A型流感病毒核蛋白泛素化/去泛素化修飾與病毒核醣核酸複製調控機制之研究

本研究成功建立一套in vitro cell-based 高效能RNAi 篩選系統，並利用此系統篩選到與A型流感病毒感染相關之宿主細胞蛋白USP11。由實驗結果顯示，USP11與抑制病毒RNA複製有關，因此當USP11表現受到抑制，病毒RNA可大量複製；反之，當USP11大量表現，則病毒RNA複製受到抑制。由進一步的實驗結果證明此抑制作用是與USP11的去泛素酵素活性有關。免疫沉澱實驗結果顯示USP11可與病毒蛋白PB2，PA及NP進行交互作用，其中更發現NP是單泛素化修飾蛋白(mono-ubiquitinated protein)，其修飾位點是胺基酸K184，而K184位於NP蛋白與病毒RNA結合位置。由實驗結果顯示當K184突變而無法進行泛素化修飾時，會影響NP與RNA的結合能力，進而使病毒的RNA複製效率下降。而宿主細胞則藉由USP11將NP蛋白去泛素化而使病毒RNA複製受到抑制。由本實驗結果推論，A型流感病毒核蛋白NP可藉由泛素化/去泛素化修飾來進行病毒RNA複製機制的調控。

關鍵字

A型流感病毒；核蛋白；泛素化； RNA複製； USP11

並列摘要

Influenza A virus RNA replication requires an intricate regulatory network involving viral and cellular proteins. Here we examined the roles of cellular ubiquitinating/deubiquitinating enzymes (DUBs). We found that down-regulation of a cellular deubiquitinating enzyme USP11 resulted in enhanced virus production, suggesting that USP11 could inhibit influenza virus replication. Conversely, over-expression of USP11 inhibited specifically viral genomic RNA replication, and this inhibition required the deubiquitinase activity. Furthermore, we showed that USP11 interacted with PB2, PA and NP of viral RNA replication complex, and that NP is a mono-ubiquitinated protein and can be deubiquitinated by USP11 in vivo. Finally, we identified K184 as the ubiquitination site on NP and this residue is crucial for virus RNA replication. We propose that ubiquitination/deubiquitination of NP can be manipulated for antiviral therapeutic purposes.

並列關鍵字

influenza A virus ； NP ； ubiquitination ； RNA replication ； USP11

參考文獻

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A型流感病毒核蛋白泛素化/去泛素化修飾與病毒核醣核酸複製調控機制之研究

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