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De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene
|Published in||Virus Research. 2009, vol. 140, no. 1-2, p. 40-48|
|Abstract||Reverse genetics has facilitated the use of non-segmented negative strand RNA viruses (NNSV) as vectors. Currently, heterologous gene expression necessitates insertion of extra-numeral transcription units (ENTUs), which may alter the NNSV polar transcription gradient and attenuate growth relative to wild-type (Wt). We hypothesized that rescuing recombinant Sendai Virus (rSeV) with a bicistronic gene might circumvent this attenuation but still allow heterologous open reading frame (ORF) expression. Therefore, we used a 9-nucleotide sequence previously described with internal ribosome entry site (IRES) activity, which, when constructed as several repeats, synergistically increased the level of expression of the second cistron [Chappell, S.A., Edelman, G.M., Mauro, V.P., 2000. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U.S.A. 97, 1536-1541]. We inserted the Renilla luciferase (rLuc) ORF, preceded by 1, 3 or 7 IRES copies, downstream of the SeV N ORF in an infectious clone. Corresponding rSeVs were successfully rescued. Interestingly, bicistronic rSeVs grew as fast as or faster than Wt rSeV. Furthermore, SeV gene transcription downstream of the N/rLuc gene was either equivalent to, or slightly enhanced, compared to Wt rSeV. Importantly, all rSeV/rLuc viruses efficiently expressed rLuc. IRES repetition increased rLuc expression at a multiplicity of infection of 0.1, although without evidence of synergistic enhancement. In conclusion, our approach provides a novel way of insertion and expression of foreign genes in NNSVs.|
|Keywords||Animals — Base Sequence — Cell Line — DNA, Complementary/genetics — Gene Expression Regulation, Viral — *Genetic Vectors — Genome, Viral — Humans — Molecular Sequence Data — Nucleocapsid Proteins/genetics — Open Reading Frames — Plasmids — RNA/*genetics — RNA, Viral/genetics — Recombinant Proteins/*biosynthesis — Sendai virus/*genetics/growth & development — Transcription, Genetic|
|Research group||Régulation traductionnelle dans les cellules de mammifère (631)|
|TOUZELET, Olivier et al. De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene. In: Virus Research, 2009, vol. 140, n° 1-2, p. 40-48. https://archive-ouverte.unige.ch/unige:20133|