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组成，而不是DNA，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，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抗药性。RNA基因组很小，通常只编码十几种蛋白质。相比之下，人类宿主细胞专用于RNA代谢的~1,500个RNA结合蛋白(RBPs)。由于病毒基因组只能编码这些蛋白质中的一小部分，它们依赖宿主蛋白质来完成其生物周期。宿主限制性商业惯例还可以在感染中扮演另一个重要角色，它充当“传感器”，检测病毒RNA及其复制中介中存在的不寻常的分子特征。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。尽管它们具有相关性，但限制性商业惯例涉及病毒感染的范围在很大程度上仍然是未知的。在这里，我们提出了一种新的策略，以典型的辛德比斯病毒为模型，以全球方式识别与感染有关的限制性商业惯例的子集。简而言之，我们将用一种名为4-硫代尿苷(4SU)的核苷酸类似物来标记病毒RNA。在365 nm的紫外光照射下，4SU被激活，充当一种“粘合剂”，将病毒RNA与与其相互作用的蛋白质共价连接起来。这些化学“冻结”的复合体将被用寡聚(DT)珠子作为“渔网”来捕捉。“粘在”病毒RNA上的蛋白质将通过蛋白质组学方法进行鉴定。将使用基因工具或药物改变关键候选者的水平或活性，以评估它们在感染辛德比斯病毒和其他人类RNA病毒方面的后果。对感染有很强影响的限制性商业惯例将被详细研究，以了解它们的生物学作用。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.