Screening And Mechanisms Study Of HP-PRRSV Non-structural Protein (s) Activating NF-κB Signaling Pathway

Porcine reproductive respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a novel infectious disease of swine. It is characterized by severe reproductive failure in sows, respiratory distress high mortality in piglets and growing pigs. The genome of PRRSV is single-stranded positive sense RNA that encodes14kinds of nonstructural proteins and7kinds of structural proteins. Among all these proteins, Nsp2is the largest and the most variable protein encoded by PRRSV.The atypical PRRS, which was caused by highly pathogenic PRRSV (HP-PRRSV), occurred in China in2006and caused tremendous economic losses due to the death of millions of pigs. Since then, the HP-PRRSV strain, which caused a more severe pneumonia and higher mortality than the classical PRRSV, has become the dominant strain in China. However, the signal transduction mechanisms involved in HP-PRRSV infection remain poorly understood. In view of the new PRRS, we analysed the mechanisms of the inflammatory responses caused by the virus in the point of signal pathway, which provide a theoretical basis for understanding molecular mechanism of PRRSV pathogenicity. The main research works were as following:1. Studies on the activation of NF-κB signal pathway induced by HP-PRRSVNF-κB is an inducible transcription factor that plays a key role in inflammation and immune responses, as well as in the regulation of cell proliferation and survival. In our study, we investigated the activation of NF-κB induced by HP-PRRSV using a luciferase reporter assay. The result showed that PRRSV activates NF-κB dependent on its replication and in a dose-dependent manner. In addition, we detect the expression pattern of various receptors and signaling molecules in MARC-145cells at diferernt time point upon PRRSV infection to evaluate the mechanisms. It was showed that the upregulated expression of receptors TLR2, TLR4and the adaptors TRAF6, TAK1was observed during PRRSV infection, which indicated that PRRSV activates NF-κB signal pathway through upregulating the expression of the TLR2and TLR4and subsequently TRAF6and TAK1signal molecules. But we also found that, the expression of other adaptors were not increased. Instead, some of them even dicreased at some certain time points, suggesting the complexity of PRRSV-induced the NF-κB activation. In this way, it was concluded that PRRSV modulates NF-κB signal pathway using a variety of different strategies, which ultimately results in the activation of NF-κB.2. Screen the specific HP-PRRSV Nsp(s) involved in NF-κB activationThe nucleocapsid (N) protein was identified as an NF-κB activator among the structural proteins encoded by PRRSV; however, it remains unclear whether the nonstructural proteins (Nsps) of PRRSV contribute to NF-κB activation. To further evaluate the mechanisms of NF-κB activation, we identified the specific PRRSV Nsp(s) involved in NF-κB activation. By screening the individual Nsps of PRRSV strain WUH3using a luciferase reporter assay, Nsp2exhibited great potential to activate NF-κB in MARC-145and HeLa cells, while this activation was not observed in cells overexpressing other viral nonstructural proteins.3. Studies on the activation of NF-κB signal pathway induced by PRRSV Nsp2In this study, overexpression of Nsp2induced IκBα degradation and nuclear translocation of NF-κB. Furthermore, Nsp2also induced NF-KB-dependent inflammatory factors, including interleukin (IL)-6, IL-8, COX-2, and RANTES. Using a series of truncated mutants of Nsp2, the functional domain responsible for NF-κB activation was located in the180-480amino acid in the HV region of Nsp2. What's more, the predicted transmembrane region in the C terminal of Nsp2play an important part in the subcellular localization of Nsp2as well as the NF-κB activation induced by Nsp2, implied that the mitochondria location of Nsp2might be necessary for the NF-κB activation. The detailed mechanism requires further investigation. Additionally, we investigated the upstream of NF-κB signal pathway activated by Nsp2. It was identified that the TLR4adaptors and TRIF and MAVS molecules was indispensable in the NF-κB activation induced by Nsp2through conducting an SiRNA technique. Moreover, the expression pattern of several adaptors were detected in HeLa cells which an increasing amount of Nsp2was transfected. It was showed that the expression of TLR4receptors and adaptors TRIF, TRAF2were upregulated upon Nsp2overexpression. All these results suggested that both the TLR4signal pathway and the RIG-I signal pathway participated in the Nsp2-induced NF-κB activation. The mitochondria localization protein MAVS was proved to play important parts in the Nsp2-induced NF-κB activation in our study, which correlated well with the previous deduction that the mitochondria location of Nsp2was necessary for its ability of NF-κB activation. Taken as a whole, these results revealed that PRRSV Nsp2and PRRSV itself activate NF-κB signal pathway in their own differents ways, which also implied the complexity of host immune response during PRRSV infection.4. The interaction of Nsp2with RAI contributes to NF-κB activationThe protein-protein interaction of between virus and host is the molecular basis of virus pathogenicity during its infection. In present study, we explored the possible mechanism of Nsp2induced NF-κB activation by searching the functional protein-protein interaction. Host proteins that interact with PRRSV Nsp2protein were screened from pig brain cDNA library through yeast two-hybrid technology. RAI was selected for further investigation due to its biological role in inhibiting the transcription activity of p65subunit of NF-κB. Then the mammalian two-hybrid assay, the co-immunoprecipitation and the fluorescent co-localization test were performed to verify the interaction of Nsp2and RAI. Using the truncations of Nsp2and RAI, we also identified that the region of interaction located in the middle HV region of Nsp2and the C-terminal of RAI. Further biological function of the interaction was explored using a luciferase reporter assay. The result showed that Nsp2could antagonize the function of RAI in the NF-κB inhibiton probablely by antagonizing its interaction with p65. What's more, Nsp2didn't inhibit the expression of RAI in a western blot assay, which also implied that Nsp2might bind to RAI in competition with p65subunit to antagonize its inhbition on NF-κB transcription.5. Study on the molecular mechanism of different degree of inflammatory response between the classical PRRSV and highly pathogenic PRRSVCompared with the Nsp2of the classical PRRSV strain, the Nsp2of highly pathogenic PRRSV (HP-PRRSV) strains that possess a30amino acid (aa) discontinuous deletion in Nsp2coding region displayed greater NF-κB activation. However, the30-aa deletion was demonstrated to not be associated with NF-κB activation in a luciferase reporter assay using a series of insertion and deletion mutants. Furthermore, a promoter luciferase reporter assay was conducted to determine the ability of different Nsp2and their mutants in the activation NF-KB-regulated target gene expression, including IL-6, IL-8, and RANTES. The results showed that Nsp2and mutants from HP-PRRSV can induce higher expression level of this inflammation factors compared to that of from classic PRRSV isolate. At tha same time, the30aa deletion in Nsp2of HP-PRRSV was not related to its different levels of viral pathogenesis. Taken together, these data indicate that PRRSV Nsp2is a multifunctional protein participating in the modulation of host inflammatory response, which suggests an important role of Nsp2in pathogenesis and disease outcomes.