Icosahedral Virion Assembly

二十面体病毒体组装

• 批准号: 0948399
• 负责人: Bentley Fane
• 金额: $ 97.5万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0948399&HistoricalAwards=false
• 关键词: Icosahedral; Virion; Assembly

The assembly of proteins and nucleic acids into viruses involves numerous molecular interactions. Often assembly is dependent on scaffolding proteins. Analogous to scaffoldings used in building construction, these proteins assist assembly, but are not found in the final product. After the virus is constructed, it can be regarded as a small machine. Its purpose is to deliver genetic material into the target cell, which is achieved by a series of programmed structural changes in viral proteins. The broad objective of this research is to elucidate the molecular mechanisms of scaffolding-mediated viral morphogenesis and DNA delivery, using the Microviridae, a family of small single stranded DNA viruses. Alterations (mutations) have been designed into the external scaffolding protein that confer dominant lethal effects. When the mutant (antiviral) protein is present in infected cells, it is able to interact with other viral proteins and unaltered scaffolding proteins (wild-type) to block viral replication in a manner similar to antiviral chemicals. If wildtype virus is continuously propagated in the presence of increasing concentrations of the antiviral protein, it selects for a multiple mutant virus that is not only highly resistant to the antiviral protein but is actually stimulated by it. Thus, the virus is able to evolve a mechanism to convert an antiviral agent into an assembly stimulator. Biochemical and genetic experiments will be conducted to test a model that seeks to explain this phenomenon. The results of this study should provide insights into the mechanism of viral assembly, protein engineering and viral evolution. After the virus recognizes its target cell, the DNA pilot protein delivers the viral genome into the cell. In a microvirus infection the DNA pilot protein and genome are first deposited in the outer membrane. The protein then directs the transport of the genome to the inner membrane, which is the site of DNA replication. The mechanism by which the protein accomplishes this function is largely unknown. During particle assembly, the internal scaffolding protein incorporates 12 individual copies of the DNA pilot protein into the forming virus. However, the results of biochemical and genetic experiments indicate that the individual DNA pilot proteins must associate with each other to deliver the viral DNA at the onset of infection. The domain within the DNA pilot protein that most likely mediates this self-association has been identified as well as the domain that initially interacts with the host cell membrane. Biochemical, genetic and structural analyses will be conducted to elucidate the mechanism of viral DNA delivery. Deciphering this mechanism should have direct applications to the fields of nanotechnology and protein engineering.Broader Impacts: The project's broader impact goes beyond the standard education of graduate and undergraduate students within the investigator's program. The impact reaches the students enrolled in the courses taught by the investigator. Incorporating research into curricula is essential at larger universities, where undergraduates who desire research opportunities far outnumber the available positions. Part of the proposed research will be conducted in a virology laboratory course in which students will conduct hypothesis-driven experiments that may uncover novel mutations and assembly mechanisms. The educational paradigm for this course has already proven successful, yielding a class-generated scientific manuscript and elevating scientific literacy.

蛋白质和核酸组装成病毒涉及许多分子相互作用。通常情况下，组装依赖于支架蛋白。类似于建筑中使用的脚手架，这些蛋白质有助于组装，但在最终产品中没有发现。病毒构建完成后，就可以看作是一台小机器。其目的是将遗传物质输送到靶细胞，这是通过病毒蛋白的一系列程序性结构变化实现的。本研究的主要目的是利用微小病毒科，一个小的单链DNA病毒家族，阐明支架介导的病毒形态发生和DNA递送的分子机制。改变(突变)已经被设计到赋予显性致死效应的外部支架蛋白中。当突变(抗病毒)蛋白存在于受感染的细胞中时，它能够与其他病毒蛋白和未改变的支架蛋白(野生型)相互作用，以类似于抗病毒化学物质的方式阻止病毒复制。如果野生型病毒在抗病毒蛋白浓度不断增加的情况下持续传播，它会选择一种不仅对抗病毒蛋白高度耐药而且实际上受到抗病毒蛋白刺激的多重突变病毒。因此，病毒能够进化出一种机制，将抗病毒剂转化为组装刺激剂。将进行生化和基因实验，以测试试图解释这一现象的模型。这项研究的结果应该为深入了解病毒组装、蛋白质工程和病毒进化的机制提供参考。在病毒识别其目标细胞后，DNA引导蛋白将病毒基因组送入细胞。在微型病毒感染中，DNA引导蛋白和基因组首先沉积在外膜。然后，这种蛋白质将基因组转移到内膜，内膜是DNA复制的地方。蛋白质如何完成这一功能的机制在很大程度上尚不清楚。在颗粒组装过程中，内部支架蛋白将12个单独的DNA引导蛋白副本合并到形成的病毒中。然而，生化和遗传学实验的结果表明，在感染开始时，单个DNA引导蛋白必须相互结合才能传递病毒DNA。DNA引导蛋白中最可能介导这种自结合的结构域已经被确定，以及最初与宿主细胞膜相互作用的结构域。将进行生化、遗传和结构分析，以阐明病毒DNA传递的机制。破译这一机制应该会直接应用于纳米技术和蛋白质工程领域。广泛的影响：该项目的更广泛影响超出了研究人员项目中研究生和本科生的标准教育。这一影响影响到了参加调查员教授的课程的学生。在规模较大的大学里，将研究纳入课程是至关重要的，因为渴望研究机会的本科生数量远远超过了空缺职位。拟议的部分研究将在病毒学实验室课程中进行，在该课程中，学生将进行假设驱动的实验，这些实验可能会发现新的突变和组装机制。这门课程的教育范例已经被证明是成功的，产生了课堂生成的科学手稿，并提高了科学素养。