| Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases that seriously affect the global swine industry. And the pathogen of this disease is porcine reproductive and respiratory syndrome virus (PRRSV), whose nonstructural proteins (nsps) play important roles in regulating viral replication and pathogenicity. Investigating the interaction between PRRSV nsps and host cell proteins, as well as exploring their biological functions are of significance for elucidating the molecular mechanism of replication and pathogenicity in PRRSV. In this study, we confirmed that viral nsp2 and nsp10 can interact with cellular DDX18, as well as the interaction between viral nspll and host IRAK1, and then the molecular mechanisms of their interaction involved in viral replication have been further investigated.Previously, we have found that the host cellular protein DDX18 has potential interaction with PRRSV nsp2 and nsp10. In this study, co-immunoprecipitation (Co-IP) assay was used to confirm their interaction, and the interaction regions were further identified as nsp2-N (PL2 domain) interacting with DDX18-N (DEXDc domain) and DDX18-C (HELICc domain) respectively, and both nsp10-N (ZBD domain) and nsp10-C (helicase domain) being able to interact with DDX18-C (HELICc domain),respectively. By utilizing indirect immunofluorescence (IFA) and confocal microscopy assays, the DDX18 was found to redistribute from the nucleus to the cytoplasm and co-localization with nsp2 and nsp10. Silencing DDX18 expressing by RNAi in MARC-145 cells can significantly reduce the viral titer and the copy number of PRRSV ORF7. Conversely, over expression of DDX18 in MARC-145 cells can slightly increase the viral titer of PRRSV and the copy number of its ORF7.These results suggest that the interaction of DDX18 with nsp2 and nsp10 play a pro-viral role in the replication of PRRSV.Furthermore, a PRRSV nspll specific monoclonal antibody, designated 3F9, was generated, and identified to bind to a PRRSV genotype-conserved epitope with the core sequence of "DCREY". By using the nsp11 monoclonal antibody, the interaction between nsp11 and host cell protein IRAK1 was determined in immunoprecipitation and tandem mass spectrometry. Besides, we also found that nspll was co-localized with IRAK1 in the cytoplasm. Silencing IRAK1 in MARC-145 cells could inhibit the production of IFN-? and significantly enhanced the replication of PRRSV, meanwhile, over expression of IRAKl in MARC-145 cells can slightly inhibit the replication of PRRSV. And the expression level of IRAK1 was found to be inhibited in the PRRSV-infected MARC-145 cells. More importantly, further studies showed that the expression of mml-miR-146a was up-regulated in PRRSV-infected MARC-145 cells, which is a host microRNA that target IRAKl's 3 'UTR and inhibits the expression of IRAK1.Inhibitor-mml-miR-146a could down-regulate mml-miR-146a to further offset the inhibition of PRRSV on IRAK1 in dose-depended manner. In addition, we found that down regulation of IRAK1 inhibits the expression of IRF7, and subsequently inhibit the production of IFN-? that provides a benefits environment for PRRSV replication. Interestingly, mml-miR-146a was also determined to target PRRSV genome directly and inhibit the replication of PRRSV. These results confirmed the interaction between nspll and IRAK1, and revealed the role of IRAK1 as well as the dual regulatory role of mml-miR-146a involved in the replication of PRRSV, which will contribute to the understanding of PRRSV replication and pathogenicity. The results of this study take the interaction of PRRSV nsp2,nsp10 and nspll with host cell proteins DDX18 and IRAK1 as the breakthrough point, and further explore the effects of DDX18 and IRAK1 on viral replication, providing a scientific basis for elucidating the replication and pathogenesis of the virus. |
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