Icosahedral Virion Scaffolding Proteins

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。本研究的主要目的是利用微小病毒科，一类小的单链DNA病毒，阐明支架介导的病毒形态发生的分子机制。微病毒在某种程度上是独一无二的：组装依赖于两种不同的支架蛋白，一种是内部的，一种是外部的。外部支架蛋白的改变(突变)通常会导致显性致死效应。当突变蛋白存在于感染细胞中时，它能够与其他病毒蛋白和未改变的(野生型)外部支架蛋白相互作用。这些相互作用以类似于抗病毒化学物质的方式阻止病毒复制。通过分析突变蛋白影响病毒组装的特定阶段，可以阐明该蛋白的功能和病毒组装的分子细节。此外，病毒还会产生额外的突变，以克服最初改变的蛋白质的致命影响，从而深入了解病毒的耐药性机制。与外部支架蛋白相反，内部支架蛋白的改变很少会对组装产生有害的后果。因此，标准的基因分析提供的洞察力有限。因此，发展了一种基于进化的范式来研究该蛋白质。一个不再需要内部支架蛋白的六重突变体已经通过五次渐进和有针对性的遗传选择而被分离出来。通过研究病毒的适应性，内部支架蛋白的功能和替代组装模式正在被阐明。虽然这种蛋白质参与了几个反应，但它的主要功能之一似乎是催化，减少在细菌细胞中形成新病毒颗粒所需的时间。广泛影响该项目的更广泛的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80名学生课程的实验室部分的病毒学/遗传学/进化生物学模块中进行。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。

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. The broad objective of this research is to elucidate the molecular mechanisms of scaffolding-mediated viral morphogenesis, using the Microviridae, a family of small single-stranded DNA viruses. Microviruses are somewhat unique: assembly is dependent on two different scaffolding proteins, an internal and an external species. Alterations (mutations) in the external scaffolding protein often result in dominant lethal effects. When the mutant protein is present in infected cells, it is able to interact with other viral proteins and unaltered (wild-type) external scaffolding proteins. These interactions block viral replication in a manner similar to anti-viral chemicals. By analyzing the specific stages of viral assembly affected by the mutant proteins, the protein's functions and the molecular details of virus assembly can be elucidated. In addition, the virus accrues additional mutations that overcome the lethal effects of the originally altered protein, providing insights into viral resistance mechanisms. In contrast to the external scaffolding protein, alterations to the internal scaffolding protein rarely have detrimental consequences to assembly. Consequently, standard genetic analyses have provided limited insights. Therefore, an evolutionary-based paradigm was developed to study this protein. A sextuple mutant that no longer requires the internal scaffolding protein has been isolated by five progressive and targeted genetic selections. By examining virus adaptation, internal scaffolding protein functions and alternate modes of assembly are being elucidated. Although this protein participates in several reactions, one of its prime functions appears to be catalytic, decreasing the time required to build new viral particles in the bacterial cell.Broader ImpactsThe 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 during a virology/genetics/evolutionary biology module in the laboratory section of an 80-student course. Students will conduct hypothesis-driven experiments that may uncover novel mutations and assembly mechanisms.