RNA Recombination at the Subgenomic Promoter in Brome Mosaic Virus: Relationship with Transcription

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The long-term goal of this research is to understand the mechanism(s) of genetic recombination in RNA viruses. The project team has previously described inter-segmental recombination within the 3' noncoding region of brome mosaic virus (BMV) RNAs, a model + strand RNA virus. More recently, the team has mapped an RNA recombination hot spot at the subgenomic promoter (sgp) region of BMV RNA3, and demonstrated a relationship between RNA recombination and transcriptional activity. To dissect the molecular mechanism of sgp-mediated homologous recombination, this project will involve both in vivo and in vitro experiments. The project focuses on the following goals: (i) study the relation between transcription and recombination in vivo through precise mapping of crossover sites, determining the role of initiation of transcription and that of sgp structure and identification of pre-detached (+) strands; and (ii) study the mechanism of recombination in vitro through determining the role of priming by nascent strands, mapping and correlating pausing and binding regions and relating the results to those in vivo. These studies represent a novel molecular approach that will help the researchers to understand the mechanism of genetic RNA recombination at the molecular level in a well-characterized model virus. The experiments will concern a new recombination system that is driven by a promoter of transcription. The investigators will determine whether recombination and transcription utilize the same RNA signals and if nascent strands can prime recombination. Although the sgp is used by many groups of RNA viruses for gene expression, the results can be extended to the understanding of RNA recombination in RNA viruses that do not use sgps for gene expression, as well as in developing strategies to engineer stable viral vectors. The BMV system of sgp-mediated recombination, developed in the PI's laboratory, is a unique system in plant virology that permits studies on the mechanism of homologous RNA recombination. A general belief exists that homologous crossovers occur at high frequency during replication of RNA viruses but escape detection. The results of the studies on the sgp-mediated recombination will contribute to our knowledge about the role of sequence/structure factors in directing homologous crossovers. They will also enhance our understanding of the relation between overlapping viral functions at the molecular level. The PI will also use this project to continue training individuals in the principles of molecular biology and biochemistry. Those currently carrying out research in his laboratory include not only undergraduate, graduate, and postdoctoral students, but also a high-school teacher.