A Chromatin Insulator-Like Element in the Herpes Simplex Virus Type 1 Latency-Associated Transcript Region Binds CCCTC-Binding Factor and Displays Enhancer-Blocking and Silencing Activities

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity

“…5C). CTRL1 consists of 46 copies of two separate, but overlapping, CTCF-binding motifs whereas CTRL2 contains 9 copies of only one of these motifs (19), perhaps explaining the greater effect of CTRL1 than CTRL2 deletion on transgene expression in the LAT locus. Recent evidence indicates that the function of insulators is dependent on additional sequences aside from CTCFbinding motifs, and thus a more complete analysis of these viral elements will be required to determine whether they have specific limitations (46).…”

“…These findings suggested that CTRLs may act as boundary elements to shield the LAT locus against the spreading of repressive epigenetic modifications (29), and it was shown in short-term plasmid transfection assays that a fragment encompassing CTRL2 combines the functions of a typical insulator (19). In the context of a complete viral genome, we found that CTRL1, along with LATP2, was essential for preventing the rapid silencing of an ectopic promoter in the LAT locus in nonneuronal cells in the absence of all viral IE gene products (Fig.…”

“…Although we do not yet know the cause of this difference, a recent report by Harkness et al noted greater persistence of transcriptional activity within repeat regions of the IE gene-depleted d109 vector than elsewhere along the genome in trigeminal ganglion neurons, but not in MRC5 human embryonic lung cells (50). The mCherry cassette in our vectors replaces the ICP4 gene in the remaining repeat bordering U S , a region flanked on each side by a cluster of CTCF-binding motifs referred to by Bloom and coworkers as CTa S (or B4) and CTRS3′ (or B6), respectively (19,29). Because the ICP4 gene is severely repressed in latently infected neurons relative to the LAT region, it seems unlikely that these elements alone are responsible for the enhanced mCherry expression we observed in rat DRGs, and thus other factors, including the nature of the transgene promoter, may play an important role.…”

“…Recent findings have shown that, during latency, the cellular CTCF protein is enriched on the two HSV CTRLs and that acetylated histone H3, an epigenetic beacon of active transcription, accumulates in the LAT region between these elements (19,(43)(44)(45). These findings suggested that CTRLs may act as boundary elements to shield the LAT locus against the spreading of repressive epigenetic modifications (29), and it was shown in short-term plasmid transfection assays that a fragment encompassing CTRL2 combines the functions of a typical insulator (19).…”

“…rich in CTCF-binding motifs, designated CTRL [CCCTC binding protein (CTCF) repeat long (CTRL)] (19), and the enhancerlike latency active promoter 2 (LAP2 or LATP2) region. Reportergene expression was also observed when the transgene cassette with LAT elements was moved to other positions in a silentvector genome, but omission of the LAT elements abolished expression.…”