Icosahedral Virion Assembly

蛋白质和核酸组装成病毒涉及许多分子相互作用。组装通常依赖于支架蛋白。类似于建筑中使用的支架，这些蛋白质有助于组装，但在最终产品中没有发现。病毒构造好后，可以看作是一台小型机器。其目的是将遗传物质输送到靶细胞中，这是通过病毒蛋白质中的一系列程序化结构变化来实现的。本研究的主要目的是阐明支架介导的病毒形态发生和DNA递送的分子机制，使用Microviridae，一个小的单链DNA病毒家族。改变（突变）已被设计到外部支架蛋白，赋予显性致死作用。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断病毒复制。如果野生型病毒在抗病毒蛋白浓度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将抗病毒剂转化为组装刺激物的机制。将进行生化和遗传实验，以测试试图解释这一现象的模型。本研究的结果将为病毒组装、蛋白质工程和病毒进化的机制提供新的见解。在病毒识别其靶细胞后，DNA先导蛋白将病毒基因组递送到细胞中。在微病毒感染中，DNA先导蛋白和基因组首先沉积在外膜中。然后，蛋白质引导基因组运输到内膜，这是DNA复制的位点。这种蛋白质实现这种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个单独的DNA先导蛋白拷贝掺入形成的病毒中。然而，生物化学和遗传学实验的结果表明，单个DNA先导蛋白必须彼此缔合以在感染开始时递送病毒DNA。最有可能介导这种自缔合的DNA先导蛋白内的结构域已经被鉴定，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输送的机制。更广泛的影响：该项目的更广泛的影响超出了研究生和本科生的标准教育范围。调查员所教课程的学生也受到影响。在规模较大的大学，将研究纳入课程是必不可少的，在那里，渴望研究机会的本科生远远超过了现有的职位。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。事实证明，本课程的教育范式是成功的，产生了课堂生成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.