GENE EXPRESSION OF HUMAN RESPIRATORY SYNCYTIAL VIRUS

这个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 幼儿呼吸道疾病的单一最重要致病因素 以及婴儿猝死综合症的可能病原体 (小岛屿发展中国家)。一种肺炎样疾病的复发原因，其特征是 鼻炎、咽炎和咽喉炎，在#年发病率很高。 人类生命的第一年。现在很明显，更好的理解 在分子水平上调节病毒的生长是必不可少的 成功控制这种人类病原体的先决条件，特别是 鉴于免疫疗法的失败。 尽管关于核苷酸的大量信息是可用的 病毒基因组及其组成基因的序列，我们的知识 关于这些基因表达的分子机制尚不清楚。 最近，我们在这个方向上迈出了重要的一步， 建立高效的呼吸道合胞病毒体外转录体系 合成所有病毒的mRNA。该系统的精确度在以下方面得到了强调 我们的发现是，mRNAs是封顶的、甲基化的和多腺化的。更多 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(S)。最近，我们解剖了呼吸道合胞病毒 更进一步的转录装置：我们已经纯化了病毒L蛋白， 磷蛋白和N-RNA模板相互分离和重组 使用这些纯化的大分子进行功能转录。 重组后的系统现在可以用来研究结构和 其组件的功能。我们最近在E. 在无磷形式的大肠杆菌中，并表明它可以被 体外细胞激活剂。细胞激酶的抑制也抑制了RSV 转录提示P蛋白磷酸化起重要作用 在它的功能上。当前的主要目标是使用重组后的 系统，以详细了解P蛋白磷酸化的作用，以及 鉴定细胞蛋白(S)的特性并测定其 在RSV转录中的确切作用。这些研究应该提供重要的 对病毒转录机制功能的洞察 是RSV生长和致病过程中的重要组成部分。

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.