Reovirus Reverse Genetics: Incorporation of the CAT Gene into the Reovirus Genome

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 98; no. 14; pp. 8036 - 8041
• Main Authors: Roner, Michael R., Joklik, Wolfgang K.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 03-07-2001; National Acad Sciences; The National Academy of Sciences
• Subjects: Biological Sciences; CAT gene; Cell lines; Chloramphenicol O-Acetyltransferase - genetics; Complementary DNA; Double stranded RNA; Gene Expression Regulation, Viral; Gene Transfer Techniques; Genetic Vectors; Genetics; Genome, Viral; Genomes; Humans; Microbiology; Nucleotides; protein 2; Proteins; Reoviridae; Reoviridae - genetics; Reovirus; Reverse genetics; Ribonucleic acid; RNA; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.131203198

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 σ2), 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.