Cell Type Differences in Human Cytomegalovirus Transcription and Epigenetic Regulation with Insights into Major Immediate-Early Enhancer-Promoter Control [RNA-seq]

Cell type differences in the human cytomegalovirus (HCMV) transcriptome may result from variations in transcription and/or post-transcription. Here we report unexpected differences in transcription and epigenetic control in late-stage HCMV infection of human differentiated NTera2 cells (D-NT2) compared to fibroblasts, using integrated functional genomic approaches (PRO-Seq, RNA-Seq, DFF 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 IE2, or viral UL87 late transcription factor (LTF). This includes transcription from the enhancer for the major immediate early (MIE) promoter where GC-box sequence mutations increase transcription and mutations in CREB and NF-kB response elements decrease transcription. The GC-box sequence mutations also alter infected cell morphology and gene expression program, whereas mutations in CREB and NF-kB response elements do not. 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 TBP-IE2-nucleosome complex, with more nucleosome than in fibroblasts, covers the MIE promoter transcription start site, potentially contributing to epigenetically silence of the promoter. Overall design: RNA-Seq library preparation is carefully detailed in the manuscript. Total RNA was isolated from biological duplicates of D-NT2, either uninfected or infected in parallel with HCMV Towne WT, N1, NB, and CK viruses for 96 h. Total RNA-Seq libraries were prepared using the Illumina TruSeq-Stranded Total RNA Prep kit with Ribo-Zero for Human/Mouse/Rat (Illumina, 200020596), according to manufacturer's instructions. Library quality was assessed on the Agilent Bioanalyzer 2100 system, after diluting 1 µL of each DNA library to 2.5 ng/µL. Once deemed acceptable, all samples were diluted to 3 ng/µL and pooled in equimolar amounts. Sequencing was performed at the Iowa Institute of Human Genetics on the Illumina NovaSeq 6000 System using SP flow cells, generating 50-bp paired-end reads.