The stability of P1 of Turnip mosaic virus impacts the HC-Pro suppression of miRNA pathway

植物病毒 Potyvirus 屬之 P1/HC-Pro 蛋白為第一個被發現之病毒抑制子 (viral suppressor)，其中 P1 蛋白雖不具有後轉錄基因沉默 (post transcriptional gene silencing; PTGS) 之功能，但 HC-Pro 須與 P1 共同作用可加強抑制轉錄後基因沉默及抑制微型核酸 (microRNA; miRNA) 調控機制之功能，目前已知 P1 病毒蛋白胺基酸序列與長度變異度高，P1 與 HC-Pro 之相互作用與詳細機制至今尚未明瞭。本研究以蕪菁嵌紋病毒 (Turnip mosaic virus, TuMV) 為材料，針對 P1 蛋白於 P1/HC-Pro 抑制調控微型核酸機制之角色探討。為瞭解 P1/HC-Pro 於病毒感染過程與轉錄後基因沉默機制之角色，所使用之血清靈敏度與專一性是相當重要的，因此本研究製備 P1、HC-N (HC-Pro 之第 1~267 個胺基酸)、HC-C (HC-Pro 之第 249~458 個胺基酸) 與 CP 之四種兔血清 (antiserum)，再利用高效能蛋白純化系統 (fast protein liquid chromatography ; FPLC) 純化血清中之免疫球蛋白 G (immunoglobulin G ; IgG)，並以蛋白濃縮管提升 IgG 之濃度，以獲得高品質之血清。本研究發現 P1 於活體中 (in vivo) 相當不穩定且極易被降解，於是利用 26S proteasome 抑制劑 (26S proteasome inhibitor) - MG132 處理 TuMV 罹病之植物或是表現 P1/HC-Pro 之轉基因植物，結果仍無法偵測到 P1 蛋白，顯示P1可能不受到 ubiquitin/26S proteasome 路徑調控。表現 P1/HC-Pro 基因之阿拉伯芥植株外表型嚴重畸型 (abnormality)，如矮化 (dwarfism)、鋸齒狀葉片 (serratedd leaves)，顯示植株體內之微型核酸路徑受到干擾。本研究於 P1 蛋白 N 端加上 FLAG 或 YFP (yellow fluorescent protein) 標記蛋白 (tags)，或於 N 端刪除 200 胺基酸並保留 C 端保守區間後，皆會使得 P1 變穩定因而被偵測到，並使得轉基因阿拉伯芥鋸齒葉之表型較輕微 (mildly serrated)。綜觀本研究結果，推測高變異度之 N 端區域對於穩定蛋白之活性佔有重要地位，因此於 N 端加上標記蛋白或是刪除部分胺基酸，都有可能破壞 N 端結構而使得 P1 變穩定，進而影響微型核酸路徑而改變轉基因植物之表型。本研究透過改變 P1 蛋白N端結構，進一步瞭解 P1 於植物發育之重要調控微型核酸路徑中所扮演之角色。The P1/HC-Pro of potyvirus is the first discovered viral suppressor and HC-Pro needs to work with P1 for enhancing the suppression effect against post transcriptional gene silencing (PTGS) and inhibiting the microRNA (miRNA) pathway; however, the mechanism of P1/HC-Pro remains unclear. Furthermore, P1 is the most divergent protein in length and amino acid sequence among potyviruses. For studying the function of P1/HC-Pro, the antisera specific to P1, HC-N (1-267 a.a. of HC-Pro), HC-C (249-458 a.a. of HC-Pro) or CP were developed. In order to improve the quality of antiserum, fast protein liquid chromatography (FPLC) was used to purify immunoglobulin G (IgG) and to concentrate IgGs by protein centricon for enhancing the sensitivity. In this study, we demonstrated that the rapid turnover rate of P1 in vivo and the stability of P1 is crucial for miRNA pathway. Moreover, TuMV-infected plants and P1/HC-Pro transgenic Arabidopsis were treated with MG132 (26S proteasome inhibitor) to demonstrate that P1 is under an ubiquitin-independent degradation process. Interestingly, the P1 protein became stabilized and detectable while its N-terminus is fused with YFP (yellow fluorescent protein) or Flag tags and it caused a mild phenotype with serrated leaves in transgenic plants. Surprisingly, the P1dN600/HC-Pro transgenic Arabidopsis, which retained the conserved regions at the C-terminus of P1, became detectable and showed mild leaf serration in Arabidopsis. It suggests that the N-terminus of P1 regarding its stability is crucial for inhibiting miRNA pathway. We conclude that the stability of P1 affects the efficiency of miRNA suppression, and the N-terminus of P1 regulates the turnover rate. Study on the instability of P1will help clarify the role of P1/HC-Pro in miRNA pathway