Single RNA sensitive probes for studying viral replication and budding

描述(申请人提供)：人类呼吸道合胞病毒(HRSV)是公认的最重要的病毒病原体，严重的儿科呼吸道疾病。在世界范围内，急性呼吸道疾病是传染病导致死亡的主要原因，HRSV仍然是疫苗和抗病毒药物开发中最重要的病原体之一，但开发针对病毒的抗病毒药物并非易事。开发抗病毒药物的困难部分是由于病毒在利用细胞的分子机制的同时在受感染的细胞内进行复制。此外，由于RNA病毒的突变率，必须确定保守的病毒特异性机制，仅涉及对其复制至关重要的病毒成分。为了发现有效的抗病毒药物，必须在我们对病毒生命周期的理解上实现重大飞跃。要做到这一点，我们需要能够以高分辨率可视化受感染细胞内vRNA和蛋白质的动态时空分布。目前，荧光融合蛋白技术能够实现病毒蛋白的活细胞成像，但还没有标准技术来成像具有单一RNA敏感性的非工程RNA。作为回应，我们开发了多标记四价RNA成像探针或MTRIP，最近发表在《自然方法》杂志上。在初步实验中，当MTRIP通过与链球菌溶血素O(SLO)的细胞膜渗透传递时，可以与RNA特异性地快速结合(10分钟)，并允许在活细胞中使用广域荧光显微镜技术进行单一RNA成像。通过每个RNA结合多个探针获得的增强的信号与背景比来鉴定目标RNA。因此，我们的短期目标是，通过优化配体亲和力和探针核心组成，创建一种探针和方法，使我们能够在单分子敏感性的活细胞内研究RNA病毒的复制和病毒颗粒在时间和空间上的萌发。我们的长期目标是使用这一方法学来确定抗病毒药物的新靶点，并将新的探针用作RSV药物筛选分析的一部分，但也将其应用于其他RNA病毒，如流感，以便在我们对RNA病毒细胞生物学的基本理解上产生重大飞跃。

DESCRIPTION (provided by applicant): Human respiratory syncytial virus (hRSV) is recognized as the most important viral agent of serious pediatric respiratory tract disease. Worldwide, acute respiratory tract disease is the leading cause of mortality due to infectious disease, and hRSV remains one of the pathogens deemed most important for vaccine and antiviral development, but the development of virus specific antiviral drugs is not easy. The difficulties of developing antivirals result, in part, from viral replication taking place inside the infected cell while utilizing the cell's molecular machinery. In addition, due to the mutation rate of RNA viruses, it is essential to identify conserved virus specific mechanisms, involving only viral components, which are vital to their replication. In order for effective antiviral drugs to be discovered, a significant leap in our understanding of viral life cycles must be achieved. To do this, we need to be able to visualize at high-resolution, the dynamic spatio-temporal distribution of vRNAs and proteins within an infected cell. Fluorescent fusion protein technology currently enables the live-cell imaging of viral proteins, but no standard technology exists to image non-engineered RNA with single RNA sensitivity. In response, we've developed multiply-labeled tetravalent RNA imaging probes or MTRIPs, published recently in Nature Methods. In preliminary experiments, MTRIPs, when delivered via cell membrane permeabilization with streptolysin O (SLO), bound specifically and rapidly to RNA (<10 minutes) and allowed for single RNA imaging using widefield epifluorescence microscopy techniques in living cells. Target RNA was identified by the enhanced signal-to-background ratio achieved through binding of multiple probes per RNA. Therefore, our short term goal is, through optimization of the ligand affinity and probe core composition, to create a probe and methodology which will allow us to study RNA virus replication and budding of viral particles in time and space within a living cell with single molecule sensitivity. Our long term goals are to use the methodology to identify new targets for antiviral drugs, and use the new probes as part of drug screening assays for RSV but also to extend their application to other RNA viruses, such as influenza, in order to generate a significant leap in our fundamental understanding of RNA virus cellular biology.