FIG. 1.

Construction and composition of HRSV cDNAs and chimeric membrane glycoprotein G^vsv. (A) Composition of HRSV cDNAs with a modified gene content in genome positions six, seven, and eight: (i) pRSΔsh lacks the SH ORF and contains instead a GFP ORF; (ii) pRSΔsh,g/G^vsv lacks the SH and G ORFs, which are replaced with those encoding GFP and G^vsv, respectively; (iii) pRSΔsh,g,f/G^vsv lacks all three transmembrane glycoprotein ORFs, SH, G, and F, and these were replaced with ORFs encoding GFP, G^vsv, and GUS, respectively. The genomic sequences in these cDNAs are flanked by a T7 promoter (T7) and an HDV self-cleaving ribozyme sequence (HDV) followed by a T7 terminator sequence (ø). In the presence of T7 polymerase, a transcript corresponding to the HRSV antigenome is produced. Numbers 1 to 10 represent the positions of each of the genes relative to the 3′ promoter of the viral genome. Sizes of the engineered viral genomes are indicated on the right in nucleotides (nt). Abbreviations: NS1/2, nonstructural proteins 1 and 2; N, nucleocapsid protein; P, phosphoprotein; M, matrix protein; M2, transcription factor; L, large (polymerase); Le, leader; Tr, trailer. (B) Strategy used for the generation of genetically modified viruses. A plasmid was generated that contained a cDNA of the prototypic HRSV A2 strain from which the SH-G-F region was deleted (pRSΔsh,g,f). A shuttle vector, pBLS-6,7,8, containing introduced restriction sites but in which the gene starts (gs), intergenic regions (ig), and gene ends (ge), as well as untranslated regions (utr) remained unaltered from the A2 virus, was used to place selected ORFs in the sixth, seventh, and eighth genome positions within engineered cDNAs. (C) G^vsv is a chimeric viral membrane glycoprotein consisting of the VSIV G ecto- and transmembrane domains (amino acids 1 to 491) coupled to the CTD of the HRSV F protein (amino acids 553 to 574) via an XbaI site, created by introduction of 3 nt (tct).