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Caulimovirus P6 protein self-association in infection

花茎病毒 P6 蛋白在感染中的自关联

基本信息

批准号：
6504682
负责人：
SCOTT M LEISNER
金额：
$ 14.2万
依托单位：
UNIVERSITY OF TOLEDO
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-01 至 2006-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6504682
关键词：
Agrobacterium DNA virus genetically modified plants genome inclusion body leaf molecular site northern blottings plant virus protein protein interaction site directed mutagenesis temperature sensitive mutant virus infection mechanism virus protein yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Viruses are serious pathogens of animals and plants. To cause disease, viruses must carry out many defined tasks. Since virus genomes are small, while the number of tasks they need to accomplish are great, it is common for viral proteins to be multifunctional. How multifunctional viral proteins integrate their activities is unknown, but it is likely that protein self-association is required. If self-association of a viral protein is important for infection then it should be possible to prevent disease by impairing that interaction. This could be accomplished by identifying the domain responsible for self-association and employing it as a dominant negative inhibitor. Here, we examine the multifunctional gene VI product (P6) of Cauliflower mosaic virus (CaMV). Inclusion body (IB) formation is one of the many functions of P6. CaMV lBs are thought to be sites of virus genome replication, protein synthesis, and virion assembly. Therefore, by studying P6, we obtain information on lBs as well as on the workings of a multifunctional protein. We have found that P6 self-associates and have identified a conserved region (ID6) important for this interaction. The work described here analyzes ID6 and examines its role in virus infection. The Specific Aims of this proposal are: 1. To Characterize The P6-P6 Interaction Domain, ID6. 2. To Elucidate The Role Of ID6 In P6 Self-Association. 3. To Determine The Role Of ID6 In Viral Infection. 4. To Test If ID6 Can Be Employed As An Inhibitor Of P6 Self-Association.To address Specific Aim 1, we will perform mutagenesis on ID6 and test these mutants for their ability to bind to P6. This will be accomplished using site-directed mutagenesis of conserved residues in ID6. We will also generate random mutations in ID6 by chemical mutagenesis. The mutants will then be tested for their ability to bind P6 by yeast two-hybrid analysis. For Specific Aim 2, we will generate mutations in the full length P6 protein equivalent to those that prevent ID6 binding to the gene VI product. These mutant proteins will then be examined for their ability to self-associate and bind to the CaMV movement protein using the yeast two-hybrid system. We will then test these mutants for their translational transactivation activity. To accomplish Specific Aim 3, we will generate the mutations defined above in gene VI of a CaMV genome. These mutant viruses will then be examined for their propagation, symptom formation, distribution, and lB formation. To accomplish Specific Aim 4, we will use a modified form of the yeast two-hybrid system to test if ID6 interferes with P6 self-association. By performing this work, we will better understand the activities of a multifunctional viral protein and provide useful training to undergraduate students.

性状（申请人提供）：病毒是动植物的严重病原体。为了引起疾病，病毒必须执行许多确定的任务。由于病毒基因组很小，而它们需要完成的任务数量却很大，因此病毒蛋白质通常是多功能的。多功能病毒蛋白如何整合其活性尚不清楚，但很可能需要蛋白质自缔合。如果病毒蛋白的自结合对感染很重要，那么通过削弱这种相互作用就有可能预防疾病。这可以通过识别负责自我关联的结构域并将其用作显性负抑制剂来实现。在这里，我们研究了花椰菜花叶病毒（CaMV）的多功能基因VI产物（P6）。包涵体（IB）的形成是P6的许多功能之一。CaMV 1B被认为是病毒基因组复制、蛋白质合成和病毒粒子组装的位点。因此，通过研究P6，我们获得了关于lBs以及多功能蛋白质工作的信息。我们已经发现P6自我关联，并确定了一个保守的区域（ID 6），这种相互作用的重要性。本文描述的工作分析了ID 6，并研究了它在病毒感染中的作用。本提案的具体目标是：1。为了表征P6-P6相互作用结构域，ID 6。2. ID 6在P6自联想中的作用3.确定ID 6在病毒感染中的作用4.测试ID 6是否可以用作P6自结合的抑制剂。为了解决特定目标1，我们将对ID 6进行突变并测试这些突变体与P6结合的能力。这将使用ID 6中保守残基的定点诱变来实现。我们还将通过化学诱变在ID 6中产生随机突变。然后通过酵母双杂交分析测试突变体结合P6的能力。对于特异性目标2，我们将在全长P6蛋白中产生突变，其等同于阻止ID 6与基因VI产物结合的突变。然后，使用酵母双杂交系统检查这些突变蛋白的自缔合和结合CaMV运动蛋白的能力。然后我们将测试这些突变体的翻译反式激活活性。为了实现特定目标3，我们将在CaMV基因组的基因VI中产生上文定义的突变。然后检查这些突变病毒的繁殖、症状形成、分布和IB形成。为了实现具体目标4，我们将使用酵母双杂交系统的修改形式来测试ID 6是否干扰P6自缔合。通过这项工作，我们将更好地了解多功能病毒蛋白的活动，并为本科生提供有用的培训。