Bacteriophage phi6 contains three pieces of double-stranded RNA (S, M and L). We have developed a scheme in which plasmid encoded viral proteins assemble in cells to form procapsids which can be used for the in vitro packaging and replication of the phi6 genome. The packaged particles are capable of infecting spheroplasts to form viable virus. RNA transcripts of cDNA copies of the genomic segments can be incorporated into virions and in this way, we have constructed virus with novel genetic insertions. We have found that the viral genome can undergo heterologous recombination at a high frequency. A system has been prepared that will lead to the rapid and simple screening for recombinational events. It is based upon the insertion of the lacz' gene into non-coding portions of the viral genome, resulting in Lac+ phage when infecting a host containing a plasmid expressing the omega fragment of beta-galactosidase. Heterologous recombinants become Lac-. This will be used to study the roles of RNA sequence in the recombination process by modifying the sequences 5' to the lac insert or both 5' and 3' to it, and examining the effect on recombination frequencies. In addition, the system will be used to screen for mutations that lead to either increased or decreased recombination rates. This will allow the determination of the roles of protein structure in the recombination process. In addition, we have prepared carrier state cells that are Lac+ due to their stably replicating phi6 constructions. These cells are particularly useful in selecting for mutants that have high or low levels of recombination. Mutants will be analyzed to determine which proteins are involved in recombination. It will also be possible to test the effects on recombination of already existing mutations in procapsid genes. Our in vitro replication reaction has been modified for studying the recombination process in vitro with purified procapsids and specifically engineered RNA molecules. Recombination products will be assayed by PCR. RNA recombination has been demonstrated in several plant and animal viruses. It is an important mechanism for both repair and genetic exchange. Heterologous recombination is a mechanism for drastic changes in viral genomes. It may be a major element in the evolution of RNA viruses. Phi6 is a model for the dsRNA viruses. Several members of the reoviridae, e.g. rotavirus and blue tongue virus, are the etiologic agents of important human and animal diseases.
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