Icosahedral Virion Assembly

蛋白质和核酸组装成病毒涉及许多分子间的相互作用。装配通常依赖于支架蛋白。类似于建筑施工中使用的脚手架，这些蛋白质有助于组装，但在最终产品中找不到。病毒构造好后，可以看作是一台小机器。它的目的是将遗传物质传递到目标细胞中，这是通过病毒蛋白的一系列程序化结构变化来实现的。本研究的主要目的是利用微病毒科（一种小单链DNA病毒）阐明支架介导的病毒形态发生和DNA传递的分子机制。改变（突变）被设计成外部支架蛋白，赋予显性致死效应。当突变（抗病毒）蛋白存在于受感染细胞中时，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繁殖，它会选择一种多重突变病毒，这种病毒不仅对抗病毒蛋白具有高度抗性，而且实际上受到抗病毒蛋白的刺激。因此，病毒能够进化出一种机制，将抗病毒药物转化为组装刺激剂。将进行生物化学和基因实验来检验一个试图解释这一现象的模型。本研究结果将为病毒组装、蛋白质工程和病毒进化机制的研究提供新的思路。病毒识别目标细胞后，DNA先导蛋白将病毒基因组传递到细胞中。在微病毒感染中，DNA先导蛋白和基因组首先沉积在外膜上。然后蛋白质引导基因组运输到内膜，这是DNA复制的地方。蛋白质实现这一功能的机制在很大程度上是未知的。在颗粒组装过程中，内部支架蛋白将12个DNA先导蛋白的独立拷贝整合到形成的病毒中。然而，生化和遗传实验的结果表明，在感染开始时，单个DNA先导蛋白必须相互关联才能传递病毒DNA。DNA先导蛋白内最有可能介导这种自结合的结构域以及最初与宿主细胞膜相互作用的结构域已被确定。将进行生化、遗传和结构分析来阐明病毒DNA传递的机制。破译这一机制将直接应用于纳米技术和蛋白质工程领域。更广泛的影响：该项目的更广泛的影响超出了研究者项目中研究生和本科生的标准教育。这种影响影响到参加研究者教授的课程的学生。在规模较大的大学，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远远超过现有的职位。部分拟议的研究将在病毒学实验室课程中进行，学生将进行假设驱动的实验，可能会发现新的突变和组装机制。这门课程的教育模式已经被证明是成功的，它产生了一篇由学生产生的科学手稿，并提高了科学素养。

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.