Requirement for a viral trans-acting factor encoded by brome mosaic virus RNA-2 provides strong selection in vivo for functional recombinants

Interaction of specific nucleotide sequences with trans-acting proteins is intrinsic to replication of viral as well as eucaryotic genomes. Brome mosaic virus RNA-2 encodes one of the two viral proteins known to be essential for replication. (R. French, M. Janda, and P. Ahlquist, Science 231:1294-1297, 1986; P. A. Kiberstis, L. S. Loesch-Fries, and T. C. Hall, Virology 112:804-808, 1981). Transfection of barley protoplasts with wild-type transcripts of brome mosaic virus RNA-1 and RNA-3 and serial dilutions of RNA-2 transcripts possessing unaltered coding sequences but bearing mutations that greatly incapacitated replication of RNA-2 revealed that trace amounts of RNA-2 are sufficient to support replication of the viral genome. In six replicate experiments containing RNA-2 transcripts devoid of the 3' 200 nucleotides that encompass the tRNA-like structure containing the minus-strand promoter, detectable levels of progeny RNA-1 and RNA-3 and subgenomic RNA-4 were present. This showed that viral p2 protein translated from the supplied RNA-2 functioned in trans to support replication of RNA-1 and RNA-3. However, in two similar experiments, progeny RNA-2 with electrophoretic mobility indistinguishable from that of wild-type RNA-2 was seen at 24 h postinoculation. Northern hybridization (RNA blot) analysis confirmed the presence of the tRNA-like 3' terminus on these progeny RNAs, indicating that recombinational restoration of the deleted sequence had occurred. This suggests that, under certain circumstances, RNA recombination may be a rapid and frequent phenomenon.