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: 7417731
• 负责人: Richard Sutton
• 金额: $ 14.15万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 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 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7417731
• 关键词: Affect; Animal Model; Candidate Disease Gene; Cell Line; Cell fusion; Cell hybridization; Cells; Chromosomes; Clone Cells; Data; Drug Delivery Systems; Endopeptidases; 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; Score; 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

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）具有血清阳性。大多数疗法用于抑制病毒逆转录酶或蛋白酶。尽管在过去的二十年中，关于艾滋病毒的复制周期和所涉及的细胞因素已经学习了很多东西，但我们的知识中仍然存在一些可以代表未来治疗靶标的差距。例如，通过表达人CD4（趋化因子共受体和细胞周期蛋白T1）来规避在小鼠进入和进入HIV复制后的块中。但是，这些小鼠细胞仍然无法完全允许HIV复制，这可能是由于病毒复制的其他障碍所致。小鼠人类细胞融合会产生传染病，表明小鼠细胞缺乏HIV复制所需的一个或多个因素。我们已经分离了几种单色小鼠 - 人类杂化细胞系，这些细胞允许传染病释放。这些细胞系并不能完全允许，因为与人类细胞融合后，病毒释放进一步增加。现在，我们寻求进一步探索小鼠细胞中复制块的性质。在第一个目的中，我们将使用相对允许的小鼠 - 人类细胞来使用各种啮齿动物 - 人类杂种细胞系进行融合实验，包括一组单色小鼠小鼠 - 人 - 人类细胞系，小鼠 - 人类微电池杂交细胞系，以及一组良好的特征性的Hamster-Hamster-Human Human Human Human Human hybrid Cell-Cellone。我们还将测试特定的候选基因，尤其是参与REV/RRE功能的基因。如果分离出允许增加HIV产生的其他嵌合细胞克隆，则将以特异性和病毒mRNA和蛋白质产生的方式进一步表征。第二个目的是集中在已经孤立的小鼠杂化细胞系的染色体工程上，这些杂化细胞系具有HIV释放，并且只有一个人类染色体。这将首先通过用逆转录病毒载体传递包括LOXP位点和转座子睡美和识别具有载体整合到人类染色体中的细胞克隆的细胞来实现。转座酶的瞬时表达应允许转座子“跳”到CIS中的另一个位置，从而形成一组细胞克隆，其中LOXP位点在人类染色体上可变。通过添加CRE重组酶，将删除间质染色体片段，并测试所得的细胞克隆释放HIV。一旦将目的的染色体区域降低到合理的间隔，将使用更多常规的方法来识别允许释放HIV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，并更坚定地迈向艾滋病毒的鼠标模型。 尽管在理解和治疗HIV方面取得了进展，但该病毒仍然没有小动物模型。在小鼠中，病毒复制有多个块，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。小鼠细胞仍然释放了很少的艾滋病毒。通过遗传方式，我们已经分离了几种释放合理量病毒的小鼠 - 人类杂化细胞系。这些仅具有单个人类染色体的细胞系不像人类细胞那样允许。该应用的目标是i）进一步评估与其他各种杂化细胞系的细胞融合，以识别可能参与HIV释放的其他人类染色体，ii）使用一种新的染色体工程方法来识别相对允许细胞系中的负责任基因。在这项研究结束时，希望将确定参与病毒生命周期的新人类基因可以作为药物靶标，还可以帮助建立艾滋病毒的小动物模型。