m6A甲基化修饰靶向J亚群禽白血病病毒基因组gRNA 3’UTR调控其复制的机制

禽白血病病毒（ALV）分为A～K不同亚群，其中J亚群ALV（ALV-J）的复制能力和致病性最强。ALV-J基因组gRNA独特的3’UTR在病毒复制和致病中发挥重要作用，但具体机制有待深入探索。前期研究发现，ALV-J 3’UTR的A7017位点发生了N6-腺苷酸甲基化（m6A）修饰，突变该位点可显著降低毒株的体外复制能力，因此我们推测ALV-J 3’UTR 的m6A甲基化修饰参与了病毒复制的调控。本项目首先测定亲本毒和A7017突变毒在体外和鸡体内的复制动态及致病性；随后利用荧光素酶报告基因试验和RIP试验明确m6A甲基化修饰对病毒基因转录和病毒miRNA加工的影响；最后通过PAR-CLIP-qPCR试验筛选与病毒3’UTR互作的m6A结合蛋白，阐明m6A甲基化修饰靶向ALV-J 3’UTR调控其复制的机制。该项目将深化对ALV-J复制调控的认识，并为病毒致病机制的解析提供理论依据。

Avian leukosis virus (ALV) can be divided into different subgroups from A to K, of which subgroup J ALV (ALV-J) is the most virulent virus with a strongest replication capacity. The unique ALV-J gRNA 3’UTR plays a critical role in virus replication and pathogenicity, but the underlying mechanism needs to be further explored. Our previous study demonstrated that ALV-J gRNA 3’UTR A7017 site was modified by N6-methyladenosine (m6A), and mutation of A7017 can weaken ALV-J replication in vitro. Thus, we hypothesize that m6A methylation of ALV-J gRNA 3’UTR may play roles in virus replication regulation. In this proposal, we will firstly investigate the replication dynamics and pathogenicity of parental and A7017 mutated ALV-J in vitro and in vivo. Secondly, we will make full use of luciferase reporter gene method and RNA immunoprecipitation to identify the regulatory effect of m6A methylation on gene transcription and viral-derived miRNA processing. Finally, we will screen the m6A binding protein which interacts with ALV-J 3’UTR via PAR-CLIP-qPCR assay to elucidate the molecular mechanism of the regulatory effect of m6A methylation on virus replication. This study is expected to deepen the understanding of ALV-J replication mechanism and provide theoretical foundation for elucidating the pathogenesis of ALV-J.