| Small ubiquitin-related modifier(SUMO) is a post-translational modification of proteins that widely exist in eukaryotic cells. It has been reported that some viruses encode proteins using the SUMO modification system to regulate host protein subcellular localization, protein stability and inhibite or escape the host’s immune response, thereby regulating the proliferation of virus. Porcine reproductive and respiratory syndrome is an immunosuppressive disease caused by porcine reproductive and respiratory syndrome virus(PRRSV). Nsp1α that encoded by PRRSV is a multifunctional non-structural proteins. Thro μ gh the biology software we predicted Nsp1α has a potential site modification by SUMO, but there is no experiment confirmed the existence of Nsp1αSUMO modification. In this study, we explore the biological function of Nsp1α from the SUMO modification of this new perspective. The main research contents are as follows:1. The interaction analysis of Nsp1α and SUMO related protein, PIAS1 and UBC9We constructed the Yeast recombinant plasmid of pGADT7-PIAS1、pGBKT7-UBC9、pGADT7-PIAS1 、 pGBKT7-UBC9. The recombinant plasmid pGADT7-PIAS1,pGBKT7-UBC9 had no toxicity and self activity in yeast. The results showed that in yeast systems, Nsp1α interact with PIAS1, but there is no interaction with the UBC9. In order to further verify whether Nsp1α can interact with PIAS1 in mammalian cells, the eukaryotic expression plasmid Nsp1α and PIAS1 were transfected into HEK293 T cells,Co-immunoprecipitation experiments showed that there is no direct interaction between Nsp1α and PIAS1.2. Detection the SUMO modification of Nsp1α.By considering the bioinformatics prediction results, we further analyzed the SUMO modification of Nsp1α althoμgh Co-immunoprecipitation experiments confirmed Nsp1αand PIAS1 have no direct interaction. In the HEK293 T cells transfected with pCAGGS-HA-Nsp1α, pCAGGS-Myc-SUMO1 and pcDNA-His-UBC9 three eukaryotic expression plasmid, by Co-immunoprecipitation to detect whether exist the SUMO modification of Nsp1α. The results confirmed that Nsp1α does not exist the SUMO modification.3. Nsp1α block intracellular SUMO modification levelSince Nsp1α SUMO modification does not exist, whether Nsp1α regulate the level of SUMO modification of host proteins? To explore this question, the HEK-293 T cells were transfected with increasing amounts of Nsp1α expression plasmid and SUMO1 expression plasmid, we found that Nsp1α hindered the intracellular SUMO modification level in a dose dependent manner, and the domain of ZF1 plays a key role.4. Nsp1α removal the SUMO modification of IRF3, promoting the degradation of IRF3Previous studies have demonstrated that PRRSV Nsp1α can antagonize RIG-I mediated interferon-beta(IFN- beta) generation. Nsp1α and RIG-I mediated signaling pathway proteins were transfection HEK293 T cells. Dual luciferase reporter assay showed that Nsp1α targeted IRF3. After over expression of Nsp1α, SUMO modification level of IRF3 were significantly reduced, at the same time, IRF3 stability decreased. This shows that Nsp1α can be destroyed IRF3 SUMO modification, and accelerate the degradation of IRF3, thus inhibiting the generation of IFN-β.5. Analysis of Nsp1α lysine sitesIn order to analyze the effect of lysine(K) residues of Nsp1α to SUMO modification function, we construct Nsp1α mutants K117 A, K150 A, K169 A, overexpress these mutants in HEK293 T cells. Western Blot results showed that the removal of intracellular SUMO modification were negatively regulated by three lysines of Nsp1α. However, dual luciferase report system test results showed that the lysine residues K117, K150, K169 of Nsp1α play negative regulatory role in the inhibition of IFN-β promoter, and inhibit the stimulation with IRF3-5D.In summary, this study demonstrated that Nsp1α can not modified by SUMO, but we found that Nsp1α can block SUMO modification in cells. More significantly, Nsp1αantagonize type I interferon production by hinder the SUMO modification of IRF3, which is a key transcription factor in the interferon signaling pathway, and promote its degradation, reveal a new mechanism of Nsp1α interferon regulation... |
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