Analysis of Nucleotide Sequences and Multimeric Forms of a Novel Satellite RNA Associated with Beet Black Scorch Virus

Abstract

The full-length sequence of a satellite RNA (sat-RNA) of Beet black scorch virus isolate X (BBSV-X) was determined. This agent is 615 nucleotides long and lacks extensive sequence homology with its helper virus or with other reported viruses. Purified virus particles contained abundant single-stranded plus-sense monomers and smaller amounts of dimers. Single-stranded RNAs from total plant RNA extracts also included primarily monomers and smaller amounts of dimers that could be revealed by hybridization, and preparations of purified double-stranded RNAs also contained monomers and dimers. Coinoculation of in vitro transcripts of sat-RNA to Chenopodium amaranticolor with BBSV RNAs was used to assess the replication and accumulation of various forms of sat-RNA, including monomers, dimers, and tetramers. Dimeric sat-RNAs with 5- or 10-base deletions or 15-base insertions within the junction regions accumulated preferentially. In contrast, the replication of monomeric sat-RNA was severely inhibited by five-nucleotide deletions in either the 5' or the 3' termini. Therefore, sequences at both the 5' and the 3' ends of the monomers or the presence of intact juxtaposed multimers is essential for the replication of sat-RNA and for the predomination of monomeric progeny. Comparisons of the time courses of replication initiated by in vitro-synthesized monomeric or multimeric sat-RNAs raised the possibility that the dimeric form has an intermediate role in replication. We propose that replication primarily involves multimers, possibly as dimeric forms. These forms may revert to monomers by a termination of replication at 5' end sequences and/or by internal initiation at the 3' ends of multimeric junctions.