Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes

The sequences involved in the replication and packaging of transmissible ga stroenteritis virus (TGEV) RNA have been studied. The structure of a TGEV d efective interfering RNA of 9.7 kb (DI-C) was described previously (A. Mend ez, C. Smerdou, A. Izeta, F. Gebamer, and L, Enjuanes, Virology 217: 495-50 7, 1996), and a cDNA with the information to encode DI-C RNA was cloned und er the control of the T7 promoter. The molecularly cloned DI-C RNA was repl icated in trans upon transfection of helper virus-infected cells and inhibi ted 20-fold the replication of the parental genome. A collection of 14 DI-C RNA deletion mutants (TGEV minigenomes) was synthetically generated and te sted for their ability to be replicated and packaged. The smallest minigeno me (M33) that was replicated by the helper virus and efficiently packaged w as 3.3 kb. A minigenome of 2.1 kb (M21) was also replicated, but it was pac kaged with much lower efficiency than the M33 minigenome, suggesting that i t had lost either the sequences containing the main packaging signal or the required secondary structure in the packaging signal due to alteration of the flanking sequences. The low packaging, efficiency of the M21 minigenome was not due to minimum size restrictions. The sequences essential for mini genome replication by the helper virus were reduced to 1,348 nt and 492 nt at the 5' and 3' ends, respectively. The TGEV-derived RNA minigenomes were successfully expressed following a two-step amplification system that coupl es pol II-driven transcription in the nucleus to replication supported by h elper virus in the cytoplasm, without any obvious splicing. This system and the use of the reporter gene P-glucuronidase (GUS) allowed minigenome dete ction at passage zero, making it possible to distinguish replication effici ency from packaging capability. The synthetic minigenomes have been used to design a helper-dependent expression system that produces around 1.0 mu g/ 10(6) cells of GUS.