Molecular mechanisms and ramifications of horizontal gene transfer into poxviruses

痘病毒水平基因转移的分子机制和后果

• 批准号: 10327724
• 负责人: Stefan Rothenburg
• 金额: $ 37.21万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-18 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10327724
• 关键词: Address; Anti-Inflammatory Agents; Architecture; Bioinformatics; Biological Models; Cell Culture Techniques; Cell Line; Cells; Communicable Diseases; Complement; DNA; DNA Viruses; Data; Elements; Essential Genes; Event; Evolution; Experimental Models; Family; Fluorescence Microscopy; Frequencies; Gene Transfer; Genes; Genetic Recombination; Genome; Genomics; Goals; Helper Viruses; Herpesviridae; Horizontal Disease Transmission; Horizontal Gene Transfer; Immune response; Immune system; In Vitro; Infection; Interleukin-10; Kinetics; Knowledge; Laboratories; Length; Location; Maps; Mediating; Mission; Modeling; Molecular; Mus; Nuclear; Nucleotides; Orthologous Gene; Outcome; Pathway interactions; Phylogenetic Analysis; Play; Poly A; Poxviridae; Process; Public Health; RNA; Research; Retrotransposon; Retroviridae; Role; Serial Passage; System; Techniques; Testing; Time; Transgenes; Transgenic Mice; United States National Institutes of Health; Vaccinia virus; Viral; Virus; Virus Replication; antagonist; base; co-infection; cytokine; deep sequencing; fitness; in vivo; integration site; novel; pressure; site-specific integration; transmission process

PROJECT SUMMARY/ABSTRACT: There is substantial phylogenetic evidence that horizontal gene transfer (HGT) of host genes into large DNA viruses is a major driver of virus evolution. The status quo understanding of HGT in viruses is based exclusively on bioinformatic and phylogenetic data; however, these approaches cannot determine the molecular or evolutionary mechanisms underlying HGT. There is a critical need to develop an experimental system to directly model HGT. The long-term goal is to define how host genes are acquired by viruses and determine how these newly acquired genes evolve to provide a selective advantage to the virus. As a step towards this goal, the objective of this project is to optimize novel in vitro and in vivo experimental models to define mechanisms of HGT into poxviruses. The central hypothesis is that poxvirus HGT is facilitated through RNA intermediates. This hypothesis is based on preliminary data using our novel cell culture-based experimental model that demonstrated multiple independent long interspersed nuclear elements (LINE)-mediated HGT events into vaccinia virus (VACV). The rationale for this proposed research is that developing a robust experimental model of HGT into viruses will permit direct experimental approaches to address questions about the initial ac- quisition and subsequent evolution of captured genes that cannot be addressed by bioinformatics. For example, our preliminary data identified an unexpected evolutionary cascade that enabled transgenes to be acquired in essential genes through a process of HGT, complementation via co-infection, and subsequent recombination. The central hypothesis will be tested by three specific aims. 1) Characterize the molecular mechanisms of, and factors influencing HGT into VACV. Long-read sequencing will map the genes and surrounding genomic archi- tecture to define the mechanism(s) of HGT in cells. Cells either expressing different levels of LINE-1 or coinfected with retroviruses will be used to determine if these conditions influence HGT. 2) Analyze the evolution of hori- zontally transferred genes. HGT viruses will be serially passaged and subjected to long-read and Illumina-based deep sequencing to determine how captured genes evolve, and if there are any adaptations driven by disruption of different VACV genes due to the gene insertion event. 3) Define the mechanisms of horizontal gene transfer in vivo. A transgenic mouse line will be generated to study HGT into VACV during infection. Moreover, mouse infection studies will be performed to investigate the newly identified mechanism for sequential virus evolution following HGT into an essential gene and to assess the fitness of viruses that acquired transgenes. The proposed research is significant because it will establish and test novel experimental systems of HGT into viruses that will facilitate a detailed understanding of HGT into poxviruses that will close a fundamental gap in our knowledge of the evolution of these important viruses. This model system will open new avenues of research to directly test questions that are currently unanswerable by bioinformatic analyses such as the frequency of HGT, how the transferred genes evolve and the impact of retrovirus coinfection on HGT.

项目总结/摘要：有大量的系统发育证据表明，水平基因转移 (HGT)将宿主基因转化为大的DNA病毒是病毒进化的主要驱动力。现状认识 病毒中的HGT完全基于生物信息学和系统发育数据;然而，这些方法不能 确定HGT的分子或进化机制。迫切需要制定一项 实验系统直接模拟HGT。长期目标是确定宿主基因是如何被 病毒，并确定这些新获得的基因如何进化，以提供选择优势的病毒。作为 作为实现这一目标的一步，本项目的目标是优化新的体外和体内实验模型 以确定HGT进入痘病毒的机制。中心假设是痘病毒HGT通过以下途径促进： RNA中间体。这一假设是基于我们的新的基于细胞培养的实验的初步数据。 一个模型，证明了多个独立的长散布核元件（LINE）介导的HGT事件 牛痘病毒（VACV）。这项研究的基本原理是，开发一个强大的实验 HGT进入病毒的模型将允许直接的实验方法来解决有关初始AC的问题， 捕获的基因的获取和随后的进化不能通过生物信息学解决。比如说， 我们的初步数据确定了一个意想不到的进化级联，使转基因能够在体内获得， 必需基因通过HGT过程、通过共感染的互补和随后的重组。 中心假设将通过三个具体目标进行检验。1)描述的分子机制，和 影响HGT转VACV的因素长读序测序将绘制基因和周围的基因组阿尔奇， 结构来定义细胞中HGT的机制。表达不同水平LINE-1或共感染的细胞 将使用逆转录病毒来确定这些条件是否影响HGT。2)分析了Hori的演变过程- 带状转移基因。HGT病毒将连续传代，并进行长读段和基于Illumina的测序。 深度测序，以确定捕获的基因如何进化，以及是否有任何由破坏驱动的适应 不同的VACV基因，由于基因插入事件。3)定义水平基因转移的机制 in vivo.将产生转基因小鼠系以研究感染期间HGT进入VACV。此外，老鼠 将进行感染研究，以调查新发现的病毒连续进化的机制 随后HGT进入一个必需的基因，并评估获得转基因的病毒的适应性。拟议 这项研究意义重大，因为它将建立和测试新的HGT病毒实验系统， 促进对痘病毒HGT的详细了解，这将缩小我们对 这些重要病毒的进化。该模型系统将开辟新的研究途径， 目前生物信息学分析无法回答的问题，如HGT的频率， 转移基因的进化和逆转录病毒共感染对HGT的影响。