Translational efficiency of Dengue 		 	Virus(DEN) and DEN variants in cells.
Download
1 / 15

Translational efficiency of Dengue Virus(DEN) and DEN variants in cells. - PowerPoint PPT Presentation


  • 112 Views
  • Uploaded on

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 .

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Translational efficiency of Dengue Virus(DEN) and DEN variants in cells. ' - tuvya


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

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

Andrew Regan - Microbiology

DREHER LAB - ALS1065


Dengue Virus variants in cells.

Vectored by mosquito


Dengue Virus variants in cells.

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


Dengue Virus variants in cells.

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 variants in cells.

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 variants in cells.

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 variants in cells.

Measure Luciferase activity in cell lysates using a luminometer

Luciferin + Luciferase + ATP + O2

Oxyluciferin + Luciferase + AMP + Light


DEN(S1) attenuated strain variants in cells.

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 variants in cells.

S1CLD

S1CLS1

S1LD

S1LS1


Circularization Sequences variants in cells.

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 variants in cells.

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 variants in cells.

DCLDm

DCmLD

DCmLDm


Initiation Context variants in cells.

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

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


Initiation Context variants in cells.


Acknowledgements variants in cells.

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


ad