Translational Control of Dengue Viral Genome: Role of 3’ UTR & CS1 Anna Carmona Mentor: Dr. Theo...
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Transcript of Translational Control of Dengue Viral Genome: Role of 3’ UTR & CS1 Anna Carmona Mentor: Dr. Theo...
Translational Control ofDengue Viral Genome:
Role of 3’ UTR & CS1
Anna CarmonaMentor: Dr. Theo Dreher
Assisted: Wei-Wei Chiu
Department of Microbiology, Oregon State University
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).
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.
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.
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.
Overall Goals of DEN Study
Translation efficiency of dengue viral gene expression. Identify features in the 5’ and 3’ regions of DEN-2 RNA Identify features in the 5’ and 3’ regions of DEN-2 RNA
genome that control translation. This will be done using genome that control translation. This will be done using a sensitive luciferase reporter mRNA.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.
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
WWC & AC
WWC & AC
AC
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.
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).
Analysis:Luciferase Expression
2.17
Initial Rate reflects the RNAstranslation efficiency.
2.0E+09
4.0E+09
6.0E+09
8.0E+09
1.0E+10
2
10
4
8
6
RL
U/m
g p
rote
in
1 2 3 4 5 6 (hr)0 1 2 3 4 5 6
Capped GCLGpolyA8.69
Maximum Accumulation illustrates the RNAs abilityto be expressed inside thecell.
Analysis:Functional ½ Life
2.0E+09
4.0E+09
6.0E+09
8.0E+09
1.0E+10
2
10
4
8
6
RL
U/m
g p
rote
in
1 2 3 4 5 6 (hr)0 1 2 3 4 5 6
2.17 3.57 hr
T1/2=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
Analysis:Accumulative ½ Life
(x109)
2.0E+09
4.0E+09
6.0E+09
8.0E+09
1.0E+10
2
10
4
8
6
RL
U/m
g p
rote
in
1 2 3 4 5 6 (hr)0 1 2 3 4 5 6
T1/2 = 1.46 hr
8.69
4.35
2.290.83
c.f. T1/2 = 1.40 hr by rates
Accumulative ½ Life shows the amount of time it takes for themRNA to reach ½ of the maximum LUC expression.
Capped GCLGpolyA
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.