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

Andrew Regan - Microbiology

DREHER LAB - ALS1065

Dengue Virus

Vectored by mosquito

WHO Regions1998 1999 2000 2001

cases deaths cases deaths cases deaths cases deathsWestern Pacific 356,554 1,470 64,066 112 45,603 167 NA NA

South-East Asia 218,859 2,075 55,405 471 57,997 542 119,707 452

Americas (DF) 708,146 0 317,040 0  394,847 0  400,875 0 

Americas (DHF) 12,426 83  5,216 98  5,667 92  5,331 44 

Americas (total) 720,572 83 322,256 98 400,514 92 406,206 44

Mediterranean No dengue cases reported to WHO

African No dengue cases reported to WHO

World 1,295,985 3,628 441,727 681 504,114 801 525,913 496

Dengue Virus

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

DEN infection causes: Dengue Fever (DF) fever, headache, rash, muscle pain

Dengue Virus

Positive-strand RNA virus Viral genome is ssRNA

around 11kbcontains 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

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

ProceduresClone 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)

ProceduresMeasure Luciferase activity in cell lysates using a luminometer

Oxyluciferin + Luciferase + AMP + Light

Luciferin + Luciferase + ATP + O2

DEN(S1) attenuated strainLive 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

S1CLS1

S1CLD

DCLS1

S1LD

S1LS1

Circularization SequencesTwo 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.

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

CS1 and cCS1 allow intramolecular circularization of the viral genome

Circularization Sequences

DCLD-wt

DCLDm

DCmLD

DCmLDm

Initiation Context

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

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

Initiation Context

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