We are exploring methods of obtaining activity for synthetic RSV vRNA made from cDNA clones. In one approach, we have constructed cDNAs which encode truncated, ca. 1000-nucleotide """"""""mini"""""""" vRNAs. These contain marker genes, such as the chloramphenicol acetyl transferase (CAT) gene, under the control of RSV transcriptive signals and flanked by the RSV 3' and 5' noncoding vRNA termini. The plan is to transfect the """"""""mini"""""""" vRNAs into tissue culture cells and superinfect with RSV to provide helper proteins to drive their replication and expression. This would provide a system for (i) performing structure-function studies of cis-acting RNA sequences and (ii) identifying and characterizing the viral proteins involved in RNA transcription and replication. In preliminary experiments, we have been able to show that transfection of two different vRNA analogs into cells results in the appearance of CAT activity dependent upon superinfection with RSV. But the success rate in these early experiments is 50%. The efficiency and success rate probably can be improved by optimizing the experimental conditions, and the fact that some success has been obtained suggests that this can be developed into a feasible experimental approach. A second line of experiments had been to construct a complete cDNA of the 15,222-nucleotide vRNA which will be used to produce (""""""""rescue"""""""") live RSV. This would be a method for introducing defined genetic changes into RSV for molecular studies and as an approach to characterizing existing attenuated strains and producing new strains that could be used as vaccines. Because existing cDNAs were synthesized from mRNA, it has necessary to reclone most of the vRNA in order to insert intergenic sequences and, in some areas, new restriction which will be useful in construction of subsequent mutants. Most of the vRNA has been recloned, and the complete cDNA is under construction.
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