Generation of VP19C NES mutants by mutagenesis. (A) Schematic representation of the HSV-1 genome containing terminal long (TRL) and short (TRS) and internal long (IRL) and short (IRS) repeats flanking the unique long (UL) and short (US) regions. (B) The NES mutations were introduced by using two-step red recombination. The sequences corresponding to the first (red and wathet) and second (blue) recombination events are shown in the indicated colors. (C) PCR products containing ~40 bp of homologous sequences upstream (red) and downstream (wathet) of the target loci, ~40 bp of complementary sequences (blue) encoding the mutations and I-SceI site, or the KanR gene were electroporated into competent GS1783 E. coli containing the HSV-1 17-37BAC. After the first recombination, the PCR product was inserted into the homologous location within the UL38 gene. After confirming that the PCR product was inserted into the correct locus by using RFLP and Southern blot hybridizations, the second recombination was performed in which the KanR gene plus one of the complimentary homologous sequences were removed. The remaining genome contained the complete VP19C ORF with the corresponding mutations.