Single RNA sensitive probes for studying viral replication and budding

Summary

Principal Investigator: Philip J Santangelo
Abstract: 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.
Funding Period: 2010-07-01 - 2015-06-30
more information: NIH RePORT

Top Publications

  1. pmc Dynamics of native β-actin mRNA transport in the cytoplasm
    Aaron W Lifland
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA
    Traffic 12:1000-11. 2011
  2. pmc Combining single RNA sensitive probes with subdiffraction-limited and live-cell imaging enables the characterization of virus dynamics in cells
    Eric Alonas
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Bldg, Atlanta, Georgia 30332, United States
    ACS Nano 8:302-15. 2014
  3. pmc Characterization of mRNA-cytoskeleton interactions in situ using FMTRIP and proximity ligation
    Jeenah Jung
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America
    PLoS ONE 8:e74598. 2013
  4. pmc Molecular mechanisms driving respiratory syncytial virus assembly
    Fyza Y Shaikh
    Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
    Future Microbiol 8:123-31. 2013
  5. pmc Quantifying RNA-protein interactions in situ using modified-MTRIPs and proximity ligation
    Jeenah Jung
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Bldg, Atlanta, GA 30332, USA
    Nucleic Acids Res 41:e12. 2013
  6. pmc Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS
    Aaron W Lifland
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA
    J Virol 86:8245-58. 2012
  7. pmc Probes for intracellular RNA imaging in live cells
    Philip J Santangelo
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA
    Methods Enzymol 505:383-99. 2012
  8. pmc Characterizing mRNA interactions with RNA granules during translation initiation inhibition
    Chiara Zurla
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America
    PLoS ONE 6:e19727. 2011
  9. pmc Structural analysis of respiratory syncytial virus reveals the position of M2-1 between the matrix protein and the ribonucleoprotein complex
    Gabriella Kiss
    Division of Pediatric Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children s Healthcare of Atlanta, Atlanta, Georgia, USA
    J Virol 88:7602-17. 2014

Detail Information

Publications12

  1. pmc Dynamics of native β-actin mRNA transport in the cytoplasm
    Aaron W Lifland
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA
    Traffic 12:1000-11. 2011
    ..These data support a model whereby processive, motor-driven motion is responsible for long-distance mRNA transport...
  2. pmc Combining single RNA sensitive probes with subdiffraction-limited and live-cell imaging enables the characterization of virus dynamics in cells
    Eric Alonas
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Bldg, Atlanta, Georgia 30332, United States
    ACS Nano 8:302-15. 2014
    ..The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses. ..
  3. pmc Characterization of mRNA-cytoskeleton interactions in situ using FMTRIP and proximity ligation
    Jeenah Jung
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America
    PLoS ONE 8:e74598. 2013
    ..Both perturbations led to a decrease in poly(A) + mRNA interactions with F-actin and an increase in the interactions with microtubules, in a time dependent manner. ..
  4. pmc Molecular mechanisms driving respiratory syncytial virus assembly
    Fyza Y Shaikh
    Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
    Future Microbiol 8:123-31. 2013
    ..Finally, a membrane scission event must occur to release the free virion. This article will review the recent literature describing the mechanisms that drive respiratory syncytial virus assembly and budding...
  5. pmc Quantifying RNA-protein interactions in situ using modified-MTRIPs and proximity ligation
    Jeenah Jung
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Bldg, Atlanta, GA 30332, USA
    Nucleic Acids Res 41:e12. 2013
    ..We also described the effects of actinomycin D (actD) on the interactions of HuR with β-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels...
  6. pmc Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS
    Aaron W Lifland
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA
    J Virol 86:8245-58. 2012
    ..To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response...
  7. pmc Probes for intracellular RNA imaging in live cells
    Philip J Santangelo
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA
    Methods Enzymol 505:383-99. 2012
    ..This chapter presents an overview of RNA imaging methodologies, and focuses on a single RNA sensitive method, employing exogenous probes, for imaging, native, nonengineered RNA in live cells...
  8. pmc Characterizing mRNA interactions with RNA granules during translation initiation inhibition
    Chiara Zurla
    Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America
    PLoS ONE 6:e19727. 2011
    ..This methodology represents a valuable tool for future studies of mRNA trafficking and regulation within living cells...
  9. pmc Structural analysis of respiratory syncytial virus reveals the position of M2-1 between the matrix protein and the ribonucleoprotein complex
    Gabriella Kiss
    Division of Pediatric Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children s Healthcare of Atlanta, Atlanta, Georgia, USA
    J Virol 88:7602-17. 2014
    ..Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization...

Research Grants30

  1. The Shelf Live Evaluation of Investigational Dosage Forms
    Jonathan White; Fiscal Year: 2013
    ..This contract is essential for continued assurance of the quality of drugs undergoing clinical investigation for different types of cancer by Cancer Therapeutics Evaluation Program. ..
  2. Pacific Southwest RCE for Biodefense &Emerging Infectious Diseases Research
    Alan G Barbour; Fiscal Year: 2013
    ..abstract_text> ..
  3. Rocky Mountain Regional Center of Excellence or Biodefense and Emerging Infectiou
    John T Belisle; Fiscal Year: 2013
    ..abstract_text> ..
  4. New England Regional Center of Excellence in Biodefense and Emerging Infectious D
    Dennis L Kasper; Fiscal Year: 2013
    ..NERCE will also continue its Developmental Projects program and Career Development in Biodefense program in an effort to initiate new research efforts and to attract new investigators to this field. ..
  5. Endothelial Injury and Repair: CardioPulmonary Vascular Biology COBRE
    SHARON IRENE SMITH ROUNDS; Fiscal Year: 2013
    ..abstract_text> ..
  6. B-cell Biology of Mucosal Immune Protection from SIV Challenge
    Eric Hunter; Fiscal Year: 2013
    ....
  7. Primary Immune Deficiency Treatment Consortium
    Morton Cowan; Fiscal Year: 2013
    ..These studies will resolve critical questions concerning HCT for these disorders and form the basis for future prospective clinical trials. ..
  8. The UPMC Sexually Transmitted Infections Cooperative Research Center
    Toni Darville; Fiscal Year: 2013
    ..abstract_text> ..
  9. Center for the Spatiotemporal Modeling of Cell Signaling (STMC)
    Bridget S Wilson; Fiscal Year: 2013
    ..The Center will strongly support translation of new technical and computational tools to other signaling systems linked to human disease, especially other immune diseases and cancer. ..
  10. University of Maryland Greenebaum Cancer Center Support Grant
    Kevin J Cullen; Fiscal Year: 2013
    ..Reflecting our remarkable and continued growth, UMGCC seeks to renew its CCSG to enhance and expand its efforts in high-quality and clinically relevant cancer research. ..
  11. Rand Center of Excellence for the Study of Appropriateness of Care in CAM
    IAN DOUGLASS COULTER; Fiscal Year: 2013
    ..From these data are abstracted about the treatment given for CCP and the number of times M/M is done for CCP is obtained. We will also interview a sample of patients attending the clinics and do a follow study for their outcomes. ..
  12. Structural studies of respiratory syncytial virus
    Elizabeth R Wright; Fiscal Year: 2013
    ..Cryo-ET, cryo-EM and helical reconstruction approaches will define the high-resolution structure of RSV M under in vitro assembly conditions. ..
  13. Host-pathogen competition in IFN mediated antiviral defense
    Jae U Jung; Fiscal Year: 2013
    ....
  14. Identification and characterization of inhibitors for hantavirus replication
    MOHAMMAD AYOUB MIR; Fiscal Year: 2013
    ....
  15. Immunobioogy for Marrow Allografts for Leukemia
    RICHARD JOHN O'REILLY; Fiscal Year: 2013
    ..The 3 cores include: Core A which provides all patient samples and evaluate grafts pre and post HSCT, Core B Biostatistics and Core C administrative support and oversight. ..
  16. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  17. Northeast Biodefense Center
    W Ian Lipkin; Fiscal Year: 2013
    ..As a Center based in a School of Public Health and a State Department of Health, the NBC has a firm commitment to and practical understanding of Emergency Preparedness. ..
  18. Immune Regulation of Virus Clearance and Tissue Injury at Sites of Infection
    Thomas J Braciale; Fiscal Year: 2013
    ..To determine the impact of viral infection on the production of Te-derived IL- 10. The proposed studies are designed to complement ongoing related studies in Projects 2, 3 and 4. ..
  19. Protein homeostasis mechanisms underlying enterovirus replication and evolution
    Raul Andino; Fiscal Year: 2013
    ..Core A: Administrative Core;and Core B: "High-throughput functional genomics and proteomics core. ..
  20. Caloric Restricted Rodent Colony
    RICK MORIN; Fiscal Year: 2013
    ..The purpose of this project is to develop, maintain and distribute a standing colony ofaged, calorically restricted rodents ofdefined strains for use by investigators in studies of aging. ..