Determining the impact of respiratory syncytial virus M2-1 protein on post-transcriptional regulation of cellular and viral mRNAs

总结 呼吸道合胞病毒（RSV）是呼吸道疾病的主要病因。它对年轻人的影响最大， 老年人和免疫力低下的人到目前为止，还没有预防感染的疫苗，也没有有效的抗病毒药物， 治疗感染者。更好地了解促进复制和影响的病毒因素 发病机制可能有助于制定有效的干预措施。 RSV是非节段负链RNA病毒的成员。它的聚合酶复合体转录 病毒基因组产生亚基因组、加帽和多腺苷酸化的mRNA。RSV转录的关键因素 机器是病毒M2-1蛋白，一种延伸因子，它是聚合酶完成 转录较长的基因，并在这样做，以访问基因组的下游区域。虽然这 M2-1的转录延伸功能及其结构和生物化学特性已得到很好的确定， 这些发现暗示了M2-1在转录后调节中的额外的、未探索的作用。 RSV在细胞质内含物中转录其基因组。高分辨率显微镜研究显示，M2-1 与新合成的RSV mRNA和细胞翻译蛋白eIF 4G一起积累， 胞质多聚腺苷酸结合蛋白，在包涵体相关颗粒内，在空间上与 RSV基因组RNA和聚合酶。这一发现表明，在RSV mRNA转录后， M2-1可能会保持与它的关联，以便于转移到翻译机器。此外，最近的工作 研究表明，除了与RSV mRNA结合外，M2-1还与细胞内的一个亚群内的特定区域结合。 mRNA，包括参与对病毒感染的免疫应答的mRNA。有趣的是，M2-1的一个关键特征是 锌指结构域，含有不寻常的半胱氨酸-组氨酸（CCCH）基序，通常 发现于参与免疫反应的mRNA转录后调节的细胞蛋白质中。 这些发现，结合已发表的数据，表明RSV M2-1与细胞蛋白相互作用， 在mRNA代谢中，导致我们假设M2-1在RSV和/或 细胞基因表达这个项目的目的就是检验这个假设。目标1将确定M2-1是否影响 RSV蛋白表达独立于其对RSV转录延伸的影响。目标2将描述 RSV M2-1对来自CXCL 5和CANX（其两个细胞mRNA靶标）的蛋白表达的影响。 总之，这些实验将对RSV M2-1蛋白的作用产生新的认识，并对以下方面产生影响： 了解RSV复制周期的关键方面和RSV发病机制。在 此外，考虑到独特的锌指CCCH基序在其他相关病毒的蛋白质中是共享的， 可能产生超越RSV生物学的影响，为丝状病毒和其他肺炎病毒提供新的见解。

SUMMARY Respiratory syncytial virus (RSV) is a major cause of respiratory disease. Its impact is greatest in the young, the elderly and the immunocompromised. As yet, there are no vaccines to prevent infection, or effective antivirals to treat those who become infected. A greater understanding of the viral factors that facilitate replication and impact pathogenesis could aid development of effective interventions. RSV is a member of the non-segmented negative strand RNA viruses. Its polymerase complex transcribes the viral genome to produce subgenomic, capped and polyadenylated mRNAs. A key factor in the RSV transcription machinery is the viral M2-1 protein, an elongation factor, which is required for the polymerase to complete transcription of longer genes and in doing so to access downstream regions of the genome. While this transcription elongation function of M2-1 and its structural and biochemical properties are well established, recent findings have hinted at an additional, unexplored role for M2-1 in post transcriptional regulation. RSV transcribes its genome in cytoplasmic inclusions. High resolution microscopy studies revealed that M2-1 accumulates together with newly synthesized RSV mRNAs and cellular translation proteins, eIF4G and cytoplasmic poly A binding protein, within inclusion body associated granules that are spatially separated from the RSV genome RNAs and polymerase. This finding suggests that following transcription of an RSV mRNA, M2-1 might remain associated with it to facilitate transfer to the translation machinery. Further, recent work revealed that in addition to binding to RSV mRNAs, M2-1 binds to specific regions within a subset of cellular mRNAs, including mRNAs involved in immune responses to viral infection. Intriguingly, a key feature of M2-1 is a Zn-finger domain containing an unusual cysteine-cysteine-cysteine-histidine (CCCH) motif, that is typically found in cellular proteins involved in post-transcriptional regulation of mRNA involved in immune responses. These findings, combined with published data that shows that RSV M2-1 interacts with cellular proteins involved in mRNA metabolism, leads us to the hypothesis that M2-1 exerts a post-transcriptional role in RSV and/or cellular gene expression. The goal of this project is to test this hypothesis. Aim 1 will determine if M2-1 affects RSV protein expression independently of its effects on RSV transcription elongation. Aim 2 will characterize the effects of RSV M2-1 on protein expression from CXCL5 and CANX, two of its cellular mRNA targets. Together, these experiments will yield new insight into the role of the RSV M2-1 protein with implications for understanding a key aspect of the RSV replication cycle and mechanisms underlying RSV pathogenesis. In addition, given that the distinctive Zn-finger CCCH motif is shared in proteins of other related viruses, this project could have an impact beyond RSV biology, providing novel insight into the filoviruses and other pneumoviruses.