GENE EXPRESSION OF HUMAN RESPIRATORY SYNCYTIAL VIRUS

该项目的长期目标是了解 人呼吸道合胞病毒（RSV）的生长和致病性， 幼儿呼吸道疾病的一个最重要因素 可能是婴儿猝死综合症的病原体 （SIDS）。一种肺炎样疾病的复发原因，特征为 鼻炎，咽炎和哮吼，它占显着的发病率， 人类生命的第一年。现在很清楚，更好地了解 在分子水平上调节病毒生长是一个重要的 成功控制这种人类病原体的先决条件，特别是 因为免疫治疗失败了 尽管有大量关于核苷酸的信息， 病毒基因组及其组成基因的序列，我们的知识 关于基因表达的分子机制仍然很差。 最近，我们朝着这个方向迈出了重要的一步， 建立了RSV的体外转录体系， 合成所有病毒的mRNA系统的保真度由以下因素强调： 我们发现mRNA被加帽、甲基化和多聚腺苷酸化。更 重要的是，我们已经证明了细胞的基本要求， 病毒转录中的蛋白质。最近，我们解剖了RSV 我们已经纯化了病毒L蛋白， 磷蛋白和N-RNA模板彼此分离并重构 使用这些纯化的大分子进行功能性转录。 重组后的系统现在可以进行结构研究， 其组成部分的功能。我们最近在大肠杆菌中表达了RSV P蛋白。 大肠杆菌中的无磷酸盐形式，并表明它可以被磷酸化， 体外细胞激酶。 细胞激酶的抑制也抑制RSV 转录提示P蛋白磷酸化的重要作用 在其功能中。当前的主要目标是利用重组后的 系统详细了解P蛋白磷酸化的作用， 以表征细胞蛋白的身份并确定其 在RSV转录中的确切作用。这些研究将提供重要的 深入了解病毒转录机制的功能， 是RSV生长和发病机制中的重要组分。

The long-term goal of this project is to understand the molecular basis of growth and pathogenicity of human respiratory syncytial virus (RSV), the single most important agent of respiratory diseases in the young children throughout the world and a probable agent of sudden infant death syndrome (SIDS). A recurring cause of pneumonia-like disease characterized by rhinitis, pharyngitis, and croup, it accounts for significant morbidity in the first year of human life. It is now clear that a better understanding of the regulation of viral growth at the molecular level is an essential prerequisite to a successful control of this human pathogen, particularly in view of the failure of immune therapy. Although considerable information is available regarding the nucleotide sequence of the viral genome and its constituent genes, our knowledge about the molecular mechanism of expression of the genes remains poor. Recently, we have taken an important step in this direction by establishing an in vitro of transcription system of RSV which efficiently synthesizes all viral mRNAs. The fidelity of the system is underscored by our finding that the mRNAs are capped, methylated and polyadenylated. More importantly, we have demonstrated an essential requirement of cellular protein(s) in viral transcription. Recently, we have dissected the RSV transcription apparatus even further: we have purified viral L protein, phosphoprotein, and the N-RNA template from one another and reconstituted functional transcription using these purified macromolecules. The reconstituted system is now amenable to studies of structure and function of its components. We have recently expressed RSV P protein in E. coli in a phosphate-free form and shown that it can be phosphorylated by cell kinase in vitro. Inhibition of the cell kinase also inhibited RSV transcription suggesting an essential role for P protein phosphorylation in its function. The immediate major goal is to use the reconstituted system to understand the role of P protein phosphorylation in detail, and to characterize the identity of the cellular protein (s) and determine its exact role in RSV transcription. These studies should provide important insights into the functioning of the viral transcription machinery which is an essential component in RSV growth and pathogenesis.