Studies On The Molecular Mechanisms That PRV Induced And TGEV Induced IFN-β Production

The mechanism of viral infection and host immune response is one of the most important frontier research fields in virology. The host innate immune system senses invading viruses through specific molecular pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and then initiate an immune response. Upon recognition, the PRRs initiates a series of signaling, this leads to expression of a number of cytokines such as type I interferon (IFN-I). The secreted IFN-I bind to the receptors and initiate signaling that activates transcription of thousands of genes. The produced proteins collaborate to inhibit viral replication and spread. On the other hand, IFN-I activate innate immune cells to induce adaptive immune response, resulting in clearance of invading virus. Thus, IFN-I play a vital role in host antiviral response.Although much progress has been made in understanding the virus-triggered IFN-I signaling pathways in recent years, there are a lot of questions remaining to be elucidated. For example, the detailed mechanisms about how the newly signal proteins recognize virus and induce IFN-I production are not known yet. Also, are there unknown proteins involved in virus-triggered IFN-I signaling? How the immune system recognizes DNA viruses to induce the expression of type I interferon, and so on. In recent years, there have been growing interests in the swine immune system, because of its potential as a model for the study of the human immune system with a lower cost and high similarity. To reveal the molecular mechanism of IFN-I induction after virus infection in swine, we cloned and characterized two important signaling proteins from porcine cells, and studied the signal pathways in innate immune responses during PRV infection and TGEV infection, as the model of DNA viruses and RNA viruses, respectively. The main research works were as following:1. Molecular cloning and functional characterization of porcine DNA-dependent activator of IFN-regulatory factors (DAI)The DNA-dependent activator of IFN-regulatory factors (DAI) is the first identified cytosolic DNA sensor for intracellular DNA that triggers a signal for the production of type I interferon. Different observations indicate that, in addition to acting in a cell type-specific way, the function of DAI might also show inter-species differences. In order to investigate the role of porcine DAI (poDAI) in the type I interferon signaling pathway, based on the porcine genomic sequence obtained from the BLAST search, we cloned and sequenced a DAI cDNA isolated from porcine peripheral blood mononuclear cells (PBMC). The full-length cDNA of poDAI contains 1320 bp and encodes 439 amino acid residues (GenBank accession number FJ455510). Structural analysis with the SMART program indicated that poDAI contains two putative N-terminal DNA-binding domains. Similar DNA-binding domains have been identified in cattle, human and mouse DAI. poDAI mRNA expression was mainly detected in spleen, lung, kidney and small intestine. To investigate whether poDAI is involved in the type I interferon signaling pathway, porcine IFN-P promoter luciferase reporter system assays showed Over-expression of poDAI activated transcription factors IRF3 and NF-κB and induced IFN-P in different porcine cell lines, but to varying degrees. Deletion mutant analysis revealed that both the DNA-binding domains and the C-terminus are required for full activation of IFN-β. siRNA targeting poDAI significantly decreased poly(dAT:dAT)-or Pseudorabies virus(PRV)-induced IFN-P activation. These results indicate that DAI is an important immuno-regulator of the porcine innate immune system.2. Molecular cloning and functional characterization of porcine stimulator of interferon genes (STING)The human stimulator of interferon genes (STING) has been proposed by three independent research groups as an adaptor that functions in cytosolic DNA signal pathway. Although the three groups concluded that STING is an important signaling sensor in the cytosolic DNA pathway, they also came to some significantly different conclusions. To investigate the role of porcine STING (poSTING) in the type I interferon signaling pathway, based on the porcine EST sequence obtained from the BLAST search, we cloned and sequenced poSTING cDNA isolated from porcine peripheral blood mononuclear cells. The full-length cDNA of poSTING contains 1137 bp and encodes 378 amino acid residues (GenBank accession number FJ455509), contains one endoplasmic reticulum(ER) retention motif, RAR. poSTING mRNA expression was mainly detected in the spleen, lymph node and lung. Structural analysis with the SMART program indicated that poSTING contains four putative transmembrane(TM) domains at its N-terminus. poSTING was found to reside predominantly in the ER, and also in the mitochondrial membrane in PK-15 cells. Over-expression of poSTING activated both IRF3 and NF-κB to induce IFN-βproduction, while knockdown of poSTING significantly inhibited DNA virus and RNA virus induced IFN-P promoter activation and IFN-P mRNA production. Altogether, these results indicate that STING is an important regulator of porcine innate immune signaling. The results will help better understand the biological role(s) of STING in innate immunity during evolution.3. Studies on the molecular mechanism that PRV induced type I interferon productionPseudorabies Virus (PRV) is a swine alpha herpesvirus owning double-stranded DNA genome. Previous studies have demonstrated PRV infection triggered efficient immune response. However, the mechanism that PRV induced type I interferon production is still unclear. Using Real-time PCR and porcine IFN-βpromoter luciferase reporter system, we demonstrated that PRV infection in PK-15 cells up-regulated IFN-βgene transcription. To evaluate the detailed molecular mechanisms, we investigated the roles of TLR signal pathway and cytosolic signal pathway in IFN-P induction during PRV infection, by RNAi technique and dominant negative mutants. Our results showed that PRV-induced IFN-βwas related with the adaptor MyD88 in TLR signal pathway and cytosolic signal proteins RIG-I, and VISA. RNAi experiments showed that PRV-induced IFN-βin PK-15 cells require IRF1, IRF5, and IRF7. Interestingly, IRF3, the very important transcription factor for type I interferon production after virus infection, is nonessential for PRV-induced IFN-βin PK-15. Further study demonstrated that IRF3 is nonessential for PRV-induced IFN-βin HEK293 cells, either. Altogether, these results suggest that a distinct pathway utilized by PRV to regulate innate immunity. However, there are many questions remain unknown, such as how the host recognize PRV to trigger IFN-I signal pathway and how about the detailed signal transduction networks.4. Studies on the molecular mechanism that TGEV induced type I interferon productionTransmissible Gastroenteritis Virus (TGEV), a member of coronavirus species, is an enveloped RNA virus. Previous studies have demonstrated TGEV is able to induce IFN-I production. However, the mechanisms that TGEV induced type I interferon production are still unclear. Using Real-time PCR and porcine IFN-βpromoter luciferase reporter system, we demonstrated that TGEV infection in PK-15 cells up-regulated IFN-βgene transcription. To evaluate the mechanisms behind this, we investigated IFN-βinduction in TLR signal pathway and cytosolic signal pathway during TGEV infection. Our results demonstrated that TGEV induced IFN-βwas related with the adaptor MyD88 in TLR signal pathway, as well as several cytosolic signal proteins, such as RIG-I, MDA5, STING, HMGB1, HMGB2, IRF5, and IRF7. Whereas unrelated with TRIF, IRF1, and IRF3. Further study demonstrated that TGEV membrance(M) protein and encleocapsid(N) protein can activate IFN-βpromoter, indicating that this two proteins may involve in TGEV induced IFN-P production.