Analysis of the subgenomic RNAs and the small open reading frames of Beet black scorch virus

Abstract

A full-length cDNA of the genome ofBeet black scorch virus(BBSV), isolate Ningxia, was constructed and modified by site-directed mutagenesis to permitin vitrotranscription of mutant viral RNAs. Two subgenomic (sg) RNAs (sgRNA1 and sgRNA2) appeared during BBSV replication. Mutagenesis revealed that sgRNA1 transcription was initiated at G2209within the P82 polymerase subunit open reading frame (ORF) and that transcription of sgRNA2 began at G2526within the nested p7b/p5′ ORF. Initiation-codon shifting or premature termination of translation of the three ORFs (P7a, P7b and P5′) encoded by sgRNA1 indicated that each of the genes was required for localized movement, accumulation of viral RNAs and formation of local lesions on the leaves ofChenopodium amaranticolor. Microscopic observations of the distribution of green fluorescent protein fused to the N-terminal portion of the capsid protein provided additional evidence that the P7a, P7b and P5′ proteins are each required for cell-to-cell movement. In contrast, elimination of sgRNA2 showed that the BBSV coat protein was not required for viral RNA accumulation or the appearance of local lesions onC. amaranticolor. In addition, disruption of the small P5 ORF previously predicted by computer analysis to originate at the C terminus of the P82 ORF had no effect on disease phenotype, suggesting that this ORF may represent a cryptic, non-essential gene. These results show that BBSV has a novel cell-to-cell movement protein organization that differs in size and sequence from that of other viruses.