Study On Interaction Of Japanese Encephalitis Virus NS3 Protein With Host Proteins

Japanese encephalitis virus(JEV) belongs to the genus Flavivirus and family Flaviviridae, it is a serious pathogen of human and animal. JEV is transmitted by mosquitoes, it causes encephalitis by targeting central nervous system. However, the molecular mechanism of JEV replication and pathogenesis still has not been elucidated. NS3 protein is one of JEV non-structural proteins, which possesses several enzyme activities, including serine protease, helicase, and RNA triphosphatase and plays a key role in viral replication and pathogenesis. Therefore, study of the interaction between JEV NS3 and host proteins will play important role in exploring of NS3 function, understanding of JEV replication and pathogenesis, and developing antiviral drugs. In this study, we screened and identified host proteins interacting with JEV NS3 protein, and in-depth analyzed of the molecular mechanisms and functions of the interactions. The main contents of this study are as follows:1. Preparation of monoclonal antibodies against JEV NS3Balb/C mice were immunized with purified prokaryotic expressed JEV NS3 protein, two specific monoclonal antibodies named 1H7 and 2D4 were prepared after cell fusion and four times subclone. Western blot and IFA analysis proved that the two monoclonal antibodies showed high reactivity and specificity.2. Screening and identification of cellular proteins interacting with JEV NS3Tandem affinity purification(TAP) combined with mass spectrometry analysis was performed to identify novel host proteins interacting with JEV NS3. Three proteins named Ribosomal Protein S11(RPS11), Adenine nucleotide translocase 2(ANT2) and Eukaryotic translation elongation factor 1-alpha(EEF1A1) were obtained. Co-immunoprecipitation showed that these proteins can interact with JEV NS3.3. The interaction between JEV NS3 and EEF1A1 and its role in JEV replicationImmunoprecipitation-RT PCR showed that EEF1A1 can interact with the components of JEV replicase complex. The intercellular co-localization of EEF1A1 with JEV NS3, NS5 and viral double strand RNA(dsRNA) was confirmed by immunofluorescence analysis. Besides, Virus proliferation assay showed that EEF1A1 can positively regulate the replication of JEV. We further explored the molecular mechanisms of EEF1A1 regulates JEV replication and found that EEF1A1 can interact with the viral proteins in replicase complex and protect them being degraded through ubituitin-proteasome system, and thus favor the viral replication.4. JEV regulates the expression of EEF1A1 and viral replication throughmiR-33a-5pFluorescence quantitative Real-time PCR and Western blot demonstrated that JEV can upregulate EEF1A1 expression. Further analysis showed that JEV upregulates EEF1A1 expression through downregulates the expression of miR-33a-5p as EEF1A1 is a target of miR-33a-5p. What’s more, JEV infection downregulates miR-33a-5p expression at the transcriptional level through inhibiting the activation of the transcription factors NF-Y and sp1 of SREBP-2.Through TAP, this study firstly discovered three host proteins: RPS11, ANT2 and EEF1A1, which can interact with JEV NS3. In-depth analysis of the interaction between EEF1A1 and NS3 reveals that EEF1A1 plays an important role in JEV replication. Meanwhile, it also clarifys the molecular mechanism of how JEV regulates the expression of EEF1A1. These results will help us understand of deep mechanism of JEV replication and provide insight to explore new antiviral drugs.