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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Sakalian, Michael
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Georgia Institute of Technology
Engineering (All Types)
Schools of Engineering
United States
Zip Code
Vanover, Daryll; Smith, Daisy V; Blanchard, Emmeline L et al. (2017) RSV glycoprotein and genomic RNA dynamics reveal filament assembly prior to the plasma membrane. Nat Commun 8:667
Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong et al. (2017) Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat Protoc 12:150-167
Alonas, Eric; Vanover, Daryll; Blanchard, Emmeline et al. (2016) Imaging viral RNA using multiply labeled tetravalent RNA imaging probes in live cells. Methods 98:91-98
Yang, Xusan; Xie, Hao; Alonas, Eric et al. (2016) Mirror-enhanced super-resolution microscopy. Light Sci Appl 5:
Groves, Benjamin; Chen, Yuan-Jyue; Zurla, Chiara et al. (2016) Computing in mammalian cells with nucleic acid strand exchange. Nat Nanotechnol 11:287-294
Wigington, Callie P; Jung, Jeenah; Rye, Emily A et al. (2015) Post-transcriptional regulation of programmed cell death 4 (PDCD4) mRNA by the RNA-binding proteins human antigen R (HuR) and T-cell intracellular antigen 1 (TIA1). J Biol Chem 290:3468-87
Kiss, Gabriella; Holl, Jens M; Williams, Grant M et al. (2014) Structural analysis of respiratory syncytial virus reveals the position of M2-1 between the matrix protein and the ribonucleoprotein complex. J Virol 88:7602-17
Alonas, Eric; Lifland, Aaron W; Gudheti, Manasa et al. (2014) Combining single RNA sensitive probes with subdiffraction-limited and live-cell imaging enables the characterization of virus dynamics in cells. ACS Nano 8:302-15
Jung, Jeenah; Lifland, Aaron W; Zurla, Chiara et al. (2013) Quantifying RNA-protein interactions in situ using modified-MTRIPs and proximity ligation. Nucleic Acids Res 41:e12
Shaikh, Fyza Y; Crowe Jr, James E (2013) Molecular mechanisms driving respiratory syncytial virus assembly. Future Microbiol 8:123-31

Showing the most recent 10 out of 22 publications