HnRNP A1 and Lamin A/C mediate nucleocytoplasmic shuttling of the nonstructural protein p17 of avian reovirus

• Main Authors: Yu-Yang Wang, 王昱揚
• Other Authors: Hung-Jen Liu
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/79737030921648341324

碩士 === 國立中興大學 === 分子生物學研究所 === 103 === Previous studies demonstrated that ARV p17 protein can regulate host cell signal transduction and it is a nuleocytoplasmic shuttling protein that continuously shuttles between the nucleus and the cytoplasm. To date, the molecular mechanisms underlying p17 protein shuttling between the nucleus and the cytoplasm is largely unknown. In this study, GST pull-down assay indicated that p17 can interact with hnRNP A1. We also demonstrate that the N-terminal (aa 19-60) of p17 is an important region for hnRNP A1 binding and the C-terminal (aa 195-268) of hnRNP A1 is an important region for p17 binding. In co-immunoprecipation assays, We also demonstrate that the N-terminal (aa 27-60) of p17 is an important region for Lamin A/C binding was defined. Immunofluorescene staining showed that p17 was translocated into the nucleus at 16 hours and released from the nucleus at 18 hours in ARV-postinfection. In addition, p17 was translocated into the nucleus at 16-18 hours and released from the nucleus at 20 hours in post-transfection. Knockdown of hnRNP A1 impeded p17 nuclear import, suggesting that it is an important carrier for mediating p17 nuclear import. Plaque formation assay supports that Lamin A/C play a critical role in mediating p17 nuclear export. Furthermore, in our bioformatic analysis, mutagenic studies and immunofluorescence staining of p17 protein, we also defined that the aa 19-26 of p17 is nuclear export signal (NES) for p17 nuclear export. By point mutation assay, we also demonstrated that amino acid residues 21L, 24L, and 26I are critical for p17 nuclear export. This is the final study to demonstrate that hnRNP A1 is a carrier protein for mediating p17 nuclear import, and Lamin A/C is an important mediator for p17 nuclear export. A clearer elucidate on the molecular basis for nucleocytoplasmic shuttling of p17 is useful for understanding the mechanisms of virus-induced signal pathways and ARV replication.