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

摘要 呼吸道合胞病毒(RSV)是引起呼吸道疾病的主要原因。它对年轻人的影响最大， 老年人和免疫力受损。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 治疗那些被感染的人。更好地了解促进复制和影响的病毒因素 发病机制有助于开发有效的干预措施。 RSV是非节段性负链RNA病毒的一员。它的聚合酶复合体转录 病毒基因组，以产生亚基因组、帽状和多腺化的mRNAs。RSV转录中的一个关键因素 机械是病毒的M2-1蛋白，是聚合酶完成所需的一种延伸因子 转录较长的基因，并在这样做时访问基因组的下游区域。虽然这件事 M2-1的转录延伸功能及其结构和生化特性已被证实，最近 研究结果暗示了M2-1在转录后调控中的另一个未被探索的角色。 RSV在细胞质内含物中转录其基因组。高分辨率显微镜研究表明，M2-1 与新合成的RSV mRNAs和细胞翻译蛋白eIF4G和 胞质Poly A结合蛋白，位于空间上分离的包涵体相关颗粒内 呼吸道合胞病毒基因组RNA和聚合酶。这一发现表明，在RSV mRNA转录后， M2-1可能仍然与之相关，以便于转移到翻译机器。此外，最近的工作 研究发现，除了与RSV mRNAs结合外，M2-1还与细胞子集中的特定区域结合 MRNAs，包括参与病毒感染免疫反应的mRNAs。有趣的是，M2-1的一个关键特征是 一个含有不寻常的半胱氨酸-组氨酸(CCCH)基序的锌指结构域，通常 在细胞蛋白中发现参与转录后调控的mRNA，参与免疫反应。 这些发现，结合公布的数据显示，RSV M2-1与相关的细胞蛋白相互作用 在mRNA代谢方面，我们提出了M2-1在RSV和/或RSV中发挥转录后作用的假设 细胞基因表达。这个项目的目标是检验这一假设。目标1将确定M2-1是否会影响 RSV蛋白的表达与其对RSV转录延伸的影响无关。目标2将描述 呼吸道合胞病毒M2-1对CXCL5和CANX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见解，并对 了解RSV复制周期的关键方面和RSV致病的潜在机制。在……里面 此外，鉴于独特的锌指CCCH基序在其他相关病毒的蛋白质中也是相同的，该项目 可能会产生超越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.