Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides that are not translated into functional proteins. Cellular lncRNAs have been shown to act as regulators by interacting with target nucleic acids or proteins and modulating their activities. We investigated the role of RNA1.2, which is one of four major lncRNAs expressed by human cytomegalovirus (HCMV), by comparing the properties of parental virus in vitro with those of deletion mutants lacking either most of the RNA1.2 gene or only the TATA element of the promoter. In comparison with parental virus, these mutants exhibited no growth defects and minimal differences in viral gene expression in human fibroblasts. In contrast, 76 cellular genes were consistently up- or down-regulated by the mutants at both the RNA and protein levels at 72 h after infection. Differential expression of the gene most highly upregulated by the mutants (Tumor protein p63-regulated gene 1-like protein; TPRG1L) was confirmed at both levels by RT-PCR and immunoblotting. Consistent with the known ability of TPRG1L to upregulate IL-6 expression via NF-κB stimulation, RNA1.2 mutant-infected fibroblasts were observed to upregulate IL-6 in addition to TPRG1L. Comparable surface expression of TNF receptors and responsiveness to TNF-α in cells infected by the parental and mutant viruses indicated that activation of signaling by TNF-α is not involved in upregulation of IL-6 by the mutants. In contrast, inhibition of NF-κB activity and knockdown of TPRG1L expression reduced the extracellular release of IL-6 by RNA1.2 mutant-infected cells, thus demonstrating that upregulation of TPRG1L activates NF-κB. The levels of MCP-1 and CXCL1 transcripts were also increased in RNA1.2 mutant-infected cells, further demonstrating the presence of active NF-κB signaling. These results suggest that RNA1.2 plays a role in manipulating intrinsic NF-κB-dependent cytokine and chemokine release during HCMV infection, thereby impacting downstream immune responses.

长链非编码 RNA (lncRNA) 是大于 200 个核苷酸的转录本，不会翻译成功能性蛋白质。细胞 lncRNA 已被证明通过与靶核酸或蛋白质相互作用并调节它们的活性来充当调节剂。我们通过比较亲本病毒的特性，研究了 RNA1.2 的作用，它是人类巨细胞病毒 (HCMV) 表达的四种主要 lncRNA 之一体外缺失突变体缺乏大部分 RNA1.2 基因或仅缺乏启动子的 TATA 元件。与亲本病毒相比，这些突变体在人类成纤维细胞中没有表现出生长缺陷和病毒基因表达的最小差异。相反，76 个细胞基因在感染后 72 小时被突变体在 RNA 和蛋白质水平上持续上调或下调。通过 RT-PCR 和免疫印迹在两个水平上证实了突变体最高度上调的基因（肿瘤蛋白 p63 调节基因 1 样蛋白；TPRG1L）的差异表达。与 TPRG1L 通过 NF-κB 刺激上调 IL-6 表达的已知能力一致，除 TPRG1L 外，还观察到 RNA1.2 突变感染的成纤维细胞上调 IL-6。在被亲本病毒和突变病毒感染的细胞中，TNF受体的可比表面表达和对TNF-α的反应性表明TNF-α对信号传导的激活不参与突变体对IL-6的上调。相反，抑制 NF-κB 活性和敲低 TPRG1L 表达会减少 RNA1.2 突变体感染细胞对 IL-6 的细胞外释放，从而证明 TPRG1L 的上调激活了 NF-κB。在 RNA1.2 突变体感染的细胞中，MCP-1 和 CXCL1 转录物的水平也增加，进一步证明了活跃的 NF-κB 信号传导的存在。这些结果表明 RNA1.2 在 HCMV 感染期间在操纵内在 NF-κB 依赖性细胞因子和趋化因子释放中发挥作用，从而影响下游免疫反应。