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Use and function of a viral translation enhancer
The first two thirds of the SFV genome (~ 12 kb) encodes the non-structural proteins (nsPs) required for the replication of the genome, and the last third codes for the structural
proteins; capsid and spikes. The virus genome functions directly as an mRNA for the nsPs when it enters the cell. The nsPs will then make a negative strand RNA using the genomic RNA as a template. This negative template is then used to make both new positive full-length RNA and large amounts of subgenomic RNA coding for a polypeptide containing the capsid and the spikes. SFV replication within the cells shuts down the host cell protein synthesis, but the synthesis of the viral structural proteins remain high in part due to an enhancer sequence located in the first part of the capsid
RNA. This enhancer sequence is commonly used to increase expression of
foreign proteins from the SFV vector. The aim of this project it to
investigate the function and requirement of this enhancer. The present model is that factors required for initiation of protein synthesis are depleted in infected cells but that the viral subgenomic RNA in some way is able to recruit the few remaining factors for its own translation.
We pose two separate questions:
1. What is the mechanism behind the enhancers function
in infected cells?
2. Can this enhancer be used to increase expression from
an unrelated promoter during infection?
Some of the methods you will use are FACS, in vitro translation and transfection of mammalian cells. You will also do some basic cloning, site directed mutagenesis and packaging of virus vectors into virus particles. There will be a possibility for you to participate in the planning and the design of the project.
SJOBERG EM, SUOMALAINEN M, GAROFF H. A significantly improved Semliki Forest virus expression system based on translation enhancer segments from the viral capsid gene. Biotechnology (N Y). 1994 Nov;12(11):1127-31.
SJOBERG EM, GAROFF H. The translation-enhancing region of the Semliki Forest virus subgenome is only functional in the virus-infected cell. J Gen Virol. 1996
Jun;77 ( Pt 6):1323-7.
FROLOV I, SCHLESINGER S. Translation of Sindbis
virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation. J Virol. 1996 Feb;70(2):1182-90.
In the Vaccine
group at SMI/MTC we develop recombinant SFV vectors to be used in
vaccination against for example Hepatitis C and HIV. We take part in two different programs for HIV vaccine development and we are in a very expansive phase. This is an examensarbete that suits a student with the ambition to continue doing a PhD in virology, biotechnology or vaccinology.
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