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

Xuefeng Yuan

Yunhe Cao

State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100094, PR China

Correspondence
Dawei Li
Lidw{at}cau.edu.cn

A full-length cDNA of the genome of Beet black scorch virus (BBSV), isolate Ningxia, was constructed and modified by site-directed mutagenesis to permit in vitro transcription of mutant viral RNAs. Two subgenomic (sg) RNAs (sgRNA1 and sgRNA2) appeared during BBSV replication. Mutagenesis revealed that sgRNA1 transcription was initiated at G²²⁰⁹ within the P82 polymerase subunit open reading frame (ORF) and that transcription of sgRNA2 began at G²⁵²⁶ within 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 of Chenopodium 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 on C. 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.

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AF452884 (BBSV-N) and AY626780 (BBSV-X).

Details of the primers used in this study are available as supplementary material in JGV Online.

These authors contributed equally to this paper.