Cell type differences in human cytomegalovirus transcription and epigenetic regulation with insights into major immediate-early enhancer-promoter control

Cell type differences in the human cytomegalovirus (HCMV) transcriptome may arise from variations in transcription or post-transcription regulation. Here we report unexpected differences in transcription and epigenetic control in late-stage HCMV infection of human differentiated NTera2 neural lineage cells (D-NT2) compared to fibroblasts, using integrated functional genomic approaches (PRO-Seq, RNA-Seq, DNA fragmentation factor-ChIP Seq, rapid viral protein degradation, and promoter mutation and function assays). In D-NT2, but not fibroblasts, RNA polymerase II initiation and elongation at several viral promoters requires viral DNA synthesis and are independent of host P-TEFb, viral immediate-early protein 2 (IE2), or viral late transcription factor (LTF). This includes transcription from the enhancer for the major immediate early (MIE) promoter where GC-box sequence mutations increase enhancer transcription, while mutations in CREB and NF- kB response elements reduce it. The GC-box mutations also alter infected D-NT2 cell morphology and gene expression program without affecting viral MIE gene expression levels, whereas mutations in CREB and NF- k B response elements do not induce these changes. In D-NT2, LTF-driven promoters constitute a smaller proportion of the viral late promoter population and are generally less active. Additionally, viral genomes have more nucleosomes, potentially restricting LTF access. A TATA-binding protein (TBP)-IE2-nucleosome complex, with more nucleosome than in fibroblasts, occupies the MIE promoter transcription start site, potentially contributing to its epigenetic silencing.