Over the last decade, RNA interference (RNAi), mediated by short interfering double-stranded RNA molecules (siRNA or dsRNA), has been gradually recognized as a major mechanism of post-transcriptional gene silencing (PTGS) in species as diverse as plants, Drosophila, and C. elegans. Recently, 21-nucleotide long dsRNA molecules corresponding to specific mRNA sequences, when introduced into mammalian cells in culture, have been shown to be highly effective in degrading the cognate mRNAs and thus abrogating the expression of the corresponding proteins . Although the exact mechanism of PTGS is currently unknown and is an area of intense research, the successful use of this phenomenon in cultured mammalian cells has raised the exciting prospect that it can be used as a simple strategy for phenotypic ablation of mammalian gene function ex vivo without the time-consuming and expensive construction of transgenic animals.
So far, the genes targeted for siRNA-mediated PTGS have been cellular in origin and thus, the mRNAs were transcribed in the nucleus by cellular DNA-dependent RNA polymerase. In contrast, the vast majority of RNA genomes are transcribed exclusively in the cytoplasm. For example, transcription of RNA viral genomes, with the exception of retroviral RNA, is catalyzed by a virally encoded RNA-dependent RNA polymerase (RdRP) . It remains untested whether the siRNA-mediated PTGS will work on mRNAs that never went through the nucleus. Interestingly, because of the potency of siRNA in some organisms, it has been proposed that they may be replicated by a cellular RdRP activity, although this has been debated . Furthermore, since dsRNA is known to be a potent inducer of the interferon pathway [5, 6], it is important to know whether this also occurs in cells in which antisense dsRNA has been introduced.
To answer these questions, we have used a nonsegmented negative-strand RNA (NNR) virus as a test target for RNA-mediated inhibition. We reasoned that, if successful, our studies would additionally contribute a reliable and simple technology for specific gene silencing in cytoplasmic RNA viruses. Traditionally, structure-function analyses of RNA genomes, including those of RNA viruses, have relied on spontaneous mutants found in natural isolates or chemically mutagenized stocks . Mutations in either case are essentially unpredictable and must be mapped by elaborate techniques such as classical complementation analyses or direct sequencing of the genome. Since conventional site-directed mutagenesis requires a DNA template, direct mutational analysis of selected RNA genes is not an option. These obstacles have been largely circumvented by the use of cloned viral cDNA that is then altered by standard DNA-based site-directed mutagenesis procedures [8, 9]. Originally designed for influenza virus minigenomes , such cDNA-based "reverse genetics" strategy has been adopted in a large number of NNR viruses, including vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), and measles, to name a few [11–14]. Recently, extension of this approach has resulted in the cloning of full-length viral cDNA capable of producing infectious recombinant virus particles upon transcription.
Despite its revolutionary effect on RNA viral reverse genetics, however, the cDNA-based strategy is not without limitations. First, as implied above, cloning and recombinant expression of the viral genomic RNA and all the viral proteins in the right proportion constitute a long and arduous task, daunting to an average laboratory. The problem is particularly acute for NNR viruses, which have large RNA genomes [8, 9]. The 10–15 kb long RNA genomes of these viruses require specific sequences at the 5' and 3' termini for transcription and replication, and must be properly encapsidated by the nucleocapsid protein (N) in order to be recognized by the viral RdRP [15, 16]. Thus, any recombinant technology must be able to faithfully reproduce these features of the genome. Moreover, the functional viral RdRP, as detailed later for RSV, is a complex holoenzyme composed of viral as well as cellular proteins [17–23]. Second, many NNR viral genomes and proteins are currently expressed from vaccinia-based cDNA clones, which generally requires superinfection by vaccinia virus [22, 23]. Unfortunately, vaccinia virus itself is a major modulator of cellular signaling, including MAP kinase pathways and the actin cytoskeleton [24–26]. It is, therefore, virtually impossible to study the interaction between cellular signaling pathways and NNR viruses in cells that are also superinfected by vaccinia virus [24–26]. Third, mutations in the recombinant DNA are "permanent", and thus, the mutational phenotype cannot be switched on at pre-determined time points in infection. For example, if a viral gene product has essential roles both early and late in infection, its mutational inactivation will fail to reveal the late function, since the mutant virus will never proceed beyond the early stage.
A member of the Paramyxoviridae family, RSV is a major causative agent of childhood respiratory disease and asthma . Pediatric RSV disease claims about a million lives annually, and no reliable antiviral or vaccine currently exists [27, 28]. A need to understand the molecular genetics of the virus and the function of the various gene products has thus been appreciated. Since our laboratory is interested in deciphering the temporal signaling pathways in host-RSV interaction and the role of RSV gene products in the process, we have sought potential alternatives to the cDNA-based approach that might allow us to study the effect of functional loss of a specific RSV gene product during the course of a standard virus infection in cell culture. Using two different RSV gene mRNAs as targets, and another NNR virus (VSV) as well as cellular mRNAs as controls, we show that synthetic dsRNA molecules are highly efficient and specific silencers of cytoplasmic RNA viral gene expression. We also provide the first direct evidence that the 21-nt long dsRNAs do not activate a general interferon response. Our results thus offer a mechanism of specific and direct ablation of RNA-based gene expression and a quicker and simpler alternative to cDNA-based reverse genetics of RNA viruses.