The role of UpA dinucleotides in regulating virus replication

UpA二核苷酸在调节病毒复制中的作用

• 批准号: 2606553
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2606553
• 关键词: role; UpA; dinucleotides; regulating; virus

What does the genetic make-up of RNA viruses look like? All genomes are composed of four bases, A, C, G and T (or U in the case of viral RNA genomes and mRNAs). If these were selected randomly, every genome would comprise 25% of each base; but this is not the case. Similarly, there are 16 possible combinations of nucleotide pairs, or dinucleotides. With random representation, each dinucleotide would occur 1/16 or 6.25% of the time, but again this is not so. In the genomes of all organisms, from bacteria to humans, TpA dinucleotides ('p' represents the phosphate bridge in the DNA backbone) are under-represented. We don't know why this is. Even more intriguingly, RNA viruses mimic this by suppressing UpA in their genomes.When a virus infects a host cell, this triggers an interferon response that results in hundreds of antiviral genes being upregulated. One such gene is RNaseL. In 1981 it was reported that mRNA is cleaved at UpA motifs by RNaseL. This offers one possible explanation for why UpAs are suppressed in the genomes of viruses and their hosts. However, when UpAs are added into virus genomes, the virus is impaired, but depletion of RNaseL from the system does not remove the impairment, suggesting other factors are at play.Alternatively, UpAs may be avoided to reduce the risk of introducing stop codons. Two of the three stop codons (UAA and UAG) include UpA motifs, and if UpAs are deselected the chances of aberrantly introducing stop codons in protein coding sequences are reduced. The purpose of this interdisciplinary project is to integrate computational (Lycett lab) and laboratory (Gaunt lab) based methods to characterise the distribution of UpAs in RNA virus genomes, and establish their importance in a virus system. Specifically you will:1. Determine the distribution of UpA motifs in selected RNA viruses by assessing whether their frequencies are different in coding and non-coding regions, whether UpAs occur within or across codon boundaries, and whether they correlate with the occurrence of stop codons or are utilised / deselected under different pressures. 2. Use this understanding to design and synthesise mutants of influenza A virus with increased UpA content, and characterise the impact of UpA introduction on virus replication. You will test whether removal of RNaseL (and other factors) from the system abrogates the anticipated defect in virus replication.3. If RNaseL restricts virus replication, you will characterise the mechanism. If RNaseL is not restrictive, you will use a small screen based approach to identify cellular factor(s) that are important for UpA recognition by the host cell. From this project, you will learn data science skills, applied to biological data - including statistical, bayesian, machine learning and evolutionary modelling, (bio)informatics, phylogenetics and phylodynamics, as well as computing skills including how to code, script and use high performance computing; and laboratory skills including how to perform virus infections and virology assays, and molecular biology techniques including CRISPR.

RNA病毒的基因组成是什么样的？所有基因组均由A、C、G和T四个碱基组成（对于病毒RNA基因组和mRNA，则为U）。如果这些是随机选择的，每个基因组将包含每个碱基的25%;但事实并非如此。类似地，有16种可能的核苷酸对或二核苷酸组合。在随机表示的情况下，每个二核苷酸将出现1/16或6.25%的时间，但同样不是这样。在所有生物体的基因组中，从细菌到人类，TpA二核苷酸（'p'代表DNA骨架中的磷酸桥）都是代表不足的。我们不知道这是为什么。更有趣的是，RNA病毒通过抑制基因组中的UpA来模拟这种情况，当病毒感染宿主细胞时，这会触发干扰素反应，导致数百个抗病毒基因上调。一个这样的基因是RNaseL。1981年，有报道称RNA酶L在UpA基序处切割mRNA。这为为什么UpAs在病毒及其宿主的基因组中受到抑制提供了一种可能的解释。然而，当将UpAs加入病毒基因组中时，病毒受到损害，但从系统中消耗RNaseL并不能消除这种损害，这表明其他因素在起作用。三个终止密码子中的两个（UAA和UAG）包括UpA基序，并且如果取消选择UpA，则在蛋白质编码序列中异常引入终止密码子的机会减少。这个跨学科项目的目的是整合基于计算（Lycett实验室）和实验室（Gaunt实验室）的方法，以确定RNA病毒基因组中UpAs的分布，并确定它们在病毒系统中的重要性。具体来说，你会：1。通过评估编码区和非编码区中UpA基序的频率是否不同，UpA是否出现在密码子边界内或跨越密码子边界，以及它们是否与终止密码子的出现相关或在不同压力下被利用/取消选择，确定UpA基序在选定RNA病毒中的分布。2.利用这种理解来设计和合成具有增加的UpA含量的甲型流感病毒的突变体，并验证UpA引入对病毒复制的影响。您将测试从系统中去除RNaseL（和其他因素）是否消除了病毒复制中的预期缺陷。如果RNaseL限制病毒复制，您将忽略该机制。如果RNaseL不受限制，您将使用基于小屏幕的方法来识别对宿主细胞识别UpA重要的细胞因子。从这个项目中，您将学习应用于生物数据的数据科学技能-包括统计，生物学，机器学习和进化建模，（生物）信息学，遗传学和生物动力学，以及计算技能，包括如何编码，脚本和使用高性能计算;和实验室技能，包括如何执行病毒感染和病毒学分析，以及分子生物学技术，包括CRISPR。