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. WHO Regions 1998199920002001 casesdeathscasesdeathscasesdeathscasesdeaths Western Pacific 356,5541,47064,06611245,603167NA South-East Asia 218,8592,07555,40547157,997542119,707452 Americas (DF) 708,1460317,0400 394,8470 400,8750 Americas (DHF) 12,42683 5,21698 5,66792 5,33144 Americas (total) 720,57283322,25698400,51492406,20644 Mediterranean No dengue cases reported to WHO African No dengue cases reported to WHO World 1,295,9853,628441,727681504,114801525,913496 Dengue Virus 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 DEN infection causes: Dengue Fever (DF) fever, headache, rash, muscle pain

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 Oxyluciferin + Luciferase + AMP + Light Luciferin + Luciferase + ATP + O2

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. S1CLS1 S1CLD DCLS1 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. The model has similarities to the protein-facilitated circularization of highly expressed cellular mRNA CS1 and cCS1 allow intramolecular circularization of the viral genome Circularization Sequences

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