Encapsidation of Adenovirus DNA

描述（由申请人提供）：腺病毒是1945年首次发现的含有线性双链DNA基因组的小型非包膜病毒。人类腺病毒与多种疾病有关，包括上呼吸道感染、胃肠道疾病和结膜炎。多年来，这些病毒一直是研究DNA复制、RNA合成、蛋白质翻译、致癌转化和细胞凋亡的杰出模型系统。最近，由于腺病毒作为疫苗接种和人类基因转移研究的载体的潜力，对腺病毒的兴趣已经扩大。病毒基因表达、基因组复制和病毒组装都发生在感染细胞的细胞核内。这个项目的重点是组装，特别是病毒DNA的封装。这个过程至少需要三个要素：病毒包装结构域，它位于基因组的左端附近，由被称为A重复的重复元素组成；与该区域所需序列基序结合的病毒IVa2蛋白；以及病毒52/55 kDa蛋白，它与IVa2蛋白结合，起着尚未确定的作用。本研究的目的是进一步阐明腺病毒封装其DNA的机制。52/55 kDa蛋白的作用将通过对不表达该蛋白的突变病毒的分析和对该蛋白的结构-功能研究来揭示。lVa2蛋白促进DNA包装的机制将通过构建和表征不表达该蛋白的突变病毒，通过确定该蛋白是否具有预测的atp酶活性，以及通过检查IVa2蛋白与其他病毒蛋白的功能相互作用来确定。最后，为了详细了解这两种蛋白以及其他因素在这一过程中的作用，我们将开发一种体外系统来封装病毒DNA。该项目的最终目标是开发一种用于完整病毒组装的体外系统。这些研究将促进我们对腺病毒如何包装其DNA并产生感染性病毒粒子的基本理解。这些知识将适用于阻断这一过程的抗病毒药物的开发，以及更安全的腺病毒载体的开发。

DESCRIPTION (provided by applicant): Adenoviruses are small, non-enveloped viruses containing a linear double stranded DNA genome that were first discovered in 1945. The human adenoviruses are associated with a variety of diseases including upper respiratory infections, gastrointestinal illness, and conjunctivitis. For many years, these viruses have been outstanding model systems for the study of DNA replication, RNA synthesis, protein translation, oncogenic transformation, and apoptosis. Most recently, interest in adenovirus has expanded due to its potential as a vector for vaccination and human gene transfer studies. Viral gene expression, genome replication, and viral assembly all take place in the nucleus of the infected cell. This project focuses on assembly and, in particular, the encapsidation of viral DNA. This process requires at least three elements: the viral packaging domain, which is located near the left end of the genome and is made up of repeated elements called A repeats; the viral IVa2 protein, which binds to required sequence motifs in this region; and the viral 52/55 kDa protein, which binds the IVa2 protein and plays an as of yet undetermined role. The goals of this proposal are to elucidate further the mechanism by which adenovirus encapsidates its DNA. The role of the 52/55 kDa protein will be uncovered through the analysis of a mutant virus that doesn't express the protein and by structure-function studies on the protein. The mechanism by which the lVa2 protein facilitates DNA packaging will be determined by construction and characterization of a mutant virus that does not express the protein, by determining whether the protein has a predicted ATPase activity, and by examining functional interactions of the IVa2 protein with other viral proteins. Finally, an in vitro system for encapsidation of viral DNA will be developed in order to obtain a detailed understanding of the roles of these two proteins, as well as other factors, in the process. The ultimate goal of the project is the development of an in vitro system for complete viral assembly. These studies will advance our basic understanding of how adenovirus packages its DNA and produces infectious virions. This knowledge will be applicable to the development of anti-viral drugs that block this process, as well as to the development of safer adenovirus vectors.