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Translational efficiency of Dengue Virus(DEN) and DEN variants in cells. . Andrew Regan - Microbiology. DREHER LAB - ALS1065. Dengue Virus. Vectored by mosquito. Dengue Virus. DEN infection causes: Dengue Fever (DF) fever, headache, rash, muscle pain .

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slide1

Translational efficiency of Dengue Virus(DEN) and DEN variants in cells.

Andrew Regan - Microbiology

DREHER LAB - ALS1065

slide2

Dengue Virus

Vectored by mosquito

slide3

Dengue Virus

DEN infection causes:

Dengue Fever (DF) fever, headache, rash, muscle pain

Four serotypes (DEN1,DEN2, DEN3, DEN4)

Co-infection by two serotypes can cause:

Dengue Hemorrhagic Fever (DHF) and/or Dengue Shock Syndrome (DSS)

can be fatal, especially in children

slide4

Dengue Virus

Positive-strand RNA virus

Viral genome is ssRNA around 11kb contains the codons for synthesis of the viral polyprotein

The 5’Untranslated Region (5’UTR) and 3’UTR are expected to control translational efficiency as they do with typical cellular mRNA

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HHMI Project

To assay changes in translational efficiency between DEN2 and DEN2 variants in cells

STRATEGY: DEN2-luciferase(luc) assay system

Assay was established by doctoral student Wei-Wei Chiu

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Procedures

Clone DEN2-luc (wt) and DEN2-luc variants into dsDNA plasmids

Linearize plasmids, cutting at the 3’-terminal cleavage site

Transcribe constructs with T7 RNApol

Transfect RNA into Vero cells (monkey kidney)

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Procedures

Measure Luciferase activity in cell lysates using a luminometer

Luciferin + Luciferase + ATP + O2

Oxyluciferin + Luciferase + AMP + Light

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DEN(S1) attenuated strain

Live virus vaccines utilize attenuated strains

Examples: Polio, Measles, Smallpox, etc…

DEN(S1) is an attenuated strain of DEN2

An attenuated virus has accumulated mutations that disable its ability to cause severe disease

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DCLS1

S1CLD

S1CLS1

S1LD

S1LS1

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Circularization Sequences

Two 11-nt sequences are conserved among all DEN serotypes, as well as all close relatives

(e.g. West Nile Virus, Yellow Fever Virus, etc…)

Shiyun, You & R. Padmanabhan. A Novel in vitro Replication System for Dengue Virus. J Biological Chem. 1999. 274:45 33714-33722

Khromykh AA, Meka H, Guyatt KJ & EG Westaway. Essential role of cyclization sequences in flavivirus RNA replication. J Virology. 2001. 75:6719-28.

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Circularization Sequences

CS1 and cCS1 allow intramolecular circularization of the viral genome

The model has similarities to the protein-facilitated circularization of highly expressed cellular mRNA

slide12

DCLD-wt

DCLDm

DCmLD

DCmLDm

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Initiation Context

40S subunit scans 5’ 3’ for the initiation codon

Nucleotides +6 through -4 are known to be important in halting ribosomal migration

slide15

Acknowledgements

Wei-Wei Chiu - OSU Microbiology

Dr. Theo W. Dreher - OSU Microbiology - OSU Center for Gene Research

Dr. Kevin G. Ahern - OSU Biochemistry & Biophysics

Howard Hughes Medical Institute

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