1. Translational Control of Dengue Viral Genome: Role of 3’ UTR & CS1 Anna Carmona Mentor: Dr. Theo Dreher Assisted: Wei-Wei Chiu Department of Microbiology, Oregon State University

2. About Dengue  Dengue is one of the most important mosquito-born viral diseases affecting humans.  Viral life cycle involves humans and the mosquito vector Aedes aegypti. In the U.S. it has been found that the mosquito Aedes albopictus also transmits the DEN virus.  The disease is caused by 4 serotypes of the Dengue virus, a member of the genus Flavivirus: DEN-1, DEN-2, DEN-3, DEN-4.  Infection with the DEN virus can result in Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS).

3. DEN-2 Serotype  Strain 16681 from Thailand.  DEN virus is an enveloped, 10.75 kb, positive, single-stranded RNA virus.  1 ORF, 380 kDa.  Structure contains a 5’ cap and a 3’ stem-loop structure (no 3’ -poly(A) tail).  Has the ability to replicate in mosquitoes and primate cells.

4. The DEN Virus  The development of a vaccine is a high priority with live attenuated virus as the preferred form.  A goal of this research is to restrict viral gene expression as a source of attenuation. Risks for this include the possibility of attenuation reversal of a vaccine strain resulting in mutations that might increase gene expression.

5. Overall Goals of DEN Study  Translation efficiency of dengue viral gene expression. Identify features in the 5’ and 3’ regions of DEN-2 RNA genome that control translation. This will be done using a sensitive luciferase reporter mRNA.  Determine whether the translation of DEN RNA is altered in the presence of viral proteins.  Understand the regulation of replication.

6. Overall Goals of DEN Study  Translation efficiency of dengue viral gene expression. Identify features in the 5’ and 3’ regions of DEN-2 RNA genome that control translation. This will be done using a sensitive luciferase reporter mRNA. Identify features in the 5’ and 3’ regions of DEN-2 RNA genome that control translation. This will be done using a sensitive luciferase reporter mRNA.  Determine whether the translation of DEN RNA is altered in the presence of viral proteins.  Understand the regulation of replication.

7. Experimentation: Series

8.  3’ UTR ∆ Series

9. Experimentation: Series  3’ UTR ∆ Series  CS1 Mutation Series

10. 3’ UTR ∆ Series: Luciferase Constructs Controls: ∆ Constructs:

11. CS1 Mutation Series

12. Experimental: General Design 2. In Vitro run-off Transcription by T7 RNA Polymerase (with cap analog) 1. Linearize Plasmid Vero Monkey Kidney Cells 4. Cell Lysis 5. Luciferase/Protein Assays LUC 3. RNA Electroporation Lysate WWC WWC & AC AC

13. Luciferase Assay  When in the presence of the substrate LAR (Luciferase Assay Reagent), luciferase will undergo an enzymatic reaction that emits light. This is measured in Relative Light Units (RLU).  Problem: This assay does not take into account the total amount of cells that were lysed.

14. Protein Assay  The protein present in the lysates cause the Protein Assay Reagent to turn blue. Light absorbance at 595 nm is measured and used as a reflection on the total amount of protein present in the lysates. Protein concentration is indicative of the lysates total cell number.  Results from the protein assay are measured in mg protein/µL of lysate. These values are then used to normalize the results from the Luciferase Assay (RLU/mg protein).

15. Analysis: Luciferase Expression 2.17 Initial Rate reflects the RNAs translation efficiency. 2.0E+09 4.0E+09 6.0E+09 8.0E+09 1.0E+10 2 10 4 8 6 RLU/mg protein 123456 (hr) 0 12 3456 Capped GCLGpolyA 8.69 Maximum Accumulation illustrates the RNAs ability to be expressed inside the cell.

16. Analysis: Functional ½ Life 2.0E+09 4.0E+09 6.0E+09 8.0E+09 1.0E+10 2 10 4 8 6 RLU/mg protein 123456 (hr) 0 12 3456 2.17 3.57 hr T 1/2 =1.40 hr Functional ½ Life shows the change over time of the RNA’s relative efficiency to be used as a template for translation. Capped GCLGpolyA

17. Analysis: Accumulative ½ Life (x10 9 ) 2.0E+09 4.0E+09 6.0E+09 8.0E+09 1.0E+10 2 10 4 8 6 RLU/mg protein 123456 (hr) 0 12 3456 T 1/2 = 1.46 hr 8.69 4.35 2.29 0.83 c.f. T 1/2 = 1.40 hr by rates Accumulative ½ Life shows the amount of time it takes for the mRNA to reach ½ of the maximum LUC expression. Capped GCLGpolyA

18. Results: 3’ UTR ∆ Series 3’UVR DB1 3’CS SLB SLA DB2

19. Results: ½ Life Analysis DCL∆ DCLG /NcoI ∆UVR ∆DB1+2 ∆DB2 ∆DB1 ∆SLB ∆SLA GCLGpA DCLD Time (hrs)

20. Results: CS1 Mutation Series

21. Results: ½ Life Analysis DCmLDm DCLDm DCmLD DCLD Time (hrs)

22. A Look Ahead…  Cap/no cap 5’ UTR ∆ series.  Examining cap dependent/independent translation.  Possible interactions between viral/cellular proteins and how they affect translation of DEN-2 genome.

23. Acknowledgements Dr. Dreher Wei-Wei Chiu Kevin Ahern HHMI NSF