EDGE CT: Genetic tools to study giant viruses

近年来，微生物学家已经意识到大量感染原生生物的“巨型病毒”。这些病毒也被认为是多细胞生物微生物组的组成部分。这些巨型病毒中的许多基因被认为对病毒来说是新的：许多基因编码的过程只与“活细胞有机体”相关，比如DNA复制或DNA修复，这些过程在这些病毒中被发现。研究表明，这些巨型病毒从所有生物中收集了这些基因，使这些病毒的基因组最多只能被描述为马赛克。此外，在巨型病毒中发现的数百个基因中，有许多对科学来说仍然是全新的。尽管有这些明显丰富的信息，我们仍然没有工具来研究这些巨大的病毒基因是如何运作的，或者它们是否必要。这项研究将开发在巨型病毒系统模型（嗜食金黄色球菌病毒）中进行基因研究所需的工具，并建立这些工具集扩展到其他巨型病毒及其宿主的能力。技术摘要：虽然15年前才被发现，但感染原生生物的巨型病毒已被证明分布在整个生态系统中。这些病毒含有编码过程的功能基因，包括依赖光和独立的DNA修复、糖代谢基因和与翻译过程相关的基因。系统发育检查表明这些基因是通过猖獗的水平基因转移收集的。这项研究将开发一种巨型病毒（AaV）的基因功能和活性测试方案，这种病毒感染嗜食金黄色葡萄球菌（pelagophyte anophagefferens）。AaV是迷你病毒科的一员，是一种具有复杂基因补体的大型病毒。pi和学员将开发体外方法来修饰和转化AaV，包括引入抗生素耐药性和/或显色/荧光蛋白，以便在随后的基因替代研究中进行遗传上可处理的选择。pi将测试其他感染藻类的迷你病毒科和藻纳病毒科的方案，以确定在其他巨型病毒宿主系统中的可加工性。随着对博士后、研究生和本科生的培训，该项目将开发工具，允许在随后的研究中进行假设驱动测试，在一个系统中，许多（60%）基因产物被注释为假设。该项目将通过培训课程和专门的研讨会交流协议，并通过protocols.io记录所有开放获取的方法。学生记者将制作供公众消费的材料，包括聚焦于病毒在生态学中的积极作用、技术可重复性在科学中的重要性的短视频，以及一些关于巨型病毒迷人本质的视频。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Non-Technical Abstract In recent years microbiologists have become aware of abundant "giant viruses" that infect protists. These viruses have also been implicated as components of the microbiomes of multicellular organisms. Many of the genes within these giants are considered novel for viruses: many genes encode for processes only associated with "living cellular organisms," like DNA replication or DNA repair, are found in these viruses. Studies suggest that these giant viruses have collected these genes from across all biology, leaving these viruses with genomes that can at best be described as mosaics. Moreover many of the hundreds of genes found in giant viruses remain completely new to science. In spite of this obvious wealth of information, we remain without tools to study how these giant virus genes function or if they are even necessary. This study will develop the tools needed to do genetic research in a model giant virus system (the Aureococcus anophagefferens Virus) and establish the ability of these tool sets to be extended to other giant viruses and their hosts.Technical AbstractWhile discovered only 15 years ago, giant viruses infecting protists have been shown to be distributed across ecosystems. These viruses contain functional genes encoding processes that can include light-dependent and independent DNA repair, sugar metabolism genes and genes associated with the process of translation. Phylogenetic examinations suggest these genes have been collected by rampant horizontal gene transfer. This study will develop protocols required to test gene function and activity in a giant virus (AaV) that infects the pelagophyte Aureococcus anophagefferens. A member of the Mimiviridae, AaV is a large virus with a complex complement of genes. The PIs and trainees will develop in vitro approaches to modify and transform AaV, including the introduction of antibiotic resistance and/or chromogenic/fluorogenic proteins for genetically tractable selections for use in subsequent gene replacement studies. The PIs will test protocols on other Mimiviridae and Phycodnaviridae that infect algae to determine tractability in other giant virus-host systems. Along with training of a postdoc, graduate and undergraduate students, this project will develop tools that allow for hypothesis driven testing in subsequent studies in a system where many ( 60%) of the gene products are annotated as hypothetical. The project will communicate protocols through training sessions as well as a dedicated workshop, and document all approaches in open access through protocols.io. Student journalists will produce material for public consumption, including short videos focusing on the positive role of viruses in ecology, the importance of technical reproducibility in science, and several on the fascinating nature of giant viruses.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.