Single RNA sensitive probes for studying viral replication and budding

描述（申请人提供）：人呼吸道合胞病毒（hRSV）是公认的儿童严重呼吸道疾病最重要的病毒病原体。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，而hRSV仍然是被认为对疫苗和抗病毒药物开发最重要的病原体之一，但开发病毒特异性抗病毒药物并不容易。开发抗病毒药物的困难部分是由于病毒在利用细胞分子机制的同时在受感染细胞内进行复制。此外，由于RNA病毒的突变率，有必要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，我们必须在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，在感染细胞内vrna和蛋白质的动态时空分布。目前，荧光融合蛋白技术能够对病毒蛋白进行活细胞成像，但目前还没有标准技术能够对具有单RNA敏感性的非工程化RNA进行成像。作为回应，我们开发了多重标记的四价RNA成像探针或MTRIPs，最近发表在《自然方法》上。在初步实验中，MTRIPs通过链溶素O （SLO）的细胞膜渗透传递，特异性和快速结合RNA（<10分钟），并允许在活细胞中使用宽视场荧光显微镜技术进行单RNA成像。通过每个RNA结合多个探针实现增强的信号与背景比来识别目标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，创造一种探针和方法，使我们能够在单分子敏感性的活细胞内研究RNA病毒复制和病毒颗粒出芽的时间和空间。我们的长期目标是利用该方法确定抗病毒药物的新靶点，并将新探针用作RSV药物筛选试验的一部分，同时也将其应用于其他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.