Methyl-CpG Binding Domain Protein 2 (MBD2) And C-AMP Responsive Element Binding Protein 1 (CREB1) Inhibit The Replication Of PRRSV

Porcine reproductive and respiratory syndrome virus(PRRSV)can induce Porcine reproductive and respiratory syndrome(PRRS),which is a highly contagious disease that causes reproductive disorders such as abortion,premature birth,stillbirth and mummified fetuses in pregnant female pigs and breathing difficulties in young pigs.Since PRRS was first discovered in the United States in 1987,it has spread rapidly to all major pig raising countries and is one of the most important diseases affecting the global pig raising industry.PRRS was first reported in China in 1996.Since a highly pathogenic HP-PRRSV induced highly pathogenic PRRS of 2006 had emerged and become prevalent that was bring huge economic losses to Chinese pig industry.The preliminary results of the laboratory research group showed that miR-373 can promote the replication of PRRSV by negatively regulating type I interferon.And the target gene prediction from software showed that methyl-CpG binding domain protein 2(MBD2)and cyclic-AMP responsive element binding protein 1(CREB1)might be the target genes of miR-373.So whether miR-373 promotes replication of PRRSV by targeting MBD2 and CREB1,and whether MBD2 and CREB1 are endogenous proteins of the host against PRRSV which not yet clear.In this study,the 3 '-UTR reporter plasmids of MBD2 and CREB1 were constructed and with miR-3 73 mimics and inhibitors and their respective controls together co-transfected into 293T cells,respectively.The results of dual-luciferase reporter experiment indicated that MBD2 and CREB1 were target genes of miR-373.Then,MARC-145 cells were infected with PPRSV(MOI=1)for 24h,36h and 48h,respectively.Real-time fluorescence quantitative PCR(qRT-PCR)and western blots results showed that PRRSV infection down-regulated the mRNA and protein expression levels of MBD2 and CREB1.To confirm whether MBD2 and CREB1 are endogenous proteins of PRRSV resistant hosts,we constructed the eukaryotic expression plasmids pcDNA3.1-Flag-MBD2 and pcDNA3.1-Flag-CREB1 of MBD2 and CREB1 using the eukaryotic expression vector pcDNA3.1-Flag.Using eukaryotic expression plasmids pcDNA3.1-Flag-MBD2 and pcDNA3.1-Flag-CREB1 were transfected into MARC-145 cells,respectively.After 24h,using PRRSV that MOI=1 to infected cells.After infected 24h,the TCID50 results showed that both MBD2 and CREB1 overexpression could reduce PRRSV titer;while qRT-PCR and western blots results showed that both MBD2 and CREB1 could reduce PRRSV load.On the contrary,siRNA experiments of MBD2 and CREB1 showed that down-regulation of both expressions was beneficial to PRRSV replication in MARC-145 cells.Finally,through gene deletion experiments,expression plasmids with partial domain deletion of MBD2 and CREB1 were constructed,and finally it was found that GR andMBD domain of MBD2 and bZIP domain of CREB1 may play a key role in inhibiting PRRSV replication of MBD2 and CREB1.In conclusion,in this study it was found that MBD2 and CREB1 is host endogenous protein of antagonism PRRSV;and MBD domain of MBD2 and bZIP domain of CREB1 may play a key role in inhibiting PRRSV replication of MBD2 and CREB1;and PRRSV may use miR-373 to down-regulate the expression of MBD2 and CREB1 to evade the host clearance.Therefore,this study further reveals that the molecular mechanism of PRRSV evade the host clearance and provides the new targets for the prevention and control of PRRSV.It provides a theoretical basis for the development of antiviral therapy based on the endogenous antiviral protein.