| Summary: | 碩士 === 國立金門大學 === 食品科學系 === 106 === The nervous necrosis virus (NNV) genome contained two small (3.1 K and 1.4 K) positive sense single strand RNAs which encoded RNA-dependent RNA polymerase, capsid protein and B2 proteins. The viruses rely on the host protein translation devices for protein synthesis and have evolved a vast array of strategies for exploiting and dominating the cellular translation machinery. In this experiment, the grouper brain (GB) cell associated proteins were obtained by the NNV virus-like particle (VLP) affinity column purification. The binding proteins were further confirmed by virus overlay protein binding assay (VOPBA), and then analyzed by LC-MS/MS. LC-MS/MS identified 118 kinds of proteins, including 64 ribosomal proteins, 3 histones, Y-box-binding protein 1, polyadenylate-binding protein (PABP), MKI67 FHA domain-interacting nucleolar phosphoprotein, complement component 1 Q subcomponent-binding protein, natural killer cell enhancement factor, heterogeneous nuclear ribonucleoproteins C1/C2, protein arginine N-methyltransferase 1 and other proteins. The function of NNV VLP binding proteins identified are mainly related to translation and transcription. However, the real role of the protein function needs further investigation. To understand the translational changes of GB cell after NNV infection, we performed the non-isotope labeled SUnSET method by puromycin. It was found that cellular translation has been slightly reduced at 12 hour after NNV infection and almost completely shutdown at 18 and 24 hour. However, some proteins were dramatically induced after NNV infection. With the specificity of anti-PBAP, we found that the distribution of PABP shifted from the cytoplasmic region to the nuclear region 6 hour post NNV infection. It is implied that PABP may be involved in the host cell translation shutdown after NNV infection.
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