| DDX3X is a RNA helicase, which belongs to the DEAD-box family of RNA helicases. DEAD-box helicases participate in many cellular processes, such as translational and transcriptional regulation, RNA splicing and nucleus export. On one hand, the results show that DDX3X takes part in the natural immune response and enhances the production of antiviral substances. On the other hand, DDX3X may be one of the means that virus achieve the purpose of the natural immune evasion.PRRSV is a single-strand RNA virus and is a member of the Arteritis virus. PRRSV causes PRRS, which brings great challenges to the pig industry around the world. Production of an effective vaccine is urgently required in the pig industry. Currently, we don't understand the functions of porcine DDX3X and the effect on the proliferation of PRRSV. So this study aimed to analyze the function of porcine DDX3X and the effect on the proliferation of PRRSV. The main contents were as follows:1. The Cloning, sequence analysis and phylogenetic tree construction of the porcine DDX3X geneThe porcine EST database was analyzed, and then the specific primers were designed. RNA of PK-15 cells was used as the template and the porcine DDX3X gene sequence was amplified via RT-PCR. The result of sequencing analysis indicated that the full length of porcine DDX3X's nucleotide was 1986bp and the open reading frame had 662 amino acids. And it had a amino acid homology of 97%,97%,98%,90%, and 53% with rodents, human, cattle, chicken and schistosoma japonicum, respectively. Although the result of phylogenetic tree analysis showed that there were some differences among the amino acid sequences of different species DDX3X, DDX3X was quite conserved in evolution, and porcine DDX3X had the closest relationship with cattle DDX3X.2. The subcellular localization of porcine DDX3XThe porcine DDX3X gene was inserted into the eukaryotic expression vector PEGFP-C2 to construct the eukaryotic expression plasmid of pEGFP-C2-DDX3X, and it was transfected into the PK-15 cells. Then we found that porcine DDX3X was mainly located in the cytoplasm, not in the nucleus.3. porcine DDX3X participated in the IFN-βproductionpCAGGS-HA-DDX3X was transfected into the PK-15 cells, then it was confirmed that porcine DDX3X could increase the levels of IFN-βmRNA expression by relative quantitative detection. Further, the siRNA named sipDDX3X which interfered with the endogenous porcine DDX3X was designed and was transfected into the PK-15 cells. Then the porcine DDX3X's effect on the IFN-βwhich was induced by SeV/poly(dA:dT) was detected. The result showed that the IFN-βmRNA levels induced by SeV/poly(dA:dT) were significantly decreased after the endogenous porcine DDX3X was knocked out. Meanwhile porcine DDX3X activated the IFN-βpromoter via dual luciferase assay system.4. The porcine DDX3X promoted TBK1/IPS-1 to activate the IFN-βpromoter in a dose-dependent mannerporcine DDX3X and TBK1/IPS-1 which were marked by the red fluorescent protein and green fluorescent protein co-localized in PK-15 cells. The result indicated that porcine DDX3X interacted with TBK1/IPS-1. Further, porcine DDX3X promoted TBK1/IPS-1 to activate the IFN-P promoter in a dose-dependent manner, which further confirmed that porcine DDX3X interacted with TBK1/IPS-1.5. The interaction between porcine DDX3X and PRRSVpEGFP-C2-DDX3X was transfected into Marc-145 cells which were then infected with PRRSV. The subcellular localization of porcine DDX3X was observed by laser confocal microscopy after DAPI staining. The result made it clear that PRRSV didn't change the porcine DDX3X's subcellular localization. Further study found that porcine DDX3X inhibited the proliferation of PRRSV, which was detected by absolute quantification and TCID50 and PRRSV could increase the expression of porcine DDX3X mRNA in PAM cells. |
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