Translational Control of Dengue Viral Genome:

1
Translational Control ofDengue 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.
• Determine whether the translation of DEN RNA is
  altered in the presence of viral proteins.
• Understand the regulation of replication.

7
Experimentation Series
8
Experimentation Series

• 3 UTR ? Series

9
Experimentation Series

• 3 UTR ? Series
• CS1 Mutation Series

10
3 UTR ? SeriesLuciferase Constructs
Controls
? Constructs
11
CS1 Mutation Series
12
ExperimentalGeneral Design
LUC
2. In Vitro run-off Transcription by T7 RNA
Polymerase (with cap analog)
1. Linearize Plasmid
WWC WWC WWC
AC WWC AC AC
3. RNA Electroporation
Vero Monkey Kidney Cells
4. Cell Lysis
Lysate
5. Luciferase/Protein Assays
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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.

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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).

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AnalysisLuciferase Expression
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AnalysisFunctional ½ Life
Capped GCLGpolyA
1.0E10
10
8
8.0E09
RLU/mg protein
6
6.0E09
4
4.0E09
2
2.0E09
0
3
4
5
6
1
2
3
4
5
6
(hr)
1
2
T1/21.40 hr
Functional ½ Life shows the change over time of
the RNAs relative efficiency to be used as a
template for translation.
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AnalysisAccumulative ½ Life
(x109)
Capped GCLGpolyA
1.0E10
10
8
8.0E09
RLU/mg protein
6
6.0E09
4
4.0E09
2
2.0E09
0
3
4
5
6
1
2
3
4
5
6
(hr)
1
2
T1/2 1.46 hr
c.f. T1/2 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.
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Results3 UTR ? Series
19
Results ½ Life Analysis
20
ResultsCS1 Mutation Series
21
Results½ Life Analysis
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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