Gene acquisition by large DNA viruses

大型DNA病毒获取基因

• 批准号: RGPIN-2020-03968
• 负责人: Evans, David
• 金额: $ 3.06万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739595
• 关键词: Gene; acquisition; large; DNA; viruses

Background - Large DNA viruses encode genes that seem to have come from cellular organisms. These have probably been acquired from their hosts as the different viruses and the organisms they infect have co-evolved over billions of years. For example, myxoma virus encodes an enzyme that protects these insect-vectored poxviruses from UV-light. The closest similar proteins are encoded in plants and insects. Where do these virus genes come from and how did they get there? We hope to find answers to this question.      "Genetic recombination" is a term used to describe the swapping and joining of different DNAs and it creates organisms with new biological properties. We've studied how poxviruses recombine DNA and this has produced commercial tools for cloning DNA and genetically-engineering viruses. We've tested how much similarity is needed for recombination between vaccinia virus (VAC) and input DNA and while there is less DNA swapping as the resemblance decreases, it doesn't drop to zero with unrelated DNAs. We've sequenced 17 VAC clones that have captured DNA bearing no resemblance to the virus DNA. These viruses exhibit complex gene rearrangements near random integration sites.      I propose that this process offers a route by which large DNA viruses can capture copies of host genes. The process could simply involve end-to-end joining of DNA fragments or might use a mechanism related to the movement of retroviruses and retrotransposons. The latter scheme has an advantage in that it can prune the gene copy (remove introns) in a way that would enable virus expression. Aims - there are three aims Transfection/mapping. We'll transfect DNA encoding a drug-selectable reporter protein into VAC-infected cells, and then isolate and sequence the resulting recombinants. These studies will determine where the DNA goes and how the virus genome is altered by these events. Double-stranded breaks. We'll introduce double-stranded breaks into replicating VAC DNA and then test if this increases the yield of viruses that have captured a reporter gene. Such studies will better define the steps underpinning DNA capture. Retroviruses/retrotransposons. Retrotransposition reactions copy RNA into DNA and then insert the copied DNA into a new site in the genome. We'll test whether reporter genes can be "retroposed" into VAC using either the proteins that catalyze movement of human LINE 1 retrotransposons or as genes packaged into retrovirus particles. Training - Trainees will acquire highly-desirable skills studying genomics, virology and molecular biology in a stimulating academic environment supported by advanced infrastructure. Conclusions - It is unknown how large DNA viruses capture genes from cellular hosts, but the process could play a key role in creating new viruses with novel properties. This work will provide original insights into the process(es) responsible for this fascinating feature of virus biology and offer HQP outstanding training in biotechnology.

背景-大型DNA病毒编码的基因似乎来自细胞生物体。这些病毒可能是从宿主那里获得的，因为不同的病毒和它们感染的生物体在数十亿年的时间里共同进化。例如，黏液瘤病毒编码一种酶，保护这些昆虫载体痘病毒免受紫外线照射。最相似的蛋白质编码在植物和昆虫中。这些病毒基因从何而来，又是如何到达那里的？我们希望找到这个问题的答案。“基因重组”是一个术语，用来描述不同dna的交换和连接，它创造了具有新的生物特性的生物体。我们已经研究了痘病毒如何重组DNA，这已经产生了用于克隆DNA和基因工程病毒的商业工具。我们已经测试了痘苗病毒（VAC）和输入DNA之间的重组需要多少相似性，虽然随着相似性的降低，DNA交换减少，但与不相关的DNA之间的交换不会降至零。我们已经对17个VAC克隆进行了测序它们捕获的DNA与病毒DNA完全不相似。这些病毒在随机整合位点附近表现出复杂的基因重排。我认为这一过程提供了一种途径，通过这种途径，大型DNA病毒可以捕获宿主基因的副本。这个过程可能只涉及DNA片段的端到端连接，或者可能使用与逆转录病毒和反转录转座子运动相关的机制。后一种方案的优势在于，它可以以一种使病毒能够表达的方式修剪基因拷贝（去除内含子）。目标-有三个目标转染/映射。我们将把编码药物选择性报告蛋白的DNA转染到vac感染的细胞中，然后分离得到的重组体并对其进行测序。这些研究将确定DNA的去向以及这些事件如何改变病毒基因组。双链断裂。我们会在复制的VAC DNA中引入双链断裂然后测试这是否会增加捕获报告基因的病毒的产量。这样的研究将更好地定义支撑DNA捕获的步骤。逆转录病毒/反转位子活动。反转录转位反应将RNA复制到DNA中，然后将复制的DNA插入基因组中的新位点。我们将测试报告基因是否可以使用催化人类LINE 1反转录转座子运动的蛋白质或作为包装成逆转录病毒颗粒的基因“反转录”到VAC中。培训-学员将在先进的基础设施支持下，在令人兴奋的学术环境中学习基因组学、病毒学和分子生物学。结论：目前尚不清楚大的DNA病毒如何从细胞宿主中捕获基因，但这一过程可能在创造具有新特性的新病毒中发挥关键作用。这项工作将提供对病毒生物学这一迷人特征的过程的原始见解，并为HQP提供生物技术方面的优秀培训。