Similar Structure and Reactivity of Satellite Tobacco Ringspot Virus RNA Obtained from Infected Tissue and by in Vitro Transcription

Abstract

The 359 nucleotide residue (nt) satellite RNA of tobacco ringspot virus increases detectably only in association with replicating tobacco ringspot virus and becomes encapsidated in the virus coat protein. Results from previous reports are consistent with participation of rolling circle transcription in replication of the satellite RNA: (i) both the more abundant plus polarity satellite RNA, s(+)RNA and the complementary s(-)RNA occur in multimeric forms that self-cleave to release the unit length, 359 nt satellite RNA, (ii) circles of both s(+)RNA and s(-)RNA are present in extracts of infected tissue, and (iii) s(-)RNA, but not S(+)RNA, spontaneously and efficiently circularizes in vitro. Our analyses of RNA in tissue extracts suggest that s(+)RNA of all forms is about 100-fold more abundant than s(-)RNA. Nucleic acids were purified rapidly to minimize interconversion of linear and circular forms. For s(+)RNA and for s(-)RNA, the circular and the linear forms were detected in about equal amounts in tissue extracts. The linear s(-)RNA from tissue extracts was found to have the same 5′-terminal sequence as previously was found for s(-)RNA self-cleaved from in vitro transcripts. Like s(-)RNA synthesized in vitro, the circular and linear s(-)RNA from tissue extracts spontaneously and readily interconverted during incubation in vitro. In contrast, the bulk of the circular and linear forms of S(+)RNA were stable. The very limited interconversion of s(+)RNA forms in vitro suggests that circularization in vivo is enzymically catalyzed. Encapsidated satellite RNA was found to be composed of linear, unit length and multimeric forms, including previously undocumented s(-)RNA present in approximately the same relative abundance compared to s(+)RNA as was observed for RNA from tissue extracts. Circles were not detected in encapsidated RNA. We interpret our results in the context of a rolling circle model for satellite RNA replication.