gga-miR-1603 and gga-miR-1794 directly target viral L gene and function as a broad-spectrum antiviral factor against NDV replication

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 <i>Gallus gallus</i> 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 <i>in vitro</i>. 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 <i>in vitro</i>. These findings will provide us with important clues for antiviral therapy against NDV infection.

作为新城疫（Newcastle disease, ND）的致病原，新城疫病毒（Newcastle disease virus, NDV）已严重制约了家禽产业的发展。既往研究表明，作为小型非编码RNA家族成员的微小RNA（microRNA, miRNA）可通过与宿主信使RNA（messenger RNA, mRNA）广泛相互作用，参与调控新城疫病毒的复制，但微小RNA是否通过直接结合新城疫病毒反义基因组来影响其复制，目前仍不明确。本研究通过在线软件RegRNA 2.0，采用生物信息学方法预测了靶向新城疫病毒反义基因组的家鸡（Gallus gallus）微小RNA，并通过双荧光素酶报告基因实验，证实gga-miR-1603与gga-miR-1794可直接靶向病毒L基因。序列比对分析显示，多基因型新城疫病毒的反义基因组中，分别在8611–8634 nt与14490–14514 nt位点处，存在gga-miR-1603与gga-miR-1794的高度保守结合位点。与此同时，本研究发现gga-miR-1603与gga-miR-1794可在RNA与蛋白水平上负向调控病毒L基因的表达，并在体外（in vitro）抑制病毒复制。此外，新城疫病毒感染对多种禽源细胞系中的内源性gga-miR-1603与gga-miR-1794表达无显著影响。综上，本研究证实gga-miR-1603与gga-miR-1794可直接结合病毒L基因，促进其降解，并在体外抑制多基因型新城疫病毒的复制。上述研究结果可为新城疫病毒感染的抗病毒治疗提供重要线索。