Functional Analysis Of Inhibition Of Prrsv Replication By Procine LSM14A Gene And Activation Of NF-KB By G4 Gene

Chapter I:Functional analysis of inhibition of PRRSV replication by procine LSM14A genePorcine reproductive and respiratory syndrome (PRRS) caused by porcine reproductive and respiratory syndrome virus (PRRSV), a single positive-strand RNA virus, leading to reproductive disturbance and respiratory disease, such as preterm birth and reproductive failure in female pigs and dyspnea of fattening pigs and piglets. PRRS has already been one of the most economically important diseases for the pig industry worldwide.Pattern recognition receptors (PRRs), including endosomal Toll-like receptors (TLRs), cytosolic RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), play an important role in the innate immune system. Emerging evidence indicates that PRRs play key roles in recognizing pathogen associated molecular patterns (PAMPs). LSM14A (also called RAP55) is identified as a novel sensor that mediates both RNA and DNA virus triggering the induction of antiviral genes during viral infection, and inhibits the replication of vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV).In the present study, Marc-145 cells and porcine alveolar macrophages (PAMs) were used to investgate the effect of porcine LSM14A on PRRSV replication. The results are as follows:(1) We cloned the complete coding sequence (CDS) of LSM14A, IFI6, IF116, PPBP, ZDHHC9 and CLU from Tongcheng pig, and inserted them into the eukaryotic expression vector pRK-flag. The recombinant plasmids were transfected into Marc-145 cells to examine the effect on PRRSV replication. The result indicated that the overexpression of LSM14A significantly inhibited PRRSV replication in Marc-145 cells.(2) The effect of LSM14A on PRRSV replication in Marc-145 cells was examined by overexpression and interference through Quantitative PCR, immunofluorescence and plaque experiment. The result indicated that the overexpression of LSM14A significantly inhibited PRRSV replication, and Knockdown of LSM14A could enhance PRRSV replication in Marc-145 cells.The overexpression of porcine LSM14A in PAMs by Ad5-LSM14A infection significantly inhibited PRRSV replication at 48 h post PRRSV infection, and knockdown of LSM14A significantly enhanced PRRSV replication at 48 h post infection.(3) We examined IFN-β and interferon stimulated response element (ISRE) promoter activities in Marc-145 cells. Promoter activities of IFN-β and ISRE were activated by LSM14A, and the activities showed a higher increase when infected with PRRSV. The expression level of IFN-β mRNA was highly significantly up-regulated by LSM14A, and the same result was obtained in RIG-I mRNA. Typical Interferon-stimulated genes (ISGs), such as IFITMs, IFITs and OAS1, were also significantly up-regulated. Overall, during PRRSV infection in Marc-145 cells, viral RNA was detected by LSM14A, and then IFN-β signaling pathway was activated and amplified, resulting in the up-regulation of the expression of ISGs to eliminate the virus.(4) The overexpression of LSM14A significantly suppressed the expression of TNF-α and IL-6, indicating that the inflammatory factor was inhibited.(5) LSM14A was down-regulated in PAMs, spleens and lungs of Tongcheng and Large White pigs in 7 days post infection.Chapter Ⅱ:Functional analysis of the activation of NF-κB by G4 geneThe major histocompatibility complex (MHC), also named leukocyte antigen, is divided into three subregions:class Ⅰ, class Ⅱ and class Ⅲ. MHC takes part in autoimmune, infectious and inflammatory diseases. MHC Ⅲ contains an inflammatory gene concentrated region, which has been suggested to be designated as the class Ⅳ region (MHC-Ⅳ), such as TNF superfamily and so on. G4, also named C6orf47, as one of the MHC-Ⅳ gene, located between Hsp70 and AIF-1, TNF, and interacted with fibroblast growth factor receptor 3(FGFR3). Few researches have been done about this gene. At the same time, expression of G4 in mononuclear cells of patients with arthritis was significantly lower than controls, this illustrated that G4 has some relationship with inflammation. Nuclear transcription factor NF-κB plays an important role in inflammation. So this research based on the novel genes in MHC-Ⅳ and the existing results we got in the early study of G4 gene, to analysis the relationship between these genes and NF-κB, and the localization characteristic of G4, thus investigate the relationship between G4 gene and inflammatory response and the signal pathway of G4 activate NF-κB at cell and individual level. The results are as follows:(1) We performed an NF-κB luciferase report assay to examine the potential NF-κB activating activity for the function-unknown MHC-IV genes, including ABHD16A、G4、 C6orf48、GPANK1、MCCD1、SAPCD1 and ZBTB12, we observed that the G4 gene activated NF-κB obviously. By co-transfection of GFP-p65 and HcRed-tagged G4 in HeLa cells, we observed that overexpression of G4 protein induced nuclear translocation of GFP-p65 significantly. This means NF-κB signaling pathway was activated and NF-κB entered the nucleus to conduct of transcriptional regulation.(2) Overexpressed G4 protein located on the plasma membrane, and partly co-localization with early endosome by immunofluorescence (IF). The endogenous G4 protein was tested by immunohistochemistry highly expressed in bladder epithelial cells and pulmonary alveolar epithelial cells, and didn’t exit in Cerebrum, cerebellum, small intestine and liver, and intriguingly, plasma membrane localization of G4 protein in bladder epithelial cells was observed.(3) G4 protein induces NF-κB activation is dependent on inhibitor of nuclear factor kappa-B kinase (IKK) complex rather than TGF-beta activated kinase 1 (TAK1) complex. TAK1 complex is in the upstream of NF-kB signaling pathway. Neither TAK1 inhibitor 5Z-7-oxozeaenol nor dominant negative mutant TAK1 (K63W) could block G4 protein induced NF-κB activation, this indicated that G4 protein induces NF-κB activation independent of TAK1 complex. G4 protein inducing NF-κB activation could be blocked by the IKK inhibitor (PS 1145), which demonstrates that G4 protein activates the canonical NF-κB pathway upstream of IKK complex.