长链非编码RNA_201363对抗猪瘟病毒蛋白MxA表达的调控作用及其机制

Long noncoding RNAs (lncRNAs) are involved in the host antiviral responses through regulating the expression of adjacent interferon-stimulated genes (ISGs). It has been shown that the ISG MxA exerts a broad antiviral activity. However, the exact regulatory mechanism of the MxA gene expression remains elusive. Recently, we have demonstrated that the porcine lncRNA_201363 is located proximally to the MxA gene and the expression of the two genes shows a positive correlation. Therefore, we hypothesize that the expression of MxA may be regulated by lncRNA_201363. In this proposal, we will firstly verify the regulatory role of lncRNA_201363 in the MxA expression and the replication of classical swine fever virus using overexpression and knockout assays. Secondly, we will determine the regulatory function of lncRNA_201363 in the MxA transcription using in situ hybridization and luciferase assays. Finally, we will reveal the regulatory mechanism of lncRNA_201363 through either lncRNA-host protein interactions or histone modification by using RNA pull-down, RNA-binding protein immunoprecipitation and chromatin immunoprecipitation assays. This project is expected to elucidate the regulatory mechanism of the MxA expression and provide a novel approach for the control and prevention of viral infections.

长链非编码RNA（lncRNAs）可通过调节邻近干扰素刺激基因（ISGs）的表达参与宿主的抗病毒应答。MxA是一个重要的广谱性抗病毒ISG蛋白，但是其表达调控机制尚不明确。我们前期的研究发现，猪lncRNA_201363在基因组中紧邻MxA基因并与MxA的表达正相关，符合lncRNAs调控ISGs表达的特征。因此，我们推测，猪MxA基因的表达可能受lncRNA_201363调控。本项目首先通过敲除和过表达lncRNA_201363，确定其对MxA基因表达和猪瘟病毒复制的影响；进一步通过原位杂交和荧光素酶试验，明确该lncRNA在细胞核内的含量变化及对MxA转录的调节作用；最后，应用RNA结合蛋白免疫沉淀和染色质免疫共沉淀等技术，从lncRNA调节组蛋白修饰和lncRNA与转录蛋白结合诱导基因表达两方面揭示其作用机制。本项目可望明确MxA表达的调控机制，为猪病的防控提供新的思路。

猪瘟（CSF）是由猪瘟病毒（CSFV）引起的一种急性、烈性传染病。CSFV感染宿主后，激活RIG-I/MDA5介导的I型IFN信号通路，诱导抗病毒蛋白、非编码RNA等多种干扰素刺激基因（ISGs）转录及表达。长链非编码RNA（lncRNAs）可被病毒和干扰素（IFN）诱导表达，目前，CSFV感染相关lncRNAs的研究尚未报道，研究CSFV感染相关lncRNAs对于CSF防控具有重要意义。.1..利用转录组测序技术筛选到lncRNA_2522和Ⅱ型2’-5’寡腺苷酸合成酶（OAS2）的mRNA转录水平均显著上调。体外试验证实CSFV感染PK-15细胞后，lncRNA_2522和OAS2的mRNA转录水平均显著增加，表明lncRNA_2522和OAS2与CSFV的感染相关。.2..利用ASO和pcDNA3.1_lncRNA-2522分别沉默和过表达lncRNA_2522，证实lncRNA_2522正向调控OAS2表达且lncRNA_2522是抗CSFV分子。.3..构建敲除OAS2基因的PK-15细胞系，过表达lncRNA_2522，感染CSFV，结果表明敲除OAS2后，lncRNA_2522丧失抗CSFV活性，即lncRNA_2522 抑制CSFV复制依赖OAS2。