Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors.
We describe replication competent retroviruses capable of expressing heterologous genes during multiple rounds of infection. An internal ribosome entry site (IRES) from encephalomyocarditis virus was inserted in the U3 region of Akv- and SL3-3-murine leukemia viruses (MLV) to direct translation of neo or the enhanced green fluorescence protein gene (EGFP). Akv-MLV's with IRES-neo and IRES-EGFP cassettes replicated with titers of about 10(6) infectious units/ml while SL3-3-MLV with IRES-neo gave about 10(3)-fold lower titers. Interestingly, RNA analysis showed a drastic reduction in the amount of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds and these revertants only slowly became dominant in the virus population.
Jespersen
T
T
Department of Molecular and Structural Biology, University of Aarhus, DK-8000, Arhus, Denmark.
Duch
M
M
Carrasco
M L
ML
Warming
S
S
Pedersen
F S
FS
D016475:3T3 Cells; D000818:Animals; D004279:DNA, Viral / Q000235:genetics*; D004686:Encephalomyocarditis virus / Q000235:genetics; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000254:growth & development; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014170:Transformation, Genetic; D014779:Virus Replication
D004279:DNA, Viral; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012313:RNA; D019868:Kanamycin Kinase