The Infectious Reovirus RNA - Reverse Genetics System: The Assembly of the Reovirus Genome
Michael R. Roner and Wolfgang K. Joklik
from: Segmented Double-stranded RNA Viruses: Structure and Molecular Biology (Edited by: John T. Patton). Caister Academic Press, U.K. (2008)
Viruses of the Reoviridae family, which encompasses nine genera and more than 200 members, possess genomes that comprise ten or twelve double-stranded (ds) RNA segments. Using reovirus as a model, we have developed a functional reverse genetics system for these viruses, by means of which alterations introduced artificially into their genomes are transferred to the genomes of viable infectious virus particles. The system consists of (a) the plus strands of nine wild type reovirus genome segments; (b) ssRNA transcripts generated from a genetically modified cDNA that replace the wild type tenth genome segment; and (c) a cell line transformed to express the protein normally encoded by the tenth genome segment. With this system, we have generated ST3 reoviruses with an engineered S2 genome segment, an M1 genome segment, and both S2 and M1 genome segments. Both the S2 and M1 engineered genes contain a functional CAT gene. The viruses are stable, replicate in cells that have been transformed to express the S2 gene product protein σ2, the M1 gene product protein μ2 or both, and express high levels of CAT activity. This technology, which we are extending to the orbiviruses and rotavirus, provides a powerful system for basic studies of dsRNA virus replication and modification read more ...