Replication Of Newcastle Disease Virus And Autophagy In Infected Cells Regulated By Toll-like Receptor 3

Newcastle disease virus (NDV) causes avian respiratory disease and death, cause great economic losses to poultry firm. It is not only an important avian disease, but also is a virus can replicate in tumor cell and destroy tumor cells. NDV has been used as an oncolytic factor in tumor therapy as it did not infect human beings. However, the mechanism of NDV oncolytic factor is still not very clear. Recently, virus induced innate immunity and interaction between virus and host immunity has become a hot point. Innate immunity is the first line in combating virus infection, so it is important to understand the innate immune response by NDV infection.The viral nucleotide is defined as pathogen-associated molecular patterns (PAMPs) which can be recognized by host innate immune system. The component recognizing PAMPs in the innate immune system is named as pattern recognition receptors (PRRs). Toll-like receptors 3 (TLR3), a member of TLR family, is a newly PRRs in the cell surface which can recognize dsRNA or dsRNA mimic (e. g. poly(I:C)). When activated, it recruit cytoplasmic adapter protein that having TIR domain (e. g. TIR-domain-containing adaptor inducing interferon-β, TRIF) to transfer signal to the down stream, leading the activation of transcription factor interferon regulators-3(IRF-3) and nuclear factor-KB (NF-κB), thus upregulate the type I interferons (interferon, IFN) and cytokines expression. It has been reported that TLR3 has an antivirus role to some virus infection. However, there were no reports about induction of TLR3 in NDV infection. Additionaly, the invasion of pathogen-induced autophagy is a hot point in recently research. It has widely known that proteins of pathogenic microorganism combined with cellular receptors can induce autophagy. TLR3 as a pattern recognition receptor can recognize viral double-stranded RNA. The relationship between activated TLR3 and autophagy is still not clear. To explore these aspects, we carried out the following studies:(1) NDV infection can activate TLR3 signaling pathway. In order to find out whether NDV infect tumor cell can activate TLR3, HeLa (Henrietta Lacks) cells were infected with NDV, Western-blot and immunofluorescence were used to detect the TLR3. The results showed that NDV Herts/33 strain could significantly upregulate TLR3 protein level at 12-24 h post infections. UV-inactivated NDV could not activate TLR3 pathway. Antibody specifically detecting dsRNA was used to detect dsRNA in the immunofluorescence. We discovered that dsRNA were produced during NDV infection, thus activating TLR3 signaling pathway. In addition, NDV avirulent strain La Sota could also activate TLR3.(2) TLR3 signaling pathway has function during NDV infection. In order to clarify whether activated TLR3 has effect on NDV infection, we constructed human TLR3 eukaryotic expression plasmid, and transfect it into HeLa cells. Western-blot、real-time PCR and TCID50 were used to detect resistance to NDV infection in transfected cells. The result showed that hTLR3 overexpression could inhibit viral protein transcription and translation significantly, virus titers in cell culture medium were greatly changed. While TLR3 siRNA transfected into HeLa cells, TLR3 expression was inhibited, viral protein transcription and translation were unregulated and virus titer increased. We also detect the interferon and cytokine change after TLR3 or siTLR3 tranfection. Real-time PCR showed that in contrast to control group, IFN-β, IL-6, IL-8 and IFIT1 mRNA increased significantly, indicating TLR3 could induce IFN-βand cytokine production. In addition, we also use luciferase report gene method to dectect IFN-β and NF-κB promoter activity. Cells transfected with TLR3 then infected with NDV, IFN-β and NF-κB promoter activity were increased.(3) TLR3 and RIG-I co-promote the production of interferon and cytokine. Beside TLR3, other PRRs like retinoic acid-inducible gene 1 (RIG-I) has important function on the innate immune. In order to clarify the contribution of TLR3 and RIG-I on the down stream signaling, we synthesized RIG-I siRNA. The siTLR3, siRIG-I and siTLR3 plus siRIG-I was transfected into cells then infected with NDV. Real-time PCR was used to detect IFN-β, IL-6, IL-8 and IFIT1 mRNA change. The result showed that IL-6 and IL-8 mRNA levels in siTLR3 transfected cells was lower than siRIG transfected cells. IFN-β mRNA level in siRIG-I cells was lower than TLR3 transfected cells. In addition, luciferase report method was used to detect the promoter activity of IFN-β、NF-κB. We found that NF-κB promoter activity in siTLR3 transfected cells was lower than siRIG transfected cells. IFN-β promoter activity in siRIG-I transfected cells was lower than siTLR3 transfected cells. These results showed that two PRRs use different strategy to regulate interferon and cytokines.(4) In order to observe whether TLR3 activation could induce autppgagy, The A549 cells (adenocarcinomic human alveolar basal epithelial cells) were treated with poly (I:C) HMW, fluorescence and laser confocal microscopy and western-blot were used to detect the formation of autophagy. A549 cells were treated with poly (I:C) and transfected with GFP-LC3 plasmid, 12-24 h post infection, dot formation was observed in the cells. Western-blot showed that LC3-I conversed to the LC3-Ⅱ. The same results were observed in the HeLa cells treated with poly (I: C). These results demonstrated that TLR3 activation will cause the formation of autophagy. In order to clarify TLR3 induced autophagy was a complete autophagy, we detect p62 degradation after poly (I:C) stimulation, and we detect LC3-II turnover after lysosome treatment, and we observe GFP-LC3-Ⅱ and LAMP1 colocalization. These results confirmed that activated poly (I: C) induced complete autophagic flux.(5) In order to clarify the mechanism of activated TLR3 inducing autophagy, we constructed a series of TRIF, TLR3 and Beclin-1 truncated and full length plasmid. The immunofluorescence method was used to observe TLR3/TRIF and Beclin-1 cocalization. The co-immunoprecipitation was used to detect the interaction between TRIF and Beclin-1. The results showed that, after TLR3 activation, adaptor protein TRIF and Beclin-1 could partially cocalization. Also, TRIF and Beclin-1 could interacted, and the interaction site was TIR domain of TRIF, BH3 domain of Beclin-1. Normally, Beclin-1 combined with B-cell lymphoma-2 (Bcl-2) to inhibit the autophagy. Once TLR3 activated, TRIF interacted with Beclin-1 to reduce the combination of Bcl-2 and Beclin-1, thus initiate autophagy.(6) NDV NP and P proteins induce autophagy via the endoplasmic reticulum stressrelated unfolded protein response. In order to explore the mechanisms of NDV induce autophagy A549 cells were transfected with plasmids expressing FLAG-tagged NDV genes.The results showed that NDV NP and P proteins trigger autophagy in A549 cells and altered ER homeostasis as well. We also demonstrated that NDV NP and P protein-induced autophagy is regulated through the PERK and ATF6 pathways of the unfolded protein response (UPR).In summary, this study demonstrated that TLR3 actively participates in the recognition of the innate pro-inflammatory response after NDV infection and leads to the consequent antiviral cytokine/interferon secretion. We also find that autophagy is detectable after activation of TLR3, its adaptor protein TRIF combined with Beclin-1 which reduce Bcl-2 combined with Beclin-1, leading the process of autophagy. Furthermore, NDV NP and P proteins are involved in NDV-induced autophagy and that this process is at least partially regulated by ER stress. We studied the relationship between TLR3 and autophagy, and combined these two biology phenomenon together, providing a possible way to disease therapy.