核因子I/A(NFIA)依赖其N末端的DNA结合结构域抑制猪繁殖与呼吸综合征病毒(PRRSV)复制

Nuclear factor I/A(NFIA) inhibits porcine reproductive and respiratory syndrome virus(PRRSV) replication by its DNA binding domain

猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)严重危害世界养猪业,目前仍无有效防控策略,因此探究宿主内源性蛋白拮抗PRRSV的分子机理意义重大。前期试验发现核因子I/A(nuclear factor I/A,NFIA)可以有效抑制PRRSV复制,故本试验以非洲绿猴胚胎肾细胞Marc-145细胞为模型,对NFIA抑制PRRSV复制的分子机理进行了深入探究。首先,构建NFIA的N末端DNA结合结构域缺失质粒pcDNA3.1-Flag-ΔN和C末端转录激活结构域缺失质粒pcDNA3.1-Flag-ΔC并分别转染Marc-145细胞,24 h后感染PRRSV,48 h后用qRT-PCR和Western blot的试验方法分别检测病毒N蛋白的RNA和蛋白表达水平。结果显示ΔC仍能有效降低PRRSV的RNA水平和N蛋白水平,但ΔN则不再能降低PRRSV的RNA水平和N蛋白水平,这表明NFIA的N末端结构域是抑制病毒的关键。其次,对NFIA全长质粒pcDNA3.1-Flag-WT进行N末端结构域内不同功能位点的双碱基突变,分别破坏掉NFIA的促腺病毒复制功能(Mut1,YR86~87WL)、DNA结合功能(Mut2,LR119~120VD)和二聚化功能(Mut3,LF135~136VD),将突变质粒分别转染Marc-145细胞,24 h后感染PRRSV,48 h后通过qRT-PCR和Western blot检测发现,Mut2和Mut3不再能降低PRRSV的RNA水平和N蛋白水平,说明N端结构域的DNA结合功能位点和二聚化功能位点是NFIA抑制PRRSV复制的关键。结果表明,核因子I/A(NFIA)主要依赖其DNA结合结构域的二聚化与DNA结合功能来抑制PRRSV复制。

Porcine reproductive and respiratory syndrome virus(PRRSV)seriously endangers the world pig industry,and there is no effective prevention and control strategy at present.Therefore,it is of great significance to explore the molecular mechanism of host endogenous protein antagonizing PRRSV.Our previous experiments showed that nuclear factor I/A(NFIA)could effectively inhibit the replication of PRRSV.Therefore,in this experiment,the Marc-145 cells of African green monkey embryonic kidney cells were used as a model to explore the molecular mechanism of NFIA inhibiting PRRSV replication.Firstly,the N-terminal DNA binding domain deletion plasmid pcDNA3.1-Flag-ΔN and C-terminal transcriptional activation domain deletion plasmid pcDNA3.1-Flag-ΔCof NFIA were constructed and transfected into Marc-145 cells respectively.24 hours later,PRRSV was added.48 hours later,the RNA and protein expression levels of viral N protein were detected by qRT-PCR and Western blot,respectively.The results showed thatΔCcould still effectively reduce the RNA level and N protein level of PRRSV,butΔN could no longer reduce the RNA level and N protein level of PRRSV.All of this showed that the N-terminal domain of NFIA is the key domain for NFIA to inhibit the virus.Secondly,the full-length plasmid pcDNA3.1-FlagWT of NFIA was mutated at different functional sites in the N-terminal domain in order to destroy the adenoviral replication function(Mut1,YR86-87 WL),DNA binding function(Mut2,LR119-120 VD)and dimerization function(Mut3,LF135-136 VD)of NFIA,respectively.Then,the mutant plasmid was transfected into Marc-145 cells respectively,and infected with PRRSV after 24 hours.48 hlater,qRT-PCR and Western blot detection showed that Mut2 and Mut3 could no longer reduce the level of RNA and N protein in PRRSV,which suggested that the DNA binding functional sites and dimerization functional sites in the N-terminal domain were the key sites for NFIA to the inhibition of PRRSV replication.In conclusion,DNA binding domain and DNA binding function were important for NFIA to inhibit PRRSV replication.