Identification of Host Genes for Retroviral Replication

描述(由申请人提供)：对模式生物酿酒酵母的细胞周期、核运输、RNA加工、染色质结构、细胞骨架和囊泡运输的研究为后生动物中类似系统的表征提供了范例。我们和其他人已经将酿酒酵母中的Ty逆转录转座子描述为逆转录病毒的模型。我们最近使用高通量筛选方法鉴定了大约300个基因，这些基因的表达影响酵母逆转录病毒样元件Ty3的逆转录转座。这些基因包括与DNA修复、RNA加工、蛋白质折叠、脂肪代谢和囊泡运输相关的基因。在酵母系统中，我们正在相当详细地研究其中少数基因的作用机制。在这里，我们建议调查19个基因的子集对一种简单的哺乳动物逆转录病毒Moloney鼠白血病病毒复制的影响。 这一应用的目的是：1)目的基因功能将被shRNA干扰。候选基因的选择将基于几个标准，包括是否存在非必要的小鼠同源基因，缺陷的严重性，以及Ty1 a逆转录转座子与Ty3的关系比逆转录病毒更远的转座可能存在的缺陷。这些基因的活性将被shRNA干扰。2)对逆转录病毒感染性和产量的影响将通过转染和感染试验来确定。3)过表达负性作用基因的效果将被确定。在筛选中发现的一些基因对Ty3转座起负面作用。这些基因的过度表达是否会减少逆转录病毒的复制将被确定。4)将确定有效候选基因影响逆转录病毒复制的生化点。对于确定了对逆转录病毒复制shRNA或过度表达的影响的基因，将检测复制中的蛋白质、RNA和DNA中间体，以确定候选基因影响复制的生命周期阶段。虽然影响可能是直接的或间接的，但了解基因的功能以及生命周期中的效应点，应该会为了解效果的机制提供重要的见解。这种方法很有可能在逆转录病毒研究中开辟意想不到的途径，并为治疗寻找新的靶点。

DESCRIPTION (provided by applicant): Studies of the cell cycle, nuclear transport, RNA processing, chromatin structure, the cytoskeleton, and vesicular trafficking in the model organism Saccharomyces cerevisiae have provided paradigms for characterization of analogous systems in metazoans. We and others have characterized Ty retrotransposons in S. cerevisiae as models of retroviruses. We have recently used high throughput screening to identify roughly 300 genes, expression of which affects retrotransposition of Ty3, a yeast retrovirus-like element. These genes include ones related to DNA repair, RNA processing, protein folding, lipid metabolism, and vesicular trafficking. In the yeast system, we are studying the mechanism of action of a small number of these genes in considerable detail. Here we propose to survey a subset of nineteen genes for effects on replication of a simple mammalian retrovirus, Moloney murine leukemia virus. The goals of this application are: 1) Target gene function will be disrupted by shRNA. Candidate genes will be chosen based on several criteria, including existence of a nonessential mouse homolog, severity of defect, and possible defects for transposition of Ty1 a retrotransposon more distantly related to Ty3 than retroviruses. Activity of these genes will be disrupted by shRNA. 2) Effects on retrovirus infectivity and production will be determined using transfection and infection assays. 3) The effect of overexpression of negatively acting genes will be determined. Some genes identified in the screen act negatively on Ty3 transposition. Whether overexpression of these genes decreases retrovirus replication will be determined. 4) The biochemical point at which retrovirus replication is affected by effective candidate genes will be determined. For genes where an effect on retrovirus replication of shRNA or overexpression is determined, protein, RNA and DNA intermediates in replication will be assayed in order to identify the stage in the lifecycle where replication is impacted by the candidate gene. Although effects could be direct or indirect, knowledge of the function of the gene together with the point of effect in the lifecycle, should offer significant insight as to the mechanism of the effect. This approach has high potential for opening unexpected avenues in retrovirus research and identifying novel targets for therapeutics.