Alphavirus replicon approach to promoterless analysis of IRES elements.
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.
Kamrud
K I
KI
AlphaVax, Inc., 2 Triangle Drive, Research Triangle Park, NC 27709-0307, USA. kamrud@alphavax.com
Custer
M
M
Dudek
J M
JM
Owens
G
G
Alterson
K D
KD
Lee
J S
JS
Groebner
J L
JL
Smith
J F
JF
D000524:Alphavirus / Q000235:genetics*; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D015152:Blotting, Northern; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000096:biosynthesis / Q000276:immunology; D001906:Botulism / Q000517:prevention & control; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D017930:Genes, Reporter; D005822:Genetic Vectors; D051379:Mice; D008967:Molecular Biology / Q000379:methods*; D014176:Protein Biosynthesis / Q000235:genetics*; D012093:Replicon; D020506:Untranslated Regions
D000907:Antibodies, Bacterial; D020506:Untranslated Regions; D015500:Chloramphenicol O-Acetyltransferase; D001905:Botulinum Toxins