G-quadruplexes regulate gene expression, recombination, packaging and latency in herpesviruses. Herpesvirus-encoded miRNAs have been linked to important biological functions. The presence and the biological role of G-quadruplexes have not been studied in the regulatory regions of virus miRNA. We hypothesized that herpesvirus-encoded miRNAs are regulated by G-quadruplexes in their promoters. We analyzed the 1 kb regulatory regions of all herpesvirus-encoded miRNAs for the presence of putative quadruplex-forming sequences (PQS). Over two-third (67%) of the regulatory regions of herpesvirus miRNAs had atleast 1 PQS. The 200 bp region of the promoter proximal to herpesvirus miRNA is particularly enriched for PQS. We chose to study the G-quadruplex motifs in the promoters of miR-K12 cluster in Kaposi's sarcoma-associated Herpesvirus (KSHV miR-K12–1-9,11) and the miR-US33 encoded by Human Cytomegalovirus (HCMV miR-US33). Biophysical characterization indicates that the G-quadruplex motifs in the promoters of the KSHV miR-K12 cluster and the HCMV miR-US33 form stable intramolecular G-quadruplexes in vitro. Mutations disrupting the G-quadruplex motif in the promoter of the KSHV miR-K12 cluster significantly inhibits promoter activity, while those disrupting the motif in the promoter of HCMV miR-US33 significantly enhance the promoter activity as compared to that of the respective wild-type promoter. Similarly, the addition of G-quadruplex binding ligands resulted in the modulation of promoter activity of the wild-type promoters (with intact G-quadruplex) but not the mutant promoters (containing quadruplex-disrupting mutations). Our findings highlight previously unknown mechanisms of regulation of virus-encoded miRNA and also shed light on new roles for G-quadruplexes in herpesvirus biology.

中文翻译：

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疱疹病毒 miRNA 上游 G 四链体分析：G 四链体介导 KSHV miR-K12–1-9,11 簇和 HCMV miR-US33 调节的证据


G-四链体调节疱疹病毒的基因表达、重组、包装和潜伏期。疱疹病毒编码的 miRNA 与重要的生物学功能有关。 G-四链体的存在及其生物学作用尚未在病毒 miRNA 的调控区域中进行研究。我们假设疱疹病毒编码的 miRNA 受其启动子中的 G 四链体调控。我们分析了所有疱疹病毒编码的 miRNA 的 1 kb 调控区域是否存在推定的四链体形成序列 (PQS)。超过三分之二 (67%) 的疱疹病毒 miRNA 调控区域至少有 1 个 PQS。靠近疱疹病毒 miRNA 的启动子的 200 bp 区域尤其富含 PQS。我们选择研究卡波西肉瘤相关疱疹病毒 (KSHV miR-K12–1-9,11) 中 miR-K12 簇启动子中的 G-四联体基序和人类巨细胞病毒 (HCMV miR-US33) 编码的 miR-US33 。生物物理表征表明，KSHV miR-K12 簇和 HCMV miR-US33 启动子中的 G 四链体基序在体外形成稳定的分子内 G 四链体。与相应野生型相比，破坏 KSHV miR-K12 簇启动子中 G-四链体基序的突变显着抑制启动子活性，而破坏 HCMV miR-US33 启动子中基序的突变显着增强启动子活性发起人。类似地，G-四链体结合配体的添加导致野生型启动子（具有完整的G-四链体）的启动子活性的调节，但不影响突变型启动子（含有四链体破坏突变）的启动子活性。 我们的研究结果强调了以前未知的病毒编码 miRNA 的调节机制，并揭示了 G-四链体在疱疹病毒生物学中的新作用。