Strategy and construction of the recombinant virus MT101, 102, and 103. (A) Schematic diagram of genome structures of wild-type YK304 and relevant domains of the recombinant viruses. Line 1, a linear representation of the YK304 genome. The YK304 genome has bacmid (BAC) in the intergenic region between UL3 and UL4. Line 2, the genomic domain encoding UL6 to UL9 open reading frames. The DNA fragment and restriction enzyme sites in the genomic domain encoding UL6 to UL9 open reading frames. Line 3, expected sizes of DNA fragments generated by cleavage of DNA. The fragment designations shown here are identical to those described in the text and in Fig. 1B. Line 4, location of the DNA fragment used as a radiolabeled probe in Fig. 1B. Line 5, an expanded section of the parts of UL6, UL8 and whole of UL7 open reading flame. Line 6, a schematic diagram of the recombinant virus genome. As a result of RecET mutagenesis, a kanamycin-resistant cassette was inserted into a truncated UL7 gene that contained an HSV-BAC maintained in an E. coli. Line 7, a schematic diagram of the recombinant virus MT102. As a result of flp-mediated site-specific recombination, the kanamycin-resistant gene was excised from the virus genome and a single FRT site remained. MT102 was reconstituted by transfection of the mutated HSV-BAC (pMT102) into rabbit skin cells. Line 8, a schematic diagram of the repaired virus MT103. The rescue of MT102 by cotransfection of its DNA was the same as that used for the radiolabeled probe. Restriction sites: H, Hind III; B, Bam HI. (B) Autoradiographic images of electrophoretically separated Bam HI and Hind III digests of YK304 (lane 1), MT102 (lane 2), and MT103 (lane 3) DNAs hybridized to the radiolabeled DNA fragment of HSV(F) described in line 4 of Figure 1A. The letters on the right refer to the digests of the DNA fragments generated by restriction endonuclease cleavage. (C) Photographic image of the immunoblots of electrophoretically separated lysates of Vero cells infected with wild-type YK304 (lane 1), MT102 (lane 2), or MT103 (lane 3). The infected cells were harvested at 18 h post-infection and subjected to immunoblotting with the rabbit polyclonal antibody to UL7 (upper panel). The same membrane was re-labeled with the rabbit polyclonal antibody to UL49 (lower panel).