FIG 2.
Construction and structure of the NgK and MgK mutant viruses. (A) The top schematic diagram shows the wt HSV-1 McKrae genome in the prototypic orientation. TRL and IRL represent the terminal and internal (or inverted) long repeats, respectively, and TRS and IRS represent the terminal and internal (or inverted) short repeats, respectively. UL and US represent the long and short unique regions, respectively. The solid rectangle represents the very stable 2-kb LAT. The start site for LAT transcription is indicated by an arrow at +1. “TATA” designates the relative location of the LAT promoter TATA box 28 nucleotides upstream of the start of transcription. (B) dLAT2903 has a deletion from LAT nucleotides −161 to +1667 in both copies of LAT and makes no LAT RNA. (C) NgK was constructed from dLAT2903 by homologous recombination between dLAT2903 DNA and a plasmid containing the complete LAT promoter and the entire structural NgK gene (including sequences for myc) signal. (D) MgK was constructed from dLAT2903 by homologous recombination between dLAT2903 DNA and a plasmid containing the complete LAT promoter and the entire structural MgK gene (including mutated sequences within the gK signal sequences and myc sequences). (E) NgK and MgK Southern blot analyses. RS cell monolayers were infected with 5 PFU of NgK, MgK, HSV-gK3, or McKrae/cell for 24 h. Viral DNAs were isolated, and 5 μg of DNA/virus was digested with EcoRV/EcoRI and hybridized to DIG-labeled HSV-1 gK (HSV-1 nucleotides 112170 to 113193). The lanes show results obtained for McKrae, HSV-gK3, NgK, and MgK. As expected, only one fragment is seen in McKrae lane. This band also is observed in HSV-gK3 and corresponds to HSV-1 nucleotides 110094 to 118640 surrounding the natural occurring gK (HSV-1 nucleotides 112170 to 113193). In contrast to the McKrae lane, in the HSV-gK3, NgK, and MgK lanes, in addition to the natural gK, there are two additional bands corresponding to two extra copies of gK gene inserted within IRL (HSV-1 nucleotides 110094 to 118640) and TRL (HSV-1 nucleotides 13 to 7727) regions of dLAT2903. Both regions within the IRL and TRL have a deletion of ∼1,600 bp of LAT.