Andrew Regan - Microbiology
Translational efficiency of Dengue
Virus(DEN) and DEN variants in cells.
DREHER LAB - ALS1065
Dengue Virus
Vectored by mosquito
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
WHO Regions
1998
1999
2000
2001
cases
deaths
cases
deaths
cases
deaths
cases
deaths
Western 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
525,913
496
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
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
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
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)
Procedures
Measure Luciferase activity in cell lysates using a luminometer
Luciferin + Luciferase + ATP + O2
Oxyluciferin + Luciferase + AMP + Light
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
DCLS1
S1CLD
S1CLS1
S1LD
S1LS1
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.
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
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