浙江大學周繼勇組揭示傳染性法氏囊病病毒聚合酶泛素化調控新機制

近日，Journal of Virology雜誌在線發表了浙江大學動物科學學院、農業部動物病毒學重點實驗室周繼勇課題組的最新進展，首次報道傳染性法氏囊病病毒聚合酶蛋白VP1泛素化修飾的分子機制。原文題為「Ubiquitination is essential for avibirnavirus replication by supporting VP1 Polymerase activity」。

傳染性法氏囊病病毒（Infectious Bursa Diseases Virus, IBDV）是雙RNA病毒科 (Birnaviridae)，禽雙RNA病毒屬 (avibirnavirus)的唯一代表成員。IBDV屬於禽類高度接觸性傳染病，主要危害15日齡左右的雛雞，雛雞中樞免疫器官法氏囊為該病毒的主要靶器官。由於該病發病突然、病程短、死亡率高，且可引起雞體免疫抑制，目前仍然是養雞業的主要傳染病之一。IBDV基因組包含雙鏈RNA (dsRNA) A片段和B片段，A片段 (Segment A)包含兩個重疊的開放閱讀框，編碼非結構蛋白VP5和多聚蛋白前體，多聚蛋白最終裂解成衣殼蛋白前體pVP2、衣殼蛋白VP2、內衣殼蛋白VP3及水解酶蛋白VP4，B片段 (Segment B) 包含一個大的開放閱讀框，編碼病毒聚合酶蛋白VP1，負責基因組的複製和轉錄。有諸多研究報道證明泛素化修飾對病毒的調節作用，特別是A型流感病毒，但是，IBDV聚合酶蛋白翻譯後修飾及其調節機制仍不清楚。

本研究首次在IBDV感染的293T、DF-1細胞以及法氏囊組織中檢測到若干條分子量偏大的VP1條帶，初步認為是泛素修飾的VP1。進一步實驗證明VP1在感染、轉染情況下均能高效發生泛素修飾（圖1A-B），通過對VP1截短髮現，VP1泛素化修飾發生在蛋白C末端，後對C末端19個賴氨酸（K）精細定位發現，K751為其泛素化修飾位點（圖1C）。為了深入研究泛素化修飾對聚合酶活性的影響，我們構建了Minigenome方法用以檢測VP1聚合酶活性，並發現泛素化修飾可顯著上調VP1聚合酶活性（圖1D），而K751R突變後，VP1聚合酶活性顯著下降（圖1E），通過反向遺傳拯救獲得了K751R突變的IBDV，且突變IBDV較比野生型IBDV，其複製能力顯著下降（圖1F）。

圖1 泛素化修飾對IBDV聚合酶蛋白VP1聚合酶活性及病毒複製能力的調節作用。

該研究首次揭示了泛素化修飾對IBDV聚合酶蛋白VP1聚合酶活性及病毒複製能力的調節作用，加深了對該病毒聚合酶調節方面的認識，也為抗病毒藥物的研發提供了潛在靶點。

ABSTRACT

Ubiquitination is critical for several cellular physical processes. However, ubiquitin modification in virus replication is poorly understood. Therefore, the present study aimed to determine the presence and effect of ubiquitination on polymerase activity of viral protein 1 (VP1) of avibirnavirus. We reported that the replication of avibirnavirus is regulated by ubiquitination of its VP1 protein, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV). In vivo detection revealed the ubiquitination of VP1 protein in IBDV infected target organs and different cells, but not in purified IBDV particles. Further analysis of ubiquitination confirms that VP1 is modified by K63-linked ubiquitin chain. Point mutation screening showed that the ubiquitination site of VP1 was at the K751 residue in the C-terminus. The K751 ubiquitination is independent of VP1』s interaction with VP3 and eukaryotic initiation factor-4A II. Polymerase activity assays indicated that the K751 ubiquitination at the C-terminus of VP1 enhanced its polymerase activity. The K751 to R mutation of VP1 protein did not block the rescueing of IBDV, but decreased the replication ability of IBDV. Our data demonstrated that the ubiquitination of VP1 is crucial to regulate its polymerase activity and IBDV replication.

IMPORTANCE

Avibirnavirus protein VP1, the RNA-dependent RNA polymerase, is responsible for IBDV genome replication, gene expression and assembly. However, little is known about its chemical modification relating to its polymerase activity. In this study, we revealed the molecular mechanism of ubiquitin modification of VP1 via a K63-linked ubiquitin chain during infection. Lysine (K) residue 751 at the C-terminus of VP1 is the target site for ubiquitin and its ubiquitination is independent of VP1』s interaction with VP3 and eukaryotic initiation factor-4A II. The K751 ubiquitination promotes the polymerase activity of VP1 and unubiquitinated VP1 mutant IBDV significantlyimpairs virus replication. We concluded that VP1 is the ubiquitin-modified protein and revealed the mechanism by which VP1 promotes avibirnavirus replication.

來源：中國病毒學（英文版）

本期編輯：Tony

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