分析B型肝炎病毒顆粒在細胞內交通運輸的調控機制

Dissection of the Modulation Mechanism of Intracellular Trafficking Activity of Human Hepatitis B Virus Capsids

黃爾毅(Er-Yi Huang)

指導教授 ： 施嘉和

共同指導教授 ： 林陽生(Young-Sun Lin)

國立臺灣大學/醫學院/分子醫學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.10038

摘要

Ｂ型肝炎病毒(HBV)的核心蛋白質(HBc) 在蛋白質序列147-183的carboxyl端上包含一塊富有arginine的區域(ARD)。在之前的研究，我們證明了ARD與HBc的交通信號有關，包含細胞核定位訊號(NLS)以及細胞核輸出訊號(NES)。在研究不同長度的HBc deletion突變型的實驗中，我們指出在交通信號之間的邊界區域對於影響HBc在細胞內的位置非常重要。在這些交通信號之間的邊界區域有共七個serines和一個threonine。以前的報告已經顯示模仿去磷酸化(dephosphorylation)的突變型S162A和S170A能強烈將HBc導向細胞核，而突變型S155A只是溫和的影響HBc在細胞核堆積。然而突變型S162A和S170A造成HBc在細胞核堆積的現象到底是透過活化NLS或者抑制NES尚不清楚。另外在交通信號之間的邊境區域中，其他的serines或者非serines序列是否調控HBc的交通運輸亦不清楚。在這裡我們的研究指出突變型S162A和S170A都能活化NLS並且同時抑制NES。相對於wild type的HBc，HBc突變型S168A在活化NLS但沒有明顯影響NES的情況下將HBc從細胞質移動到細胞核的趨勢增加200倍。突變型S178A則在強烈的抑制NES和較小程度影響NLS的情況下將HBc從細胞質移動到細胞核的趨勢增加117倍。除了serines和threonines之外，另外一個具有明顯表型的突變P163Q讓超過百分之70的細胞具有HBc在細胞核堆積的現象。這個結果可以在hydrodynamic delivery的老鼠模型中被確認。不過，HBc在細胞核堆積的現象在雙突變型S162D/P163Q中減弱了10 倍，這個結果顯示S162D對HBc的影響超越P163Q。總而言之，我們證明了HBc在細胞內的分佈可以受到NLS、NES及其邊境區域的調節。然而在整個HBc ARD區域中缺乏負電荷(serine phosphorylation)強烈促進HBV核心蛋白質在細胞核堆積。

關鍵字

Ｂ型肝炎病毒 ； 核心蛋白質 ； arginine-rich區域 ； 細胞核定位訊號 ； 細胞核輸出訊號 ； 磷酸化

並列摘要

Hepatitis B virus (HBV) core protein (HBc) contains an arginine-rich domain (ARD) at the carboxyl terminus of HBc 147-183. Previously, we demonstrated that ARD is associated with trafficking signals, including nuclear localization signals (NLS) and nuclear export signals (NES). In our studies of serially truncated HBc mutants, we noted that border regions between the trafficking signals are very important in affecting the subcellular localization. There are a total of 7 serines and one threonine at the borders between these trafficking signals. Previous reports have shown that dephosphorylation-mimicking mutations S162A and S170A can strongly target HBc to nucleus, while S155A exhibited only mild effect on nuclear accumulation of HBc. Mechanistically, it is unclear if nuclear import of HBc of mutants S162A and S170A is mediated through activation of NLS or inactivation of NES. Furthermore, it remains unclear if other serine or non-serine residues at the borders could also be subject to modulation of trafficking activity. In our studies here, both S162A and S170A can activate NLS and inactivate NES simultaneously. Relative to wild type HBV, HBc mutant S168A exhibited 200 fold increased tendency to shift HBc from cytoplasm to nucleus by activating NLS, with no apparent effect on NES. Mutant S178A exhibited 117 fold increased tendency to shift HBc from cytoplasm to nucleus by strongly inactivating NES and to a lesser extent on NLS. In addition to serines and threonines, a striking phenotypic effect was observed in mutant P163Q with more than 70 % of cells containing nuclear HBc. This result was confirmed by a mouse model with hydrodynamic delivery. However, double mutant S162D/P163Q exhibited a 10-fold reduction of HBc accumulation in the nucleus, suggesting that S162D is dominant over P163Q. In summary, we demonstrated that the subcellular localization of HBc can be regulated by both NLS and NES. Lack of negative charge (serine phosphorylation) spanning the entire HBc ARD strongly promoted nuclear accumulation of HBV core protein.

並列關鍵字

Hepatitis B virus ； HBV ； core protein ； HBc ； arginine-rich domain ； ARD ； nuclear localization signals ； NLS ； nuclear export signals ； NES ； phosphorylation

參考文獻

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