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. 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.

人类巨细胞病毒（human cytomegalovirus, HCMV）转录组的细胞类型差异可能源于转录和/或转录后调控的变化。本研究通过整合功能基因组学方法（精确核运行测序（PRO-Seq）、RNA测序（RNA-Seq）、DFF染色质免疫沉淀测序（DFF ChIP-Seq）、快速病毒蛋白降解实验以及启动子突变与功能验证实验），对比分析了人分化NTera2细胞（D-NT2）与成纤维细胞在HCMV感染晚期的转录与表观遗传调控差异，揭示了此前未被报道的调控特征。
在D-NT2细胞而非成纤维细胞中，多个病毒启动子区域的RNA聚合酶II起始与延伸过程依赖病毒DNA合成，且不依赖宿主P-TEFb、病毒IE2或病毒UL87晚期转录因子（late transcription factor, LTF）。
这包括主要即刻早期（major immediate early, MIE）启动子增强子区域的转录：其中GC盒序列突变可增强转录活性，而CREB与NF-κB应答元件突变则会减弱转录。GC盒序列突变还会改变感染细胞的形态与基因表达程序，而CREB与NF-κB应答元件突变则无此类效应。
在D-NT2细胞中，受LTF调控的启动子在病毒晚期启动子群体中占比更低，且整体活性更弱。
此外，病毒基因组结合的核小体数量更多，可能会限制LTF的结合。一种相较于成纤维细胞包含更多核小体的TBP-IE2-核小体复合物覆盖了MIE启动子的转录起始位点，可能参与该启动子的表观遗传沉默。
本研究详细记载了RNA测序文库的构建流程：从未感染或并行感染HCMV Towne WT、N1、NB及CK毒株96小时的D-NT2细胞生物学重复样本中提取总RNA；采用Illumina TruSeq链特异性总RNA建库试剂盒（搭配Human/Mouse/Rat Ribo-Zero试剂，Illumina，货号200020596），严格遵循制造商说明书完成总RNA-Seq文库制备；将每份DNA文库取1μL稀释至2.5 ng/μL后，使用Agilent Bioanalyzer 2100生物分析仪评估文库质量；质量合格的样本将被稀释至3 ng/μL，并以等摩尔浓度混合。测序工作在美国爱荷华大学人类遗传学研究所完成，采用Illumina NovaSeq 6000测序系统搭配SP流动槽，生成50 bp双端测序读段。