Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up The ability to recover infectious virus from cDNA clones of RNA virus genomes facilitates molecular analyses of every aspect of the viral replicative cycle. For example, the demonstration that a plasmid encoding bacteriophage Q13 RNA gave rise to infectious phage when introduced into Escherichia coli allowed the experimental manipulation of the genomes of Qf3 and other RNA phages (1). The subsequent finding that a cDNA clone of the entire genome of poliovirus gave rise to infectious virus particles when introduced into mammalian cells in culture extended these developments to animal viruses (2). Since then, infectious cDNA clones of a variety of positivesense RNA virus genomes have been constructed, permitting the application of recombinant DNA technology to all aspects of the biology of these viruses (for a review, see ref.3). In contrast to this success with positive-strand RNA viruses, attempts to generate infectious cDNA clones of negativestrand RNA viruses have until recently been unsuccessful. The genomes of negative-strand RNA viruses must be associated with their nucleocapsid protein to form the active ribonucleoprotein template for their RNA-dependent RNA polymerase (4). As a consequence of this and in contrast to positive-strand RNA viruses, naked genomic RNAs of negative-strand viruses are not infectious. Therefore the development of a system in which cDNA transcripts could be associated with the nucleocapsid protein and the viral polymerase was a prerequisite for the successful manipulation of the genomes of negative-strand RNA viruses. Several developments have permitted the manipulation of incomplete genomes of some of these viruses (5-15) and have led to the generation of infectious rabies virus from a positive-sense transcript of a complete genome-length cDNA clone (16).Working with the prototypic nonsegmented negative-strand RNA virus, vesicular stomatitis virus (VSV), our laboratory developed a system for the recovery of infectious defective interfering (DI) particles and VSV subgenomic replicons from cDNA clones (12,17). In these experiments the production of infectious particles was supported entirely by viral proteins expressed in cells from plasmid vectors. These vectors were transcribed following transfection into cultured baby hamster kidney (BHK21) cells that were infected with the vaccinia virus (VV)-T7 RNA polymerase recombinant ref. 18) to provide a source of transcriptase. Negative-sense RNAs were generated from transcription plasmids that encoded subgenomic analogs or naturally occurr...

Homology-dependent RNA silencing occurs in many eukaryotic cells. We reported recently that nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Here, we present further evidence to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway, as the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions as a natural antiviral immunity in mosquito cells. We further show that vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. We found that the double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells. R NA silencing is a unique RNA-guided gene regulatory mechanism that operates in a wide range of eukaryotic organisms from plants to mammals (1). A feature common to all RNA silencing processes is the production of 21-to 26-nt small RNAs from structured or double-stranded RNA (dsRNA) by the endoribonuclease Dicer (2-6). These small interfering RNAs (siRNAs) control the specificity of RNA silencing in a homology-dependent manner by means of an RNA-induced silencing complex (RISC), of which Argonaute-2 (AGO2) is an essential protein component (1,7,8). RNA silencing in fungi, plants, and worms involves a cellular RNA-dependent RNA polymerase (RdRP); however, the multiple-turnover RISC may mediate RNA silencing in absence of a cellular RdRP in Drosophila and mammalian cells (1, 9-11).We reported recently that infection of cultured Drosophila cells with the plus-strand RNA Nodavirus flock house virus (FHV), triggers specific silencing of FHV RNAs that is associated with accumulation of 22-nt siRNAs (12). Silencing of the replicating viral RNAs is RISC-dependent and sensitive to inhibition by the FHV B2 protein, as shown by the observation that B2 is essential for FHV infection of WT Drosophila cells but dispensable in cells depleted for AGO2 (12). These findings provided an example indicating an antiviral role for RNA silencing in the animal kingdom (12, 13), as has been established in higher plants (14)(15)(16)(17)(18).In this article, we report that specific RNA silencing was induced in mosquito cells in response to viral RNA replication and show that this mosquito antiviral immunity is RISCdependent and sensitive to suppression by the B2 protein encoded by either FHV or nodamura virus (NoV). We demonstrate th...

The major surface glycoprotein (G) of human respiratory syncytial (RS) virus has an estimated mature Mr of 84,000-90,000. Among a library of cDNA clones prepared from RS virus mRNAs, we identified clones that hybridized to a message that encoded a M, 36,000 polypeptide that was specifically immunoprecipitated with anti-G antiserum. The amino acid sequence of the G protein backbone was determined by nucleotide sequence analysis of several of the cDNA clones. It contains a combination of structural features that make it unique among the known viral glycoproteins. The G mRNA is 918 nucleotides long and contains a single major open reading frame that encodes a polypeptide having 298 amino acid residues with a Mr of 32,587, a finding consistent with the Mr 36,000 estimate for the in vitro translation product of the G mRNA. This suggests that >50% of the molecular weight ofthe mature glycoprotein may be contributed by carbohydrate. Glycosylation of G is largely resistant to tunicamycin, an inhibitor of the attachment of N-linked oligosaccharides, suggesting that the majority of the carbohydrate residues are attached via O-glycosidic bonds. In accordance with this, serine and threonine residues, the acceptor sites for O-linked oligosaccharides, comprise 30.6% of the total amino acid composition. There are also four potential acceptor sites for N-linked oligosaccharides. The amino acid sequence lacks both an