Study On Interaction Of Japanese Encephalitis Virus NS5 Protein With Host Proteins

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a serious pathogen of human and animal. It causes Japanese encephalitis (JE) which is the most prevalent viral encephalitis in Asia. JE is also a veterinary problem, from which the pig industry suffered serious losses because of the reproduce failure. The excessive activation of inflammatory response induced by JEV infection is the key factor causing JE. However, the molecular mechanism of JEV pathogenesis still has not been elucidated. Studies have demonstrated that the protein interactions between JEV and hosts cellular factors contribute to viral replication, neurotropic virulence, neuronal apoptosis, immune evasion and immune activation. The non-structural protein NS5 of JEV comprised of an N-terminal methyltransferase domain and a C-terminal RNA polymerase domain plays a key role in viral replication and pathogenesis. Therefore, study of the interaction between JEV NS5 and host proteins will play important role in understanding of JEV replication and pathogenesis. Due to the unclear mechanism of JEV pathogenesis, no specific and effective therapy for the disease has been developed yet. So the exploration of new drugs against JE is of great importance. In this study, the interactions of JEV NS5 with host proteins and its role on JEV replication and immune evasion was investigated, and the effect of the commercial drug etanercept on JE was evaluated by using mouse model. The main contents of this study are as follows:1. Interaction between JEV NS5 and Hsp70Tandem affinity purification (TAP) followed by mass spectrometry analysis was performed to identify novel host proteins that interact with NS5. Heat shock protein 70 (Hsp70), eukaryotic elongation factor 1-alpha (eEF-la) and ras-related nuclear protein (Ran) were demonstrated to interact with NS5. In addition to NS5, Hsp70 was also found to interact with NS3 which is another important member of the replicase complex. It was observed that the cytoplasmic Hsp70 partially colocalizes with the components of viral replicase complex including NS3, NS5 and viral dsRNA during JEV infection. Knockdown of Hsp70 resulted in a significantly reduced JEV genome replication. Further analysis reveals that Hsp70 enhances the stability of viral proteins in JEV replicase complex. These results suggest an important role for Hsp70 in regulating JEV replication, which provides a potential target for the development of anti-JEV therapies.2. Inhibitory effect of JEV NS5 on nuclear import pathway of host cellsTrafficking of cellular molecules between the nucleus and the cytoplasm is essential to maintain cell homeostasis. By using the reporter system, we demonstrate that JEV NS5 protein is able to inhibit the classical nuclear import pathway in host cells, and this inhibitory function requires the nuclear localization signal (NLS) within NS5. We found that NS5 interacts with karyopherins, KPNA2, KPNA3 and KPNA4, through its NLS. Furthermore, NS5 was shown to block the nuclear localization of NF-κB subunits p50 and p65, which are cargo proteins of KPNA3 and KPNA4, and suppress the IFN-β production induced by poly(I:C). The immunoprecipitation assay revealed that the presence of NS5 inhibits the interaction of p50 and p65 with KPNA3 and KPNA4. Overexpression of KPNA2, KPNA3 and KPNA4 increases the production of IFN-βinduced JEV and inhibits the viral propagation. Collectively, our data suggest that JEV NS5 can block nuclear import pathway of host cells by targeting KPNA2, KPNA3 and KPNA4, which leads to the suppression of type Ⅰ IFN production and facilitates viral replication, suggesting a novel strategy for JEV to evade host antiviral response.3. Therapeutic effect of etanercept on Japanese encephalitisTumor necrosis factor alpha (TNF-a) is a key factor that mediates immunopathology in the central nervous system (CNS) during JE. Etanercept is a safe anti-TNF-a drug that has been commonly used in the treatment of various human autoimmune diseases. In this study, the effect of etanercept on JE was investigated with mouse neuron/glia cultures and JEV-infected mouse model. The in vitro assay with mouse neuron/glia cultures showed that etanercept treatment reduced the inflammatory response induced by JEV infection. In vivo experiments further demonstrated that administration of etanercept protected mice from JEV-induced lethality. Neuronal damage, glial activation, and secretion of proinflammatory cytokines were found to be markedly decreased in JEV-infected mice that received etanercept treatment. Additionally, etanercept treatment restored the integrity of the blood-brain barrier and reduced viral load in mouse brains. In conclusions, our data demonstrates that etanercept effectively reduces the inflammation and provides protection against acute encephalitis in a JEV-infected mouse model.