The Role Of DICER1 In PRRSV Replication

Porcine reproductive and respiratory syndrome(PRRS),one of the most important infectious diseases,is caused by PRRS virus(PRRSV)that leads to reproductive failure in sows and respiratory disease patterns in pigs of all ages.And it has affected the swine industry worldwide for almost 30 years and caused huge economic losses to the swine industry.Due to its high speed of transmission,genetic diversity,severe infection of multiple pathogens,and limited protection of current commercial vaccines and antiviral drugs,the prevention and control of PRRSV remains a major problem for the swine industry.Therefore,it is of great theoretical and practical significance to understand the mechanism of interaction between PRRSV and host factor for the study of viral biology and the development of effective vaccines.In previous research,we found that there was interaction between Dicer protein and the PRRSV nucleocapsid protein.It is suggested that Dicer may have an effect on PRRSV replication,but exactly how influence is unclear.This study is to further verify the interaction between Dicer protein and PRRSV nucleocapsid protein and clarify the effect of DICER1 on PRRSV replication.The specific work and results were as follows:1.Construction of pEGFP-C1-PRRSV N and p CAGEN-myc-Dicer eukaryotic expression plasmidsThe entire ORF7 gene of PRRSV and the CDS region of Sus scrofa DICER1 were amplified by PCR and Overlap PCR,respectively.Then they were cloned into pEGFP-C1 and p CAGEN eukaryotic expression vectors respectively to successfully construct pEGFP-C1-PRRSV N and p CAGEN-myc-DICER1 recombinant expression plasmids.2.The interaction between Homo sapiens Dicer and PRRSV nucleocapsid proteinThe interaction between Homo sapiens Dicer and PRRSV nucleocapsid protein after transfection of HEK293 T with eukaryotic expression plasmid of pEGFP-C1-PRRSV N was detected by co-immunoprecipitation.The results showed that there was an interaction between Homo sapiens Dicer and PRRSV nucleocapsid protein,suggesting that Dicer may have an impact on PRRSV replication.3.The interaction between Sus scrofa DICER1 and PRRSV nucleocapsid proteinTo further clarify whether the host protein DICER1 interacts with PRRSV nucleocapsid protein,the interaction between Sus scrofa DICER1 and PRRSV nucleocapsid protein after co-transfection of HEK293 T with eukaryotic expression plasmids of pEGFP-C1-PRRSV N and p CAGEN-myc-DICER1 was detected by co-immunoprecipitation.The results showed that there was an interaction between Sus scrofa DICER1 and PRRSV nucleocapsid protein,further suggesting that Dicer may have an impact on PRRSV replication.4.Knockdown the expression of DICER1 in PAMs supressed replication of different PRRSV strainsThe si RNA-mediated RNA interference was used to deplete DICER1 in PAMs,and the effect on replication of different PRRSV strains was examined by q RT-PCR and TCID50.The results showed that knockdown of DICER1 expression significantly reduced the expression level of PRRSV ORF7 gene and virus loads in the supernatants,demonstrating that reducing basal expression level of DICER1 in PAMs could significantly inhibit replication of different PRRSV strains.In summary,this study has demonstrated the interaction between the Homo sapiens Dicer protein or Sus scrofa Dicer protein and the PRRSV nucleocapsid protein from the perspective of interaction between host factor and viruse,and has clarified that reducing the basal expression of DICER1 could inhibit replication of different PRRSV strains.These results of this study not only provide a theoretical basis for a better understanding of the biological characteristics of PRRSV and its interaction with the host,but also provide a new perspective for prevention and control of PRRSV.