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

1. Linearize Plasmid

2. In Vitro run-off Transcription

by T7 RNA Polymerase
(with cap analog)

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/L 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

Maximum Accumulation
illustrates the RNAs ability
to be expressed inside the
cell.

6.0E+09

4.0E+09

2.0E+09

2.17

Initial Rate reflects the RNAs

translation efficiency.

(hr)

Analysis:

Functional Life
Capped GCLGpolyA

1.0E+10

RLU/mg protein

10

8.0E+09

6.0E+09

4.0E+09

2.0E+09

2.17

(hr)

3.57 hr

T1/2=1.40 hr

Functional Life shows the change over time of the RNAs

relative efficiency to be used as a template for translation.

Analysis:

Accumulative Life
(x109)

Capped GCLGpolyA

1.0E+10

RLU/mg protein

10

8.69

8.0E+09

6.0E+09

4.35

4.0E+09

2.0E+09

1
0.83

(hr)

2.29

T1/2 = 1.46 hr

c.f. T1/2 = 1.40 hr by rates

Accumulative Life shows the amount of time it takes for the

mRNA to reach of the maximum LUC expression.

Results:

3 UTR Series
3UVR

DB2

DB1

3CS
SLB SLA

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

Results:

CS1 Mutation Series

Results:

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