De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene.

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.

Suppression of the Sendai virus M protein through a novel short interfering RNA approach inhibits viral particle production but does not affect viral RNA synthesis.

Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3' untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50- to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.

High throughput screening assay for negative single stranded RNA virus polymerase inhibitors.

The Paramyxoviridae form a large family of viruses containing many human and veterinary pathogens for which a need for antiviral treatment is emphasized, particularly following the recent emergence of new viruses. The viral RNA-dependent RNA polymerase constitutes an obvious target for antiviral compounds. An in vitro assay was developed that allows high throughput screening of compounds potentially inhibiting the Sendai virus RNA-dependent RNA polymerase. Screening relies on the detection of the Photinus pyralis luciferase produced in a transcription/translation coupled assay using a mini-replicon virus. It contains an internal control for possible adverse effects of the tested compounds on translation or on luciferase activity. It is estimated that the mini-replicon template produced in one fertilized egg is sufficient to run 5000-10,000 reactions. This assay constitutes a simple, sensitive and easily automated method to perform high throughput screening of Paramyxoviridae RNA-dependent RNA polymerase inhibitors.

Identification of a cis-acting element required for shunt-mediated translational initiation of the Sendai virus Y proteins.

Shunting is a mechanism that permits translational initiation at internal codons positioned in proximity to a ribosome acceptor sequence. Sendai virus exploits shunting to express a series of proteins that initiate at the fourth and fifth start sites on the P/C mRNA (namely, the Y1 and Y2 proteins, respectively). Shunt-mediated initiation at these sites is codon independent. In an attempt to characterise the acceptor site, an extensive deletion analysis was performed spanning the entire C ORF. Only mutants flanking the Y1/Y2 start sites exhibited altered shunt phenotypes. Some of these significantly enhanced shunting efficiency to the point where the Y1/Y2 proteins were the major translational products of the mRNA. Additionally, removal of a short region just downstream of the Y2 start codon (referred to as Delta10) ablated all Y protein initiation via shunting but had no effect on Y expression when the AUG codons were viewed by a scanning ribosome. Point mutations introduced into this Delta10 sequence severely perturbed shunt-mediated initiation. We also provide evidence that changes in this region of the P/C mRNA may be used to modulate Y protein expression levels in different viral strains.