Alphavirus replicon approach to promoterless analysis of IRES elements

Alphavirus replicon approach to promoterless analysis of IRES elements

Abstract Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae ) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region up...

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Bibliographic Details
Published in:Virology (New York, N.Y.) Vol. 360; no. 2; pp. 376 - 387
Main Authors: Kamrud, K.I, Custer, M, Dudek, J.M, Owens, G, Alterson, K.D, Lee, J.S, Groebner, J.L, Smith, J.F
Format: Journal Article
Language:English
Published: United States Elsevier Inc 10-04-2007
Subjects:
Alphavirus
Alphavirus - genetics
Animals
Antibodies, Bacterial - blood
Blotting, Northern
Blotting, Western
Botulinum Toxins - biosynthesis
Botulinum Toxins - immunology
Botulism - prevention & control
Chloramphenicol O-Acetyltransferase - biosynthesis
Chloramphenicol O-Acetyltransferase - genetics
Dicistronic vector
EMCV
Enzyme-Linked Immunosorbent Assay
EV71
Genes, Reporter
Genetic Vectors
Infectious Disease
internal ribosome entry site
IRES
Mice
Molecular Biology - methods
promoter regions
Promoterless
Protein Biosynthesis - genetics
Replicon
reporter genes
structural genes
Togaviridae
Untranslated Regions
VEE
IRES
Promoterless
VEE
Alphavirus
Dicistronic vector
EMCV
EV71
Replicon
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Description
Summary:Abstract Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae ) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced > 95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.
Bibliography:http://dx.doi.org/10.1016/j.virol.2006.10.049
ObjectType-Article-1
SourceType-Scholarly Journals-1
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ISSN:0042-6822
1096-0341
DOI:10.1016/j.virol.2006.10.049