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
3. RNA Electroporation

WWC

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
1.0E+10

10 8

Capped GCLGpolyA
8.69

RLU/mg protein

8.0E+09

6.0E+09

4.0E+09

4 2

2.0E+09

Maximum Accumulation illustrates the RNAs ability to be expressed inside the cell.
0

(hr)

2.17

Initial Rate reflects the RNAs translation efficiency.

Analysis:
Functional Life
1.0E+10

10 8

Capped GCLGpolyA

RLU/mg protein

8.0E+09

6.0E+09

4.0E+09

4 2

2.0E+09

4
3.57 hr

(hr)

2.17

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) 1.0E+10
10 8

Capped GCLGpolyA
8.69

RLU/mg protein

8.0E+09

6.0E+09

4.0E+09

4.35

4 2

2.0E+09

1
0.83

(hr)

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

T1/2 = 1.46 hr

Accumulative Life shows the amount of time it takes for the mRNA to reach of the maximum LUC expression.

Results:
3UVR

3 UTR Series
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