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Translational efficiency of Dengue Virus(DEN) and DEN variants in cells. PowerPoint Presentation
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Translational efficiency of Dengue Virus(DEN) and DEN variants in cells.

Translational efficiency of Dengue Virus(DEN) and DEN variants in cells.

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Translational efficiency of Dengue Virus(DEN) and DEN variants in cells.

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  1. Translational efficiency of Dengue Virus(DEN) and DEN variants in cells. Andrew Regan - Microbiology DREHER LAB - ALS1065

  2. Dengue Virus Vectored by mosquito

  3. Dengue Virus DEN infection causes: Dengue Fever (DF) fever, headache, rash, muscle pain Four serotypes (DEN1,DEN2, DEN3, DEN4) Co-infection by two serotypes can cause: Dengue Hemorrhagic Fever (DHF) and/or Dengue Shock Syndrome (DSS) can be fatal, especially in children

  4. Dengue Virus Positive-strand RNA virus Viral genome is ssRNA around 11kb contains the codons for synthesis of the viral polyprotein The 5’Untranslated Region (5’UTR) and 3’UTR are expected to control translational efficiency as they do with typical cellular mRNA

  5. HHMI Project To assay changes in translational efficiency between DEN2 and DEN2 variants in cells STRATEGY: DEN2-luciferase(luc) assay system Assay was established by doctoral student Wei-Wei Chiu

  6. Procedures Clone DEN2-luc (wt) and DEN2-luc variants into dsDNA plasmids Linearize plasmids, cutting at the 3’-terminal cleavage site Transcribe constructs with T7 RNApol Transfect RNA into Vero cells (monkey kidney)

  7. Procedures Measure Luciferase activity in cell lysates using a luminometer Luciferin + Luciferase + ATP + O2 Oxyluciferin + Luciferase + AMP + Light

  8. DEN(S1) attenuated strain Live virus vaccines utilize attenuated strains Examples: Polio, Measles, Smallpox, etc… DEN(S1) is an attenuated strain of DEN2 An attenuated virus has accumulated mutations that disable its ability to cause severe disease

  9. DCLS1 S1CLD S1CLS1 S1LD S1LS1

  10. Circularization Sequences Two 11-nt sequences are conserved among all DEN serotypes, as well as all close relatives (e.g. West Nile Virus, Yellow Fever Virus, etc…) Shiyun, You & R. Padmanabhan. A Novel in vitro Replication System for Dengue Virus. J Biological Chem. 1999. 274:45 33714-33722 Khromykh AA, Meka H, Guyatt KJ & EG Westaway. Essential role of cyclization sequences in flavivirus RNA replication. J Virology. 2001. 75:6719-28.

  11. Circularization Sequences CS1 and cCS1 allow intramolecular circularization of the viral genome The model has similarities to the protein-facilitated circularization of highly expressed cellular mRNA

  12. DCLD-wt DCLDm DCmLD DCmLDm

  13. Initiation Context 40S subunit scans 5’ 3’ for the initiation codon Nucleotides +6 through -4 are known to be important in halting ribosomal migration

  14. Initiation Context

  15. Acknowledgements Wei-Wei Chiu - OSU Microbiology Dr. Theo W. Dreher - OSU Microbiology - OSU Center for Gene Research Dr. Kevin G. Ahern - OSU Biochemistry & Biophysics Howard Hughes Medical Institute