Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (−)-sense genomic RNA

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (−)-strand of genomic RNA 1,2 , (2) premature termination during (−)-strand synthesis, followed by independent replication of the subgenomic RNA 3 and (3) processing by nuclease cleavage of genome-length RNA 4 . Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) 5–7 to synthesize the viral subgenomic RNA in vitro , we now provide evidence that subgenomic RNA arises by internal initiation on the (−)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.