Single RNA sensitive probes for studying viral replication and budding

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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.