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.

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

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

Results:3’ UTR ∆ Series

3’UVR DB1 3’CSSLB SLA

DB2

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