Competing endogenous RNA network profiling reveals novel host dependency factors required for MERS-CoV propagation

Circular RNAs (circRNAs) are an integral component of the host competitive endogenous RNA (ceRNA) network. These noncoding RNAs are characterized by their unique splicing reactions to form covalently closed loop structures and play important RNA regulatory roles in cells. Recent studies showed that circRNA expressions were perturbed in viral infections and circRNAs might serve as potential antiviral targets. We investigated the host ceRNA network changes and biological relevance of circRNAs in human lung adenocarcinoma epithelial (Calu-3) cells infected with the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV). A total of ≥49337 putative circRNAs were predicted. Among the 7845 genes which generated putative circRNAs, 147 (1.9%) of them each generated ≥30 putative circRNAs and were involved in various biological, cellular, and metabolic processes, including viral infections. Differential expression (DE) analysis showed that the proportion of DE circRNAs significantly (<i>P</i> &lt; 0.001) increased at 24 h-post infection. These DE circRNAs were clustered into 4 groups according to their time-course expression patterns and demonstrated inter-cluster and intra-cluster variations in the predicted functions of their host genes. Our comprehensive circRNA-miRNA-mRNA network identified 7 key DE circRNAs involved in various biological processes upon MERS-CoV infection. Specific siRNA knockdown of two selected DE circRNAs (circFNDC3B and circCNOT1) significantly reduced MERS-CoV load and their target mRNA expression which modulates various biological pathways, including the mitogen-activated protein kinase (MAPK) and ubiquitination pathways. These results provided novel insights into the ceRNA network perturbations, biological relevance of circRNAs, and potential host-targeting antiviral strategies for MERS-CoV infection.

环状RNA（circRNAs）是宿主竞争性内源RNA（ceRNA）网络的核心组成部分。这类非编码RNA（noncoding RNAs）以其独特的剪接反应形成共价闭合环状结构为特征，并在细胞中发挥重要的RNA调控功能。近期研究表明，病毒感染会干扰环状RNA的表达，且环状RNA有望成为潜在的抗病毒靶点。本研究针对感染高致病性中东呼吸综合征冠状病毒（MERS-CoV）的人肺腺癌上皮（Calu-3）细胞，探究了其宿主ceRNA网络的变化以及环状RNA的生物学意义。研究共预测得到至少49337个潜在环状RNA。在产生潜在环状RNA的7845个基因中，有147个（占比1.9%）各自可产生至少30个潜在环状RNA，且这些基因参与包括病毒感染在内的多种生物学、细胞及代谢过程。差异表达（DE）分析结果表明，感染后24小时，差异表达环状RNA的占比显著升高（*P* < 0.001）。根据时序表达模式，这些差异表达环状RNA可被分为4个簇，且其宿主基因的预测功能在簇间与簇内均存在差异。本研究构建的完整circRNA-miRNA-mRNA调控网络共筛选出7个关键差异表达环状RNA，它们参与了MERS-CoV感染后的多种生物学过程。对筛选出的2个差异表达环状RNA（circFNDC3B与circCNOT1）进行特异性小干扰RNA（siRNA）敲低后，MERS-CoV的载量显著降低，其调控的靶信使RNA（mRNA）的表达也随之下降，这些靶mRNA参与包括丝裂原活化蛋白激酶（MAPK）通路与泛素化通路在内的多种生物学通路。本研究结果为MERS-CoV感染中的ceRNA网络紊乱、环状RNA的生物学意义以及潜在的靶向宿主抗病毒策略提供了全新的认识。