| Transmissible gastroenteritis virus(TGEV) is a coronavirus which causes an acute enteric disease of swine, with clinical symptoms typified by lethal watery diarrhea and severe dehydration in piglets. Because mortality can be up to 100% in infected piglets less than 2 weeks old, this disease always causes high economic losses once outbreaks occur and poses a threat to swine industry worldwide. Several previous studies have described that TGEV is a good type I interferon(IFN-I) inducer. In addition, previous research has demonstrated that virulent TGEV caused significant inflammatory response in intestinal tissues, and animal death was mainly attributed to the severe unbalance of Na+and K+ions caused by the clinical manifestation of the infection. Thus, the pathogenesis of TGEV is strongly associated with the powerful induction of innate immune response during TGEV infection in host cells. Nevertheless, there is only limited information on the mechanisms of TGEV pathogenesis and TGEV-mediated innate immune response.Given that proteomic methods have become important tools with which to study host cellular responses to viral infection, we used proteomic technique to reveal the relationship between TGEV and host cells in this study. Moreover, the molecular mechanism of inflammatory response was also analyzed in TGEV-infected host cells. Our study provides a better understanding of TGEV pathogenicity and was useful for the design of new prophylactic and therapeutic strategies. The main research works were as following:1. Proteomic analysis on TGEV-infected host cellsQuantitative proteomic methods are widely accepted tools with which to study virus-host cell interactions. In this study, isobaric tags for relative and absolute quanti?cation(i TRAQ) approach was used to identify the di?erentially expressed proteins in PK-15 cells after TGEV infection. In total, 162 di?erentially regulated proteins were identi?ed, including 60 upregulated proteins and 102 downregulated proteins. A bioinformatic analysis showed that these di?erentially expressed cellular proteins were involved in various biological systems and processes. Most importantly,many upregulated proteins were associated with interferon signaling, especially signal transducer and activator of transcription 1(STAT1) and interferon-stimulated genes(ISGs). Immunoblotting and q RT-PCR demonstrated that TGEV infection induces STAT1 phosphorylation and nuclear translocation, as well as ISG expression.2. The molecular mechanism of NF-κB activation by TGEV infectionNF-κB is an inducible transcription factor involved in promoting inflammatory response, as well as the regulation of cell proliferation and survival. The pathogenesis of TGEV is strongly associated with inflammation, in which NF-κB plays a pivotal role.Firstly, our study demonstrated that TGEV infection activated NF-κB by using luciferase reporter system. And we also found that TGEV infection induced phosphorylation and nuclear translocation of p65 and degradation of IκBα. Moreover, the levels of IL-6, IL-8,TNF-α, and RANTES m RNAs were signi?cantly raised in TGEV-infected cells, which were blocked by a specific NF-κB inhibitor. Further studies investigated the underlying mechanisms of TGEV-induced NF-κB activation and showed that the expression of phosphorylated p65 and proinflammatory cytokines were greatly reduced after knockdown of the receptors RIG-I or MDA5. Similar results showed that adaptor proteins MAVS and STING, rather than My D88 or TRIF, were critical for this inflammatory response. These data indicated that RIG-I-like receptors pathway is involved in TGEV sensing and NF-κB signaling represents a major contribution to the TGEV-induced inflammatory response.3. Studies on TGEV-induced inflammation through JNK signalingMAPK signaling pathway is involved in many cellular processes such as inflammation, proliferation, cell differentiation and death. MAPK signaling can be activated by various inflammatory stimulis, as well contributes to inflammatory response.In our study, the three MAPKs(ERK1/2, JNK1/2, p38) were all phosphorylated after TGEV infection, and TGEV activated the transcription factor AP-1 by dual luciferase assay. To further determine which signaling pathway was involved in TGEV-induced the production of proinflammatory cytokines, the specific inhibitors of ERK, JNK and p38 were respectively added to cells during infection. The results showed that inhibition of JNK1/2 signaling pathway markedly decreased the expression of IL-6, IL-8, TNF-α, and RANTES in TGEV-infected cells. In addition, our study found that inhibition of JNK1/2impaired TGEV replication. |
