Single RNA sensitive probes for studying viral replication and budding

性状（由申请方提供）：人呼吸道合胞病毒（hRSV）被认为是严重儿科呼吸道疾病的最重要病毒因子。在世界范围内，急性呼吸道疾病是由于感染性疾病导致死亡的主要原因，并且hRSV仍然是被认为对于疫苗和抗病毒开发最重要的病原体之一，但是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复制发生在受感染的细胞内，同时利用细胞的分子机制。此外，由于RNA病毒的突变率，必须鉴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组分。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受感染细胞内vRNA和蛋白质的动态时空分布。荧光融合蛋白技术目前能够实现病毒蛋白的活细胞成像，但不存在标准技术以单个RNA灵敏度成像非工程化RNA。作为回应，我们开发了多重标记的四价RNA成像探针或MTRIP，最近发表在Nature Methods上。在初步实验中，MTRIPs，当通过细胞膜透化与链球菌溶血素O（SLO），特异性和快速结合到RNA（<10分钟），并允许在活细胞中使用宽视野落射荧光显微镜技术的单个RNA成像。通过每个RNA结合多个探针实现的增强的信号背景比来鉴定靶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，使我们能够研究RNA病毒复制和病毒颗粒在活细胞内的时间和空间中的出芽，具有单分子灵敏度。我们的长期目标是使用该方法来确定抗病毒药物的新靶点，并使用新探针作为RSV药物筛选测定的一部分，但也将其应用扩展到其他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方面产生重大飞跃。 公共卫生相关性：人呼吸道合胞病毒（hRSV）是一种RNA病毒，是美国和世界范围内婴儿病毒性肺炎、细支气管炎、呼吸衰竭、机械通气和病毒性死亡的主要原因，造成的婴儿死亡人数是流感病毒的9倍。目前，没有针对hRSV疾病的有效疫苗，并且迫切需要用于抗病毒筛选的新技术和靶点。在这项资助申请中，通过探针开发人员和成熟的病毒学家的合作，我们将开发，优化和验证我们的单一RNA敏感的活细胞成像探针和方法，允许在不干扰病毒过程的情况下识别病毒复制位点和定量病毒的复制和出芽;这将导致RNA病毒复制的更精确的时空视图和筛选抑制基本病毒特异性过程的分子的能力。

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. PUBLIC HEALTH RELEVANCE: Human respiratory syncytial virus (hRSV), an RNA virus, is the leading cause of viral pneumonia, bronchiolitis, respiratory failure, mechanical ventilation, and viral death in infants in the USA and worldwide, and causes nine times as many infant deaths as influenza virus. Currently, there are no effective vaccines for hRSV disease, and new techniques and targets for antiviral screening are badly needed. In this grant application, through the collaboration of a probe developer and well established virologist, we will develop, optimize, and validate our single RNA-sensitive, live-cell imaging probes and methodology, allowing for the identification of viral replication sites and quantification of replication and budding of the virus without interfering with viral processes; this will lead to a more accurate spatio-temporal view of RNA virus replication and the ability to screen molecules that inhibit essential virus-specific processes.