Initiation and regulation of RSV mRNA transcription and genome replication

描述（由申请方提供）：呼吸道合胞病毒（RSV）是一种重要的公共卫生问题。它是婴儿呼吸道疾病的主要原因。它还导致免疫功能低下、老年人或患有潜在肺部疾病（如哮喘或慢性阻塞性肺病）的个体患严重疾病。目前没有疫苗或有效的抗病毒药物来治疗RSV感染的患者。治疗仅限于支持性护理，在严重的情况下需要住院治疗和重症监护。因此，显然需要限制RSV疾病的严重程度以降低与RSV相关的死亡率的抗RSV药物，以及需要强化治疗。抗病毒药物的理想靶标是RSV RNA依赖性RNA聚合酶（RdRp），因为它具有独特和必需的酶活性。RdRp转录并复制非节段负义RNA基因组，并且能够从相同的病毒启动子启动两种活性。该项目的目标是生成关于RSV RdRp的关键功能和结构特性的详细分子信息。这项工作分为三个主要目标。目标1中的实验将阐明RdRp从同一启动子元件内的两个位点启动转录和RNA复制的机制，并确定使用小分子成功抑制这些步骤的可能性。目标2将确定RdRp如何组装成完全有能力的转录酶和复制酶复合物，并研究这些复合物如何受到调节。目标3中的研究将利用负染和冷冻电子显微镜来深入了解RdRp单独的结构特征以及与启动子RNA和核衣壳的结构特征。这项研究将显著提高我们对RdRp的功能和结构特性的理解，并为合理开发抗病毒药物提供见解。

DESCRIPTION (provided by applicant): Respiratory syncytial virus (RSV) is a significant public health concern. It is the major cause of respiratory disease in infants. It also causes severe disease in individuals who are immunocompromised, elderly or have underlying lung disorders, such as asthma or chronic obstructive pulmonary disease. Currently there is no vaccine or effective antiviral drug to treat patients infected with RSV. Treatment is limited to supportive care, which in severe cases requires hospitalization and intensive care. Thus, there is a clear need for anti-RSV drugs that limit the severity of RSV disease to reduce the mortality associated with RSV, as well as the need for intensive treatment. An ideal target for antiviral drugs is the RSV RNA dependent RNA polymerase (RdRp), as it has unique and essential enzymatic activities. The RdRp transcribes and replicates the non-segmented negative sense RNA genome and is capable of initiating both activities from the same viral promoter. The goal of this project is to generate detailed molecular information about key functional and structural properties of the RSV RdRp. The work is divided into three major aims. The experiments in Aim 1 will elucidate the mechanisms by which the RdRp initiates transcription and RNA replication from two sites within the same promoter element, and determine the likelihood of using small molecules to successfully inhibit these steps. Aim 2 will determine how the RdRp is assembled into fully-competent transcriptase and replicase complexes, and will investigate how these complexes are regulated. The research in Aim 3 will utilize negative stain and cryo-electron microscopy to gain insight into the structural characteristics of the RdRp alone and in association with promoter RNA and the nucleocapsid. This research will significantly improve our understanding of the functional and structural properties of the RdRp and provide insights that could be exploited for rational development of antiviral drugs.