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
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
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.
Experimentation:
Series
Experimentation:
Series
 3’ UTR ∆ Series
Experimentation:
Series
 3’ UTR ∆ Series
 CS1 Mutation Series
3’ UTR ∆ Series:
Luciferase Constructs
Controls:
∆ Constructs:
CS1 Mutation Series
Experimental:
General Design
LUC
2. In Vitro run-off Transcription
by T7 RNA Polymerase
(with cap analog)
1. Linearize Plasmid
WWC
WWC
3. RNA Electroporation
Vero Monkey Kidney Cells
WWC & AC
4. Cell Lysis
WWC & AC
Lysate
5. Luciferase/Protein Assays
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
Capped GCLGpolyA
1.0E+10
RLU/mg protein
10
8.69
8.0E+09
8
Maximum Accumulation
illustrates the RNAs ability
to be expressed inside the
cell.
6.0E+09
6
4.0E+09
4
2.0E+09
2
0
1
1
2
2
3
4
5
6
3
4
5
6
2.17
Initial Rate reflects the RNAs
translation efficiency.
(hr)
Analysis:
Functional ½ Life
Capped GCLGpolyA
1.0E+10
RLU/mg protein
10
8.0E+09
8
6.0E+09
6
4.0E+09
4
2.0E+09
2
0
1
1
2
2
2.17
3
4
5
6
3
4
5
6
(hr)
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.
Analysis:
Accumulative ½ Life
9)
(x10
1.0E+10
RLU/mg protein
10
Capped GCLGpolyA
8.69
8.0E+09
8
6.0E+09
6
4.35
4.0E+09
4
2.0E+09
2
0
1
1
0.83
2
2
3
4
5
6
3
4
5
6
(hr)
2.29
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.
Results:
3’ UTR ∆ Series
3’UVR
DB2
DB1
3’CS
SLB SLA
Results:
½ Life Analysis
DCL∆
DCLG
/NcoI
∆UVR
∆DB1+2
∆DB2
∆DB1
∆SLB
∆SLA
GCLGpA
DCLD
Time (hrs)
Results:
CS1 Mutation Series
Results:
½ Life Analysis
DCmLDm
DCLDm
DCmLD
DCLD
Time (hrs)
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.
Acknowledgements
Dr. Dreher
Wei-Wei Chiu
Kevin Ahern
HHMI
NSF