Several RNA viruses have a 3' sequence that, like tRNA, can accept a specific amino acid. We have shown that modifications that interfere with the aminoacylation function severely decrease infectivity. Our overall research objective is to understand the role of aminoacylation of viral RNAs in infection processes. The replicase formed on infection of E. coli with phage O-beta is known to have four subunits, two of which are host cell elongation factors. By analogy, because tyrosylated (but not uncharged) brome mosaic virus (BMV) RNA can bind eukaryotic elongation factor EF 1, it is cogent to consider if its 3' structure facilitates the formation of a replicase complex. An important step towards this goal has been our recent development of techniques for the isolation of an enzyme from BMV-infected barley leaves that actively incorporates UMP into high molecular weight material that co-migrates with the replicative forms of each of the four RNA components of BMV. The reaction is stimulated over 15-fold by the addition of BMV RNA, but poorly or not at all by the addition of other viral RNAs or cellular RNAs (rRNA, tRNA, mRNA); i.e., it is not only a template-dependent, but also a template-specific polymerase. We propose to further purify this enzyme. Native and modified BMV RNAs, singly and in combination, will be tested for their ability to serve as templates for this enzyme. Electrophoretic analysis of the purified enzyme should reveal if host-coded EF 1 is present. Techniques for the isolation of barley protoplasts and their infection with purified BMV RNA have been established. Electrophoresis of extracts of BMV RNA-inoculated protoplasts incubated in radioactive media will be used to analyze the synthesis of viral RNA and protein. Protoplasts will thus be used to follow in vivo the transcription, translation, and infectivity of native and modified BMV RNA components. A combination of the information obtained from the protoplast experiments and the cell-free reactions is expected to yield insight as to how viral RNA is replicated in eukaryotic cells and how the 3' aminoacylatable sequence is involved in this aspect of infection processes.