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

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复制的病毒酶显示出高突变率，允许病毒快速进化，这有助于它逃避免疫防御，并允许出现对抗病毒药物的耐药性。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种RNA结合蛋白（RBP）用于RNA代谢。由于病毒基因组只能编码这些蛋白质中的一小部分，因此它们依赖宿主蛋白质来完成其生物循环。宿主RBP还可以在感染中发挥另一个重要作用，作为“传感器”，检测病毒RNA及其复制中介体中存在的不寻常分子特征。一旦结合，这些“传感器”就会触发抗病毒反应，提醒邻近细胞，并提供阻止病毒感染的机会。尽管它们的相关性，涉及病毒感染的限制性商业惯例的范围在很大程度上仍然是未知的。在这里，我们提出了一个新的策略，以确定在全球范围内的RBPs的子集涉及感染的原型辛德毕斯病毒作为一个模型。简而言之，我们将用称为4-硫尿苷（4SU）的核苷酸类似物标记病毒RNA。在365 nm紫外线照射下，4SU被激活，作为一种“胶水”，将病毒RNA共价连接到与其相互作用的蛋白质上。这些化学“冷冻”的复合物将被oligo（dT）珠作为“渔网”捕获。将通过蛋白质组学方法鉴定“粘附”在病毒RNA上的蛋白质。将使用遗传工具或药物改变关键候选物的水平或活性，以评估其在辛德毕斯病毒和其他人类RNA病毒感染中的后果。将详细研究在感染中具有强烈影响的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。

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.