ABSTRACT

As the causative agent of Newcastle disease (ND), Newcastle disease virus (NDV) has seriously restricted the development of the poultry industry. Previous research has shown that miRNAs, members of the small noncoding RNA family, are implicated in the regulation NDV replication through extensive interactions with host mRNAs, but whether miRNAs affect NDV replication by directly binding to the NDV antigenome remains unclear. In this study, potential Gallus gallus miRNAs targeting the antigenome of NDV were bioinformatically predicted using the online software RegRNA 2.0, and gga-miR-1603 and gga-miR-1794 were identified as targeting the viral L gene directly through dual-luciferase reporter assays. Sequence alignment analysis demonstrated that multiple genotypes of NDVs harbored highly conserved binding sites for gga-miR-1603 and gga-miR-1794 in the viral antigenome located at 8611–8634 nt and 14,490–14,514 nt, respectively. Meanwhile, we found that gga-miR-1603 and gga-miR-1794 negatively regulated the expression of viral L gene at both the RNA and protein levels, as well as viral replication in vitro. Furthermore, NDV infection had no effect on endogenous gga-miR-1603 and gga-miR-1794 expression in various avian cell lines. Overall, our present study demonstrated that gga-miR-1603 and gga-miR-1794 directly bind to the viral L gene to facilitate ts degradation and inhibit the replication of multiple genotypes of NDVs in vitro. These findings will provide us with important clues for antiviral therapy against NDV infection.

摘要

作为新城疫（ND）的病原体，新城疫病毒（NDV）严重制约了家禽业的发展。先前的研究表明，作为非编码小 RNA 家族成员的 miRNA，通过与宿主 mRNA 的广泛相互作用参与了 NDV 复制的调控，但 miRNA 是否通过直接结合 NDV 反基因组影响 NDV 复制仍不清楚。在这项研究中，潜在的鸡使用在线软件 RegRNA 2.0 对靶向 NDV 反基因组的 miRNA 进行生物信息学预测，并通过双荧光素酶报告基因检测确定 gga-miR-1603 和 gga-miR-1794 直接靶向病毒 L 基因。序列比对分析表明，NDV 的多个基因型在病毒反基因组中分别在位于 8611-8634 nt 和 14,490-14,514 nt 处具有高度保守的 gga-miR-1603 和 gga-miR-1794 结合位点。同时，我们发现gga-miR-1603和gga-miR-1794在RNA和蛋白质水平上负调节病毒L基因的表达，以及体外病毒复制. 此外，NDV 感染对各种禽类细胞系中的内源性 gga-miR-1603 和 gga-miR-1794 表达没有影响。总体而言，我们目前的研究表明，gga-miR-1603 和 gga-miR-1794 直接与病毒 L 基因结合，以促进 ts 降解并抑制体外多种基因型 NDV 的复制。这些发现将为我们提供针对 NDV 感染的抗病毒治疗的重要线索。