Recombinant viruses. Schematic representation of the sequence arrangements in the recombinant viruses used in this study. Line 1, the HSV-1(F)Δ 305 genome lacks the 501-bp Bgl II-Sac I sequence from the tk gene domain in the Bam HI Q fragment of HSV-1(F). Line 2, the domain of the γ134.5 gene in the inverted repeat b'a' flanking UL sequence. The identical sequence in inverted orientation in the ab repeat is shown with dashed lines. Lines 4 and 5, sequence arrangement of the relevant domain of the R3659 recombinant. The Stu I-Bst EII fragment from the γ134.5 domain was replaced with the chimeric α 27-tk gene. The substitution was made in both the ab and a'b' domains of the recombinant genome (not shown diagrammatically). Lines 7 and 8, sequence arrangement of the relevant domain of the R5225 recombinant. The α 27-tk gene of the R3659 recombinant was replaced with a cassette containing the first 100 bases of the HPV-16 E7 ORF in an antisense orientation under the egr-1 promoter, and the hepatitis B virus polyadenylation signal. An additional deoxythymidine nucleotide was inserted at the nucleotide 571 of the HPV-16 E7 ORF ("T") in order to introduce a frameshift and two stop codons in the sense orientation of the E7 ORF. The corresponding tk+ recombinant R5226 was constructed by placing the tk gene back to its natural location ("Δ ", line 1). The R3616 recombinant has a 1 kb deletion in both copies of the γ134.5 gene, and the rest of the genome is intact. R3617 is similar to R3616 but is tk negative, having a 501 bp deletion in its tk gene similarly as in line 1. Lines 3, 6, and 9, expected sizes of bands generated by restriction enzyme digestion with Nco I and hybridized with digoxigenin-labeled 1.8 kb Nco I fragment of the Bam HI S; HSV-1(F), R3659, and R5225 would yield bands A, B, and C, respectively (see Fig. 2).