FIG 1.
JΔNI10 genome engineering. (Top) Compared to the wild-type HSV-1 KOS genome represented at the top, JΔNI10GW (7) has the entire internal repeat (joint) region (IRL and IRS); the remaining ICP0 (Δ0), LAT (ΔLAT), and ICP4 (Δ4) loci; and the single-copy ICP27 (Δ27) gene deleted (red crosses). The promoter and translation initiation codon of the ICP47 (Δ47) gene are also deleted, and the ICP22 promoter acts as an early (β22) promoter due to deletion of its regulatory sequences. The glycoprotein B gene contains a pair of entry-accelerating mutations (gB:NT) (58), and a GW destination cassette is located between the UL50 and UL51 genes, whose directions of transcription are indicated by arrows. A ubiquitin C promoter (UbCp)-mCherry gene cassette is located at the position of the deleted ICP4 gene (Δ4). UL and US, unique long and short segments; TRL and TRS, terminal repeats flanking UL and US, respectively; IRL and IRS, the cognate internal repeats of UL and US, respectively. (Bottom) CAG promoter-EGFP cassettes flanked by antisilencing elements in different configurations were inserted into JΔNI10GW through GW recombination. vCAG contains a CAG promoter-controlled EGFP cassette and the rabbit β-globin polyadenylation (pA) region. vLAT contains the EGFP expression cassette embedded in sequences from the LAT locus between the LATP2 enhancer-type element and CTRL2, with CTRL1 included upstream of the LAT promoter (LAP1). vHS4-S contains two tandem copies of the cHS4 core (core*2) at both sides of the expression cassette, while vHS4-L contains the cassette flanked on each side by a single copy of the full-length (1.2-kb) cHS4. vUCOE1 contains the complete 4.1-kb A2UCOE (black boxes; exons) and a 218-bp cloning sequence (wavy line) upstream of CAG. In vUCOE2, the linker sequence was deleted. vUCOE3 lacks both the linker and the first intron of HNRPA2B1. In vUCOE4 and vUCOE5, the EGFP expression cassette is located in the divergent promoter region separating the CBX3 and HNRPA2B1 transcription start sites, and in vUCOE5, the first intron of HNRPA2B1 is deleted. Black boxes, UCOE exons; thick horizontal lines, UCOE introns and dual-promoter region; arrows, transcription start sites and directions.