Characterization of cell clones resistant to HIV

描述(由申请人提供)：‘据估计，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，啮齿动物细胞仍然不能完全允许HIV复制，这表明还需要其他未知的宿主因素。我们对部分单倍体中国仓鼠卵巢(CHO)K1细胞株进行了诱变，用HIV-EGFP(VSV G)进行重复转导，然后进行负细胞分选，鉴定了大约40个对HIV高度抵抗但对小鼠白血病病毒(MLV)感染敏感的细胞克隆。被测试的子集的所有克隆也对猿猴免疫缺陷病毒(SIV)具有抗药性，并且该区块似乎不在转录水平上。在这个十几个克隆的子集中，表型似乎是隐性的，根据成对的细胞融合，存在四个互补组。在这项为期两年的应用中，我们希望进一步鉴定这些抗HIV的CHO克隆。在第一个目标中，我们将确定每个克隆的表型是隐性的还是显性的。对于隐性无性系，我们将进行配对融合，以确定互补组的总数。在第二个目标中，我们将测试来自每个互补组的代表性克隆和所有优势克隆，以确定对其他逆转录病毒和海绵病毒的敏感性，包括禽白血病病毒、人泡沫病毒、牛免疫缺陷病毒、马传染性贫血病毒、猫免疫缺陷病毒和SIV。在第三个目标中，将对相同的克隆进行测试，以确定阻止艾滋病毒复制的分子性质。早期和晚期逆转录产物、一个和两个LTR环以及前病毒整合子将被量化，病毒长末端重复序列的活性也将被量化。在这些研究完成后，一组特征良好的细胞系将免费提供，以进行更详细的生化或遗传互补研究。

DESCRIPTION (provided by applicant): ' By the end of this year, it has been 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. Rodent cells, however, are still not fully permissive for HIV replication, suggesting additional unknown host factors are required. We mutagenized the partially haploid Chinese hamster ovary (CHO) K1 cell line, performed repeated transductions using HIV-eGFP(VSV G) followed by negative cell sorting, and identified approximately 40 cell clones that are highly resistant to HIV and yet permissive to murine leukemia virus (MLV) infection. All clones of a tested subset were also resistant to simian immunodeficiency virus (SIV) and the block did not appear to be at the level of transcription. In this subset of a dozen clones the phenotype appeared to be recessive and four complementation groups were present, based upon pair-wise cell fusions. In this 2-year application we wish to further characterize these HIV-resistant CHO clones. In the first aim for each clone we will determine whether the phenotype is recessive or dominant. For the recessive clones we will perform pair-wise fusions to determine the overall number of complementation groups. In the second aim we will test representative clones from each complementation group and all dominant clones to determine susceptibility to other retroviruses and a spumavirus, including avian leucosis virus, human foamy virus, bovine immunodeficiency virus, equine infectious anemia virus, feline immunodeficiency virus, and SIV. In the third aim, the same clones will be tested to determine the molecular nature of the block to HIV replication. Early and late reverse transcription products, one and two- LTR circles, and proviral integrants will be quantified, as will the activity of the viral long terminal repeat. At the completion of these investigations, a well-characterized panel of cell lines will be freely available to perform more detailed biochemical or genetic complementation studies.