Mutational analysis of the core and modulator sequences of the BMV RNA3 subgenomic promoter

Abstract

The subgenomic promoter of a (+)-stranded RNA virus, brome mosaic virus (BMV) controlling synthesis of subgenomic RNA4 has been defined in vitro. Truncated and mutant (-)-strand RNA templates were produced by in vitro transcription of cloned RNA3 cDNA. Subgenomic (+)-sense RNA was synthesized in vitro from these templates by a replicase (RNA-dependent RNA polymerase) preparation extracted from infected barley leaves. The activities of templates with truncations and deletions surrounding the RNA4 initiation site revealed a promoter of approximately 62 bases grouped into four functional domains. The core sequence consists of about twenty bases immediately upstream of, and including, the initiation nucleotide. In addition to the core sequence, a domain overlapping the 5' untranslated end of RNA4 apparently determines correct initiation. Two domains immediately upstream of the promoter core consist of the internal poly(A) tract of RNA3, which probably serves as an non base-paired spacer facilitating access of the replicase to the promoter, and a sequence, UUAUUAUU, that is required for high levels of promoter activity. Homologies to sequences surrounding the initiation sites of subgenomic RNAs from several plant RNA viruses, and from alphaviruses, have been detected.