We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which the CAT gene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein 2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens. T he reovirus genome comprises 10 segments of doublestranded (ds)RNA that are transcribed nonconservatively within cores into plus strands which are extruded from virus particles by means of 12 icosahedrally distributed projections͞ spikes. These plus strands exercise two functions: they are translated into the reovirus-encoded proteins and they are transcribed into minus strands with which they remain associated, thereby generating the progeny dsRNA genome segments (1, 2).Several years ago, we demonstrated that reovirus RNA is infectious (3). The basic features of the infectious reovirus RNA system are as follows. Monolayers of L929 mouse fibroblasts are lipofected with the 10 species of plus strands [single-stranded (ss)RNA] or dsRNA of reovirus serotype (ST)3, with a rabbit reticulocyte lysate in which ssRNA or melted dsRNA has been translated for 60 min. Eight hours later, the cells are infected with reovirus ST1 or ST2 as helper viruses. Virus yields are harvested 24 or 48 h later and plaqued either in the presence of antiserum against ST1 virus if it was the helper virus, or alone if the helper virus was ST2 virus. (ST3 virus forms plaques after 5 days, whereas ST2 virus forms plaques only after 12 days.) No reassortants are formed. All 10 species of RNA are necessary. Virus yields are 20 times higher if ds-rather than ssRNA is used, and if both are used, they are 10 times higher still, because dsRNA increases the infectivity of ssRNA. Lipofection of RNA translation mixtures is not essential but increases virus yields 100-fold.This system can be used to generate novel viruses by controlling the nature of the 10 RNA species that are lipofected into cells. Thus, we have constructed a double temperature-sensitive (ts) mutant of ST3 virus by using (together with 8 ssRNA species of ST3 virus) ssRNA species that contain ts mutat...
Background: Understanding how an organism replicates and assembles a multi-segmented genome with fidelity previously measured at 100% presents a model system for exploring questions involving genome assortment and RNA/ protein interactions in general. The virus family Reoviridae, containing nine genera and more than 200 members, are unique in that they possess a segmented double-stranded (ds) RNA genome. Using reovirus as a model member of this family, we have developed the only functional reverse genetics system for a member of this family with ten or more genome segments.
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