MOLECULAR CHARACTERIZATION OF TRANSMISSIBLE GASTROENTERITIS CORONAVIRUS DEFECTIVE INTERFERING GENOMES - PACKAGING AND HETEROGENEITY

Three transmissible gastroenteritis virus (TGEV) defective RNAs were s elected by serial undiluted passage of the PUR46 strain in ST cells. T hese RNAs of 22, 10.6, and 9.7 kb (DI-A, DI-B, and DI-C, respectively) were detected at passage 30, remained stable upon further passage in cell culture, and significantly interfered with helper mRNA synthesis. RNA analysis from purified virions showed that the three defective RN As were efficiently packaged. Virions of different densities containin g either full-length or defective RNAs were sorted in sucrose gradient s, indicating that defective and full-length genomes were independentl y encapsidated. DI-B and DI-C RNAs were amplified by the reverse trans cription-polymerase chain reaction, cloned, and sequenced. DI-B and DI -C genomes are formed by three and four discontinuous regions of the w ild-type genome, respectively. DI-C contains 2144 nucleotides (nt) fro m the 5'-end of the genome, two fragments of 4540 and 2531 nt mostly f rom gene 1b, and 493 nt from the 3' end of the genome. DI-B and DI-C R NAs include sequences with the pseudoknot motif and encoding the polym erase, metal ion binding, and helicase motifs. DI-B RNA has a structur e closely related to DI-C RNA with two main differences: it maintains the entire ORF-1b and shows heterogeneity in the sire of the 3' end de letion. This heterogeneity maps at the beginning of the S gene, where other natural TGEV recombination events have been observed, suggesting that either a process of template switching occurs with high frequenc y at this point or that the derived genomes have a selective advantage . (C) 1996 Academic Press, Inc.