| Brome Mosaic Virus (BMV) is a positive-strand RNA virus that belongs to the alphavirus-like superfamily. It has a genome divided among three 5'-capped RNAs. RNA1 encodes the helicase and capping protein 1a, and RNA2 expresses the RNA polymerase 2a. RNA3 encodes the 3a protein and a coat protein (expressed from a subgenomic RNA4), both of which are required for systemic infection, but not genomic replication. BMV replication in S. cerevisiae has been shown to parallel all aspects of normal plant infection. Therefore, yeast is used as a model system to study individual steps in the viral life cycle. RNA3 contains a replication enhancer (RE) within a larger intergenic region (IGR) that interacts with 1a and is required for replication and subgenomic RNA synthesis. This interaction is possibly host factor mediated and results in recruitment of RNA3 to the endoplasmic reticulum (ER) membrane, the site of replication complex assembly. At the 3'-end, RNA3 exhibits an extensive tRNA-like structure (TLS) that interacts with several host tRNA binding proteins. In the presence of one such protein, GCD10, RNA3 becomes stabilized in the cytoplasm. This effect is independent of the IGR, but rather requires an area near the 3 '-end including the TLS. Stabilization of GCD10 was seen by the use of RNA3 deletion constructs lacking the RE, and thus lacking la stabilization. By transforming yeast cells with plasmid constructs for BMV RNA, interactions with GCD10 were studied in vivo and in cellular extracts. Chemical mapping of the 3'-end of RNA3 was performed using DMS, CMCT, kethoxal, iron(II), and lead acetate to plot accessible nucleotides. This method was then applied to the investigation of a possible GCD10 footprint at the 3'-end of RNA3. |
