胞与多种啮齿动物-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小鼠-人杂交细胞系。一组经过充分表征的仓鼠-人类辐射杂交细胞克隆。我们还将测试特定的候选基因，特别是那些涉及 Rev/RRE 功能的基因。如果分离出额外的嵌合细胞克隆来增加 HIV 的产量，这些克隆将在特异性以及病毒 mRNA 和蛋白质的产量方面得到进一步的表征。第二个目标是对已经分离的小鼠-人类杂交细胞系进行染色体工程，这些细胞系允许艾滋病毒释放并且只有一条人类染色体。这将首先通过使用包含 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”到顺式的另一个位置，从而产生一组细胞克隆，其LoxP位点在人类染色体上不同地分开。通过添加 Cre 重组酶将删除间质染色体片段，并测试所得细胞克隆的 HIV 释放情况。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艾滋病毒释放的因素。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，为艾滋病毒小鼠模型奠定更坚实的基础。 尽管在认识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。在小鼠中，病毒复制存在多种障碍，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。小鼠细胞释放的艾滋病毒仍然很少。通过遗传手段，我们分离出了几种释放适量病毒的小鼠-人类杂交细胞系。这些细胞系只有一条人类染色体，不像人类细胞那样受到限制。本申请的目标是 i) 通过与多种其他杂交细胞系进行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) 使用染色体工程的新方法鉴定相对允许的细胞系中的负责基因。在这项研究结束时，希望能够识别出参与病毒生命周期的新人类基因，这些基因可以作为药物靶标，并有助于建立艾滋病毒小动物模型。