Template recruitment, replication and recombination of brome mosaic virus genomic RNAs

Selected steps of brome mosaic virus (BMV) RNA replication were studied in Saccharomyces cerevisiae. BMV RNA replication occurs in endoplasmic reticulum membrane-bound compartments induced by BMV replication protein 1a. Recruitment of BMV RNAs into replication compartments is accomplished by 1a through a cis-acting template recruitment element on each of the BMV genomic RNAs. In these studies, an inducible system for BMV intermolecular RNA recombination was established and used to determine the role of the recruitment element in RNA recombination. The results imply that most BMV RNA recombination occurs as a byproduct of RNA replication, and support an independent template recruitment model in which (i) parental RNA templates do not interact outside replication compartments, (ii) parental RNA templates are independently recruited into replication compartments, (iii) most viral RNA replication compartments receive only one, and only occasionally two or more RNA templates, and (iv) intermolecular RNA recombinants are formed in heterozygous replication compartments. To further understand the mechanisms of recruitment of BMV RNAs to replication compartments, chimeric RNA2-RNA3 molecules were made and characterized. Biochemical and genetic data show that the RNA2 and RNA3 recruitment elements are not equivalent, that the RNA2 recruitment element is dominant over that of RNA3, and that RNA2 recruitment into replication compartments is modulated by host factors, including Ded1p, that do not influence RNA3 recruitment.