Discovering patterns in the genome of RNA viruses that drive evolution and regulate replication

所有基因组都由四个碱基组成，A，C，G和T（或U，在RNA基因组和mRNA中）。如果这些是随机编码的，每个基因组将包含每个碱基的约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。随机表示，每个二核苷酸将出现1/16或6.25%的时间，但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）代表不足。原因尚不清楚，但有趣的是，RNA病毒通过抑制基因组中的UpA来模仿宿主[1]。当病毒感染细胞时，会引发抗病毒反应，导致数百个基因上调。一个这样的基因编码核糖核酸酶L（RNaseL）酶。1981年，有报道称RNA酶L在UpA基序处切割mRNA [2]，这可能解释了为什么UpA在病毒及其宿主的基因组中受到抑制。然而，当UpAs被添加到病毒基因组中时，病毒生长受到损害，但RNaseL的消耗并不能消除这种损害（来自我们实验室的未发表数据），这表明可能涉及其他因素。RNaseL是已知具有抗病毒活性的，其活化极大地改变了细胞基因翻译，导致新蛋白质的合成[3]。我们假设RNaseL激活也可能改变感染过程中产生的病毒蛋白质的分布。在这个项目中，您将研究UpA二核苷酸如何影响病毒复制，并确定RNAseL是否会改变病毒感染过程中的病毒蛋白质产生。具体来说，你会：1。设计和合成UpA含量增加的甲型流感病毒突变体，并研究UpA引入对病毒复制的影响。您将测试RNaseL耗竭是否恢复病毒适应性。2.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细胞UpA识别重要的细胞因子。3.产生敲除的RNAseL细胞并用甲型流感病毒感染它们，然后进行质谱分析以确定RNAseL是否影响感染期间产生的病毒肽谱。您将学习实验室技能，包括如何进行病毒感染，分子生物学技术，包括CRISPR，质谱，以及病毒基因组重新编码的计算机方法。[1]冈特和迪加德，2022年。RNA病毒的成分偏差：原因，后果和应用。WIREs RNA，e1679。[2]Wreschner等人，1981.干扰素作用-ppp（A2 'p）nA依赖性核糖核酸酶的序列特异性。Nature，289：414-7. [3]Karasik等人，2021.抗病毒因子RNase L的激活触发非编码mRNA序列的翻译。Nucleic Acids Research，49（11）：6007-26.

All genomes are composed of four bases, A, C, G and T (or U, in RNA genomes and mRNAs). If these were encoded 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 in the genomes of all organisms, from bacteria to humans, TpA dinucleotides ('p' represents the phosphate bridge in the DNA backbone) are under-represented. The reason(s) are unknown, but intriguingly, RNA viruses mimic their hosts by suppressing UpA in their genomes [1].When a virus infects a cell, this triggers an antiviral response resulting in hundreds of genes being upregulated. One such gene encodes the Ribonuclease L (RNaseL) enzyme. In 1981 it was reported that mRNA is cleaved at UpA motifs by RNaseL [2], potentially explaining why UpAs are suppressed in the genomes of viruses and their hosts. However, when UpAs are added into virus genomes, virus growth is impaired, but depletion of RNaseL does not remove the impairment (unpublished data from our lab), suggesting that other factors may be involved.RNAseL is a known to have antiviral activity, and its activation drastically alters cellular gene translation, leading to the synthesis of novel proteins [3]. We hypothesise that RNaseL activation may also change the profile of viral proteins produced during infection. In this project you will characterise how UpA dinucleotides influence virus replication, and determine whether RNAseL alters viral protein production during virus infection. Specifically you will:1. 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 RNaseL depletion restores virus fitness.2. 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 cellular UpA recognition. 3. Generate knockout RNAseL cells and infect them with influenza A virus, then perform mass spectrometry to determine whether RNAseL impacts the profile of viral peptides produced during infection. You will learn laboratory skills including how to perform virus infections, molecular biology techniques including CRISPR, mass spectrometry, and in silico methods for virus genome recoding. [1] Gaunt and Digard, 2022. Compositional biases in RNA viruses: Causes, consequences and applications. WIREs RNA, e1679.[2] Wreschner et al., 1981. Interferon action - sequence specificity of the ppp(A2'p)nA-dependent ribonuclease. Nature, 289: 414-7.[3] Karasik et al., 2021. Activation of the antiviral factor RNase L triggers translation of non-coding mRNA sequences. Nucleic Acids Research, 49 (11): 6007-26.