PROTEOME-WIDE IDENTIFICATION OF RNA-BINDING PROTEINS PLAYING CRITICAL ROLES IN VIRUS INFECTION

对在病毒感染中发挥关键作用的 RNA 结合蛋白进行全蛋白质组鉴定

• 批准号: MR/R021562/2
• 负责人: Alfredo Castello Palomares
• 金额: $ 10.44万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 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=MR%2FR021562%2F2
• 关键词: PROTEOME; WIDE; IDENTIFICATION; RNA; BINDING

Many viruses that infect humans have a genome made of RNA instead of DNA, including human immunodeficiency virus, hepatitis C virus and influenza virus. The viral enzymes involved in RNA replication display a high mutation rate, allowing rapid evolution of the virus, which helps it to evade immune defences and allows the emergence of resistance to antiviral drugs. RNA genomes are small, often encoding just a dozen proteins. By contrast, the human host cell dedicates ~1,500 RNA-binding proteins (RBPs) to RNA metabolism. Since viral genomes can only encode a handful of these proteins they rely on host proteins to complete their biological cycle. Host RBPs can also play another important role in infection, by acting as "sensors" that detect unusual molecular signatures present in viral RNAs and their replication intermediaries. Upon binding, these "sensors" trigger the antiviral response to alert neighbouring cells and provide an opportunity to block virus infection. Despite their relevance, the scope of RBPs involved in virus infection remains largely unknown. We propose, here, a new strategy to identify in a global manner the subset of RBPs implicated in infection using the prototypical Sindbis virus as a model. In brief, we will label viral RNA with a nucleotide analogue called 4-thiouridine (4SU). Upon irradiation, with 365 nm ultraviolet light, 4SU is activated acting as a "glue" that covalently links the viral RNA to the proteins interacting with it. These chemically "frozen" complexes will be captured using oligo(dT) beads as a "fishing net". The proteins "stuck" to the viral RNA will be identified by proteomic approaches. The levels or activity of key candidates will be altered using genetic tools or drugs to assess their consequences in the infection of Sindbis virus and other human RNA viruses. RBPs with a strong influence in infection will be studied in detail to understand their biological role. Our approach will identify targets for new antiviral strategies.

许多感染人类的​​病毒的基因组由RNA而不是DNA组成，包括人类免疫缺陷病毒、丙型肝炎病毒和流感病毒。参与RNA复制的病毒酶表现出很高的突变率，使得病毒能够快速进化，从而帮助其逃避免疫防御并出现抗病毒药物耐药性。 RNA 基因组很小，通常只编码十几种蛋白质。相比之下，人类宿主细胞将约 1,500 个 RNA 结合蛋白 (RBP) 专门用于 RNA 代谢。由于病毒基因组只能编码其中的少数蛋白质，因此它们依赖宿主蛋白质来完成其生物周期。宿主 RBP 还可以在感染中发挥另一个重要作用，通过充当“传感器”来检测病毒 RNA 及其复制中间体中存在的异常分子特征。结合后，这些“传感器”会触发抗病毒反应，向邻近细胞发出警报，并提供阻止病毒感染的机会。尽管具有相关性，但 RBP 参与病毒感染的范围仍然很大程度上未知。我们在这里提出了一种新策略，以使用典型辛德比斯病毒作为模型，以全局方式识别与感染有关的 RBP 子集。简而言之，我们将用一种称为 4-硫尿苷 (4SU) 的核苷酸类似物来标记病毒 RNA。在 365 nm 紫外线照射下，4SU 被激活，充当“胶水”，将病毒 RNA 与与其相互作用的蛋白质共价连接。这些化学“冷冻”复合物将使用oligo(dT) 珠作为“渔网”捕获。 “粘”在病毒 RNA 上的蛋白质将通过蛋白质组学方法进行鉴定。将使用遗传工具或药物改变关键候选者的水平或活性，以评估其在辛德比斯病毒和其他人类 RNA 病毒感染中的后果。将详细研究对感染有强烈影响的 RBP，以​​了解其生物学作用。我们的方法将确定新的抗病毒策略的目标。

项目成果

The molecular dissection of TRIM25's RNA-binding mechanism provides key insights into its antiviral activity

TRIM25 RNA 结合机制的分子剖析为其抗病毒活性提供了重要见解

• DOI: 10.21203/rs.3.rs-3692619/v1
• 发表时间: 2023
• 作者: Álvarez L

Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences.

• DOI: 10.7554/elife.74153
• 发表时间: 2022-01-20
• 期刊: eLife
• 影响因子: 7.7
• 作者: Lee JY;Wing PAC;Gala DS;Noerenberg M;Järvelin AI;Titlow J;Zhuang X;Palmalux N;Iselin L;Thompson MK;Parton RM;Prange-Barczynska M;Wainman A;Salguero FJ;Bishop T;Agranoff D;James W;Castello A;McKeating JA;Davis I
• 通讯作者: Davis I