Flock house virus (FHV),Flock house virus (FHV) is a positive-strand RNA virus whose small genome, high-level replication, and other characteristics make it an attractive model for analyzing positivestrand RNA virus replication and assembly (6). FHV is the best-studied member of the Nodaviridae, the family with the smallest genomes of any animal positive-strand RNA virus. The Nodaviridae family contains viruses infecting a variety of invertebrates and vertebrates, including some viruses that cause lethal neuropathologies in fish (9,30,31). FHV was originally isolated from the grass grub Costelytra zealandica, near the Flock House agricultural research station in Bulls, New Zealand (13). FHV also replicates robustly in Drosophila melanogaster cells, converting 20% of total cell protein to viral protein (14, 16), and directs RNA replication in mammalian cells (5).The FHV genome consists of two RNAs, which are both packaged in a single, nonenveloped, icosahedral virion (17, 37). RNA1 (3.1 kb) serves as mRNA for protein A (112 kDa), which contains the Gly-Asp-Asp (GDD) amino acid motif characteristic of RNA polymerases and which is essential for FHV RNA replication (4, 15). FHV RNA2 (1.4 kb) encodes the capsid protein precursor (43 kDa) (11,15). RNA2 thus replicates only in the presence of RNA1, while RNA1 replicates efficiently in the absence of RNA2.RNA1 (see Fig. 1) also encodes a subgenomic RNA3 (0.4 kb) containing two overlapping open reading frames (ORFs) encoding proteins B1 and B2 (20). The protein B1 ORF is the 3Ј end of the protein A ORF, which extends into RNA3. Translation of this B1 fragment (10.8 kDa) of protein A is not required for any step of the FHV life cycle, and the presence of an initiation codon in the corresponding region of the protein A ORF is not conserved in other nodaviruses (22). Protein B1 production is also inefficient, perhaps because the 7-nucleotide (nt) 5Ј untranslated region preceding its initiation codon is too short for efficient translation. Protein B2, translated from the second AUG in RNA3, accumulates to 20-fold-higher levels than B1 and can represent up to 5% of total cell protein (14). Protein B2 is not required for RNA1 replication or RNA3 synthesis in single-cycle replication assays but is required for maintenance of RNA1 replication upon serial RNA passaging (4). Based on these results, it was suggested that protein B2 might contribute to the fidelity of RNA replication or to regulating the balance of RNA1 translation and replication (4).We previously showed that, following transfection of FHV virion RNA into the yeast Saccharomyces cerevisiae, FHV undergoes its complete replication cycle, including the production of infectious virions (34). Furthermore, we showed that an FHV RNA2-derived replicon could express a yeast reporter gene (34). However, the free RNA replicons are readily lost from dividing yeast populations, which would greatly inhibit

Here we describe the first instances to our knowledge of animal virus genome replication, and of de novo synthesis of infectious virions by a nonendogenous virus, in the yeast Saccharomyces cerevisiae, whose versatile genetics offers significant advantages for studying viral replication and virus-host interactions. Flock house virus (FHV) is the most extensively studied member of the Nodaviridae family of (+) strand RNA animal viruses. (5), and mammalian (6) cells, suggesting that host factors necessary for its replication are highly conserved. An FHV plaque assay (7) and neutralizing antibodies (8) allow sensitive detection of FHV virions. Infectious transcripts from full-length cDNAs can be produced in vitro (9) and in vivo (10). The cis elements needed for genome replication have been characterized (11, 12) and FHV RNAs have been used to carry and express heterologous sequences (13). In addition, a replicase capable of full in vitro replication of added FHV RNA has been isolated from infected cells (14,15).The FHV genome is bipartite (Fig. IA) MATERIALS AND METHODSPlasmids. To construct plR and pl(fs)R (see Results), the 5.3-kb ScaI-NarI fragment of pFHV1 [1,0] (10), containing an FHV RNA1 cDNA flanked 1 nt from its 5' end by a T7 promoter and 0 nt from its 3' end by a hepatitis delta virus ribozyme and a T7 terminator, was transferred to the 3.0-kb NarI-ScaI fragment of the yeast shuttle plasmid YEplac12 (24). For pl(fs)R, the EagI site at position 373 of the FHV RNA1 cDNA was digested, filled in, and religated. The 0.17-kb Sacl fragment containing the T7 terminator was deleted from plR and pl(fs)R.To construct the plasmid pDU (see

During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.Nodamura virus (NoV) is the type species of the genus Alphanodavirus of the Nodaviridae, a family of small riboviruses with bipartite, positive-strand RNA genomes that also includes Flock House virus (FHV). NoV is unique among alphanodaviruses in its ability to lethally infect both insects and mammals, including the mosquitoes Aedes aegypti, Aedes albopictus, and Toxorhynchites amboinensis (4, 36, 41), suckling mice, and suckling hamsters (17,35,36). NoV infects cultured mosquito cells from Aedes pseudoscutellaris, A. aegypti, and A. albopictus (1, 3, 41) and cultured baby hamster kidney BHK21 cells (3,23,30). When NoV genomic RNAs are introduced by transfection, they can replicate in a wide range of cultured cells (6).The divided nodavirus genome naturally separates the replicative and packaging functions onto two different positivesense RNA molecules, RNA1 and RNA2, respectively. These two genomic RNAs are copackaged into the same virion, and both are required for infectivity (24,30,38). RNA1 encodes protein A, the RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of both genome segments. RNA2 encodes the viral capsid precursor protein, ␣. RNA2 and protein ␣ are dispensable for RNA1 replication. Protein A also catalyzes the synthesis of a single subgenomic RNA3 from an RNA1 template. RNA3 is not packaged into virus particles. For FHV, RNA3 encodes two small proteins, B1 and B2, in overlapping reading frames. Protein B1 is in the same reading frame as protein A and thus represents its C-terminal fragment, whereas protein B2 is in the ϩ1 reading frame relative to protein A (11). For NoV, the first and second AUG codons of RNA3 initiate the translation of two forms of B2 (B2-137 and B2-134) that differ only at the N terminus, whereas B1 initiates at the third AUG codon. As for FHV, the B2 proteins are in the ϩ1 reading frame relative to the A/B1 open reading frame (ORF) (23). All three proteins are detected in cells transfected with NoV RNA1 (NoV1), which replicates autonomously and leads to the synthesis of RNA3 (23).The functions of the nodavirus B1 and B2 prote...

Nodamura virus (NoV) was the first isolated member of the Nodaviridae, and is the type species of the alphanodavirus genus. The alphanodaviruses infect insects; NoV is unique in that it can also lethally infect mammals. Nodaviruses have bipartite positive-sense RNA genomes in which RNA1 encodes the RNA-dependent RNA polymerase and the smaller genome segment, RNA2, encodes the capsid protein precursor. To facilitate the study of NoV, we generated infectious cDNA clones of its two genomic RNAs. Transcription of these NoV1 and NoV2 cDNAs in mammalian cells led to viral RNA replication, protein synthesis, and production of infectious virus. Subgenomic RNA3 was produced during RNA replication and encodes nonstructural proteins B1 and B2 in overlapping ORFs. Site-directed mutagenesis of these ORFs, followed by SDS-PAGE and MALDI-TOF mass spectrometry analyses, showed synthesis of B1 and two forms of B2 (B2-134 and B2-137) during viral replication. We also characterized a point mutation in RNA1 far upstream of the RNA3 region that resulted in decreased RNA3 synthesis and RNA2 replication, and a reduced yield of infectious particles. The ability to reproduce the entire life cycle of this unusual nodavirus from cDNA clones will facilitate further analysis of NoV RNA replication and pathogenesis.

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