Integration and Expression of Retrovirus DNA

病毒与宿主细胞DNA的整合是逆转录病毒复制的核心和独特特征，并直接导致了这类重要病毒的许多特殊特征，包括它们通过激活和转导癌基因而致癌的能力，它们作为宿主生殖系内源性病毒的进化持久性，它们非常广泛的致病谱，以及它们作为基因治疗师载体的能力。尽管整合过程的生化和结构方面已经得到了很好的研究，但许多重要的特征仍然未知，包括什么定义整合靶标，染色质和DNA结构之间的关系以及整合特异性，以及整合前病毒的位置对其随后表达的影响。这个项目的总体目标是提高我们对整合在病毒-宿主相互作用中作用的关键方面的理解。具体地说，我们将解决以下问题：1.我们能否在体外为整合酶定义一个最佳的靶序列(或结构)？整合酶靶向的结构相关性是什么？细胞内环境如何影响某些序列(或结构)作为靶标的使用？2.细胞DNA区域的结构、位置和活性如何影响其作为整合靶标的使用？特别是，我们将在我们先前工作的基础上检验这一假设，即整合没有强烈的区域偏好，并且(与先前的想法相反)，由于结合转录因子的干扰，基因转录活性的增加并不会增强基因的转录活性，而是会降低整合靶点。3.整合如何影响前病毒的后续表达。为什么有效的表达显然需要整合？整合位点在整合前病毒转录水平中的作用是什么？尤其是，“位置效应”导致整合前病毒表达的克隆间差异的基础是什么？

Integration of viral into host cell DNA is a central and unique feature of retrovirus replication, and is directly responsible for many of the special characteristics of this important group of viruses, including their ability to cause cancer by activating and transducing oncogenes, their evolutionary persistence as endogenous viruses in the host germline, their very broad pathogenic spectrum, and their ability to serve as vectors for the gene therapist. Although the biochemical and structural aspects of the integration process are becoming quite well worked out, many important features remain unknown, including what defines an integration target, the relationship between chromatin and DNA structure and integration specificity, and the effect of the location of the integrated provirus on its subsequent expression. The overall goal of this project is to improve our understanding of key aspects of the role of integration in the virus-host interaction. Specifically, we will address the following questions: 1. Can we define an optimal target sequence (or structure) for integrase in vitro? What are the structural correlates of integrase targeting? How does the intracellular environment affect the use of certain sequences (or structures) as targets? 2. How does the structure, location, and activity of a region of cellular DNA affect its use as an integration target? In particular, we will test the hypothesis, based on our prior work, that integration has no strong regional preference, and that (contrary to prior ideas), increasing transcriptional activity of a gene does not enhance is use as an integration target, but rather reduces it, as a consequence of interference of bound transcription factors. 3. How does integration affect subsequent expression of the provirus. Why is integration apparently required for efficient expression? What is the role of the integration site in the level of transcription of the integrated provirus? In particular, what is the basis for "position effects" leading to clone-to-clone variation in expression of integrated proviruses?