FIG. 3.

Verification of the UL46 mutations produced by recombineering of the KOS-37 BAC. (A) Genomic structures of wild-type and UL46 mutant viruses. Below the schematic diagram of the HSV-1 KOS-37 genome is an expanded view of the UL46 locus. The location and orientation of the UL46 ORF are indicated, along with the locations of cleavage sites for BamHI and MluI. “Flanks” indicates the probe that detects the UL46 5′ and 3′ flanking sequences. ΔUL46galK bears a deletion/substitution mutation that replaces the UL46 ORF with the E. coli galK gene; ΔUL46 bears a precise deletion of the UL46 ORF; and RUL46 is a UL46 repair virus derived from ΔUL46galK. (B) Southern blot analysis of viral DNA. Total cellular DNA extracted from Vero cells infected with the indicated viruses was cleaved with BamHI or MluI and analyzed by Southern blot hybridization using the indicated probes. Mobilities of molecular weight markers (in nucleotides) are indicated.