Insertion of the G and F ORFs of RSV subgroup A or B into rB/HPIV3 as additional, promoter-proximal genes. The rB/HPIV3 antigenomic cDNA was modified previously by introduction of a unique BlpI site (boldface italic type) in the nontranslated region of the N gene preceding the start codon of the N ORF (49). PCR mutagenesis was used to add a PIV3 gene end (GE) (5′-AAGTAAGAAAAA), intergenic region (5′-CTT), and gene start (GS) (5′-AGGATTAAAG) signal immediately downstream of the stop codon of the G and F ORFs of subgroups A and B. An NheI site was added to the downstream end of each G ORF and on either side of each F ORF, and BlpI sites were added on either side of each insert. The modifications to the subgroup A ORFs were performed in previous work (49), and the subgroup B ORFs were modified in the same way in the present study. Each insert was cloned into the BlpI site in the rB/HPIV3 cDNA to create constructs containing the four single-gene insert viruses, namely, rB/HPIV3-GA, rB/HPIV3-FA, rB/HPIV3-GB, and rB/HPIV3-FB. To create the double-insert constructs rB/HPIV3-GAFAand rB/HPIV3-GBFB the NheI fragment bearing each F ORF was excised from the rB/HPIV3-FA and rB/HPIV3-FB antigenomic cDNA and inserted into the NheI site of the rB/HPIV3-GA and rB/HPIV3-GB cDNAs, respectively. Elements of the viral promoter that, by analogy to Sendai virus (56), are present in the N gene nontranslated region are indicated by the three boxed hexamers, with important G residues shown in boldface type. Nucleotides that differ between the subgroup A and subgroup B constructs are shown on separate lines (top line for subgroup A constructs and bottom line for subgroup B constructs).