GENES REQUIRED FOR REPLICATION OF THE 15.5-KILOBASE RNA GENOME OF A PLANT CLOSTEROVIRUS

A full-length cDNA clone of beet yellows closterovirus (BW) was engine ered and used to map functions involved in the replication of the vira l RNA genome and subgenomic RNA formation. Among 10 open reading frame s (ORFs) present in BYV, ORFs 1a and 1b suffice for RNA replication an d transcription. The proteins encoded in these ORFs harbor putative me thyltransferase, RNA helicase, and RNA polymerase domains common to Si ndbis virus-like viruses and a large interdomain region that is unique to closteroviruses. The papain-like leader proteinase (L-Pro) encoded in the 5'-proximal region of ORF la was found to have a dual function in genome amplification. First, the autocatalytic cleavage between L- Pro and the remainder of the ORF la product was essential for replicat ion of RNA. Second, an additional L-Pro function that was separable fr om proteolytic activity was required for efficient RNA accumulation. T he deletion of a large, similar to 5.6-kb, 3'-terminal region coding f or a 6-kDa hydrophobic protein, an HSP70 homolog, a 64-kDa protein, mi nor and major capsid proteins, a 20-kDa protein, and a 21-kDa protein (p21) resulted in replication-competent RNA. However, examination of m utants,vith replacements of start codons in each of these seven 3'-ter minal ORFs revealed that p21 functions as an enhancer of genome amplif ication. The intriguing analogies between the genome organization and replicational requirements of plant closteroviruses and animal coronav irus-like viruses are discussed.