Schematic for generation of recombinant viruses that specify epitope-tagged UL20p and gK. (A) The top line represents the prototypic arrangement of the HSV-1 genome with the unique long (UL) and unique short (US) regions flanked by the terminal repeat (TR) and internal repeat (IR) regions. (B) An expanded genomic region between map units 0.25 and 0.3 containing the UL19, UL20, UL20.5, UL21, and UL22 genes (left) and the region between map units 0.7 and 0.8 containing the UL52, UL53, and UL54 open reading frames (right). (C) Diagram of the UL20-null virus, which contains an EGFP gene cassette within the UL20p open reading frame. (D) Homologous recombination plasmids that encode the indicated 3× FLAG epitope-tagged UL20 genes as well as flanking sequences for recombination were used to rescue the UL20 deletion and transfer the UL20p genes with their 3× FLAG epitope tags into the virus. (E) Schematic depicting the experimentally determined gK and UL20p membrane topologies, as well as the sites of insertion of each of the epitope tags and ER retention motifs.