Identification Of Interferon-Stimulated Genes Against Porcine Reproductive And Respiratory Syndrome Virus And Uncovering Of Their Antiviral Mechanism

Porcine reproductive and respiratory syndrome(PRRS)is a contact infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).Since the outbreak of PRRS in 1987,it has been threatening the development of the world pig industry.The highly pathogenic PRRS(HP-PRRS)that broke out in 2006 has caused huge economic losses to the world pig industry.In recent years,with the development and use of vaccines,the PRRS epidemic has been controlled to a certain extent,but under the pressure of immune pressure,PRRSV has also accelerated its own variation and evolution.Therefore,finding and developing new anti-PRRSV drugs has become an urgent problem to be solved.In order to solve this problem,we constructed a number of eukaryotic expression vectors expressing interferon-stimulated genes(ISGs),and identified three ISGs with anti-PRRSV effects,namely MOV10,TRIM25 and cGAS.MOV10 is a member of the RNA helicase superfamily-1,which is one of the components of the RNA-induced silencing complex and has a wide range of antiviral effects.In this study,a MARC-145 cell line stably expressing porcine MOV10 was constructed.It was found that MARC-145 cells stably expressing MOV10 can reduce the expression level of PRRSV N protein,and the viral titer and viral RNA copy number in cells were significantly lower than that in wild-type MARC-145 cell,so MOV10 is an ISG with anti-PRRSV effect.Adsorption experiments revealed that MOV10 did not affect the attachment and internalization of PRRSV.Co-immunoprecipitation and immunofluorescence co-localization analyses showed that MOV10 interacted and co-localized with the PRRSV N protein in the cytoplasm.Notably,MOV10 affected the distribution of N protein in the cytoplasm and nucleus,leading to the retention of N protein in the former.Taken together,these findings demonstrate for the first time that MOV10 inhibits PRRSV replication by restricting the nuclear import of N protein.The tripartite motif protein 25(TRIM25)is an E3 ubiquitin ligase involved in various cellular processes including regulating the innate immune response against viruses.When the viral RNA is recognized by RIG-I,the N-terminal caspase recruitment domains(CARDs)of RIG-I are modified by ubiquitin,which is mediated by TRIM25,and this is essential to active a signaling cascade ultimately resulting in the transcriptional activation of type I interferons.In this study,a new host-restricted factor,TRIM25,was identified as an inhibitor of PRRSV replication.Co-immunoprecipitation assay showed that the PRRSV N protein interferes with TRIM25–RIG-I interactions by competitively interacting with TRIM25.Furthermore,N protein inhibits the expression of TRIM25 and the TRIM25-mediated ubiquitination of RIG-I to suppress IFN-? production.Moreover,as TRIM25 expression increased,the inhibitory effect of N protein on the ubiquitination of RIG-I was diminished.These results indicate for the first time that TRIM25 inhibits PRRSV replication and the N protein antagonizes the antiviral activity by interfering with TRIM25-mediated RIG-I ubiquitination.As a DNA receptor in the cytoplasm,cGAS not only plays a major role in the natural immune process induced by DNA virus infection,but also plays an important role in the anti-RNA virus process.In this study,cGAS was verified inhibiting the replication of PRRSV on MARC-145 cells through overexpression and knockout experiments.At the same time,overexpression of cGAS can also stimulate the expression of IFN-?,ISG15 and OASL.Besides,cGAMP can also inhibit the replication of PRRSV on MARC-145 cells.In addition,infection with the virus after transfection of cGAS can significantly increase the activity of cGAMP in cells.Electron microscopy and indirect immunofluorescence experiments suggested that PRRSV infection can cause mitochondrial damage and the releasing mitochondrial DNA to the cytoplasm.The mitochondrial DNA released into the cytoplasm can bind to cGAS,thereby activating the host's innate immune system and cGAMP activity and then exerts antiviral effect.Moreover,cGAS not only inhibits HP-PRRSV,but also inhibits both European-type PRRSV and classical PRRSV.In summary,three ISGs with anti-PRRSV function were discovered in this study,and the mechanisms by which these three ISGs exert anti-PRRSV effects were elucidated experimentally.This not only provides a theoretical basis for the development of drugs to prevent and control PRRSV,but also further clarifies the host's new mechanism of antagonizing PRRSV replication.