title: De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene.
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.
authors.affiliation: Centre of Infection & Immunity, School of Medicine, Dentistry & Biomedical Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland.
authors.forename: Olivier
authors.initials: O
authors.lastname: Touzelet
authors.forename: Noureddine
authors.initials: N
authors.lastname: Loukili
authors.forename: Thierry
authors.initials: T
authors.lastname: Pelet
authors.forename: Derek
authors.initials: D
authors.lastname: Fairley
authors.forename: Joseph
authors.initials: J
authors.lastname: Curran
authors.forename: Ultan F
authors.initials: UF
authors.lastname: Power
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics; D016366:Open Reading Frames; D010957:Plasmids; D012313:RNA / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D029082:Sendai virus / Q000235:genetics* / Q000254:growth & development; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; C039919:RNA, recombinant; D011994:Recombinant Proteins; D012313:RNA