translational efficiency of dengue virus(den) and den variants in cells
DESCRIPTION
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 . - PowerPoint PPT PresentationTRANSCRIPT
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