EB病毒BGLF4蛋白質激酶與IQGAP1的交互作用

The interaction of EBV BGLF4 with IQGAP1

黃浩雲(Hao-Yun Huang)

指導教授 ： 陳美如

國立臺灣大學/醫學院/微生物學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10428

摘要

BGLF4為EB病毒的絲胺酸/蘇胺酸激酶，具有與Cdc2相似的激酶活性。目前已知其可磷酸化許多病毒及細胞內蛋白質。前人利用酵母菌雙雜交實驗發現BGLF4可與IQGAP2產生交互作用。IQGAP (IQ motif-containing GTPase activating protein)家族成員中以IQGAP1被研究得較為清楚，不但可作為細胞骨架調控蛋白，亦可作為ERK訊息傳遞路徑的鷹架蛋白。本研究的目的在確認BGLF4與IQGAP family的交互作用，並進一步探討這樣的交互作用對EB病毒顆粒的釋出有何重要性。本研究首先以共同免疫沉澱方式確認外源性IQGAPs能與BGLF4產生交互作用。此外以TPA/SB刺激NA細胞，使EB病毒進入溶裂期後，BGLF4可與內生性IQGAP1共同免疫沉澱。透過免疫螢光染色也發現當表現外源性BGLF4時，有13%細胞中BGLF4會與內生性IQGAP1共同聚集在細胞核旁的凹陷區域。進一步利用免疫螢光染色發現當EB病毒複製活化時IQGAP1與BGLF4也會聚集在細胞核旁凹陷區域，此區域與內質網位於相同位置。另外的研究發現IQGAP1能與MMLV的MA蛋白有交互作用，且對於MMLV的進入及釋出細胞都是重要的，因此本研究接著探討IQGAP1是否參與在EBV釋出的過程當中。利用帶有shIQGAP1的慢病毒knockdown細胞中IQGAP1表現後，再誘使EB病毒進入溶裂期，並以PCR或Q-PCR觀察在細胞培養上清液中EB病毒含量。在EREV8細胞中發現抑制IQGAP1表現對於EB病毒釋出並無太大影響，反而會影響細胞的存活率。而在NA細胞中觀察發現在抑制IQGAP1表現後培養液中的EB病毒有減少的趨勢，因此我們認為IQGAP1可能在EB病毒釋出的過程中扮演重要的角色。

關鍵字

EB病毒 ； IQGAP1 ； BGLF4 ； 病毒釋出

並列摘要

BGLF4 of Epstein-Barr virus (EBV) is a proline dependent Serine/threonine protein kinase which shares similar kinase activity with Cdc2. BGLF4 can phosphorylate many viral and cellular proteins. It was found that BGLF4 interacts with IQGAP2 in yeast-two-hybrid system in a previous study. The functions of IQGAP1 (IQ motif-containing GTPase activating protein 1) are the most characterized one in IQGAP family. IQGAP1 functions as a cytoskeleton regulator, and as a scaffold protein for ERK signaling pathway. This study aimed to confirm the interaction between BGLF4 and IQGAP protein, and to further study the contribution of this interaction in EBV viron release. The interactions between exogenous IQGAPs and BGLF4 were first confirmed in co-immunoprecipitation assay. By using Immuno- fluorescence staining, ectopic BGLF4 colocalized with endogenous IQGAP1 at the concave site of perinuclear region in 13% of BGLF4 positive cells. In addition, BGLF4 was co-immunoprecipated with endogenous IQGAP1 in TPA/SB treated NA cells. Immunofluorescence staining showed that IQGAP1 also co-localized with BGLF4 at concave site of perinuclear region in EBV replicating cells. The interaction sites of IQGAP1 and BGLF4 were associated with ER marker. Additionaly, IQGAP1 was reported to interact with the MA protein of MMLV, and this interaction is required for trafficking of MMLV both into and out of cells. Therefore it was addressed whether IQGAP1 participates in the process of EBV production. Using lentivirus carrying shIQGAP1 to knockdown IQGAP1 in cells, we detected EBV DNA in culture supernatant by PCR or Q-PCR after EBV reactivation. In IQGAP1 knockdown EREV8 cells, the release of EBV did not decrease after EBV reactivation, whereas preliminary data showed that the EBV DNA in culture supernatant decreased in IQGAP1 knockdown NA cells. It suggested that IQGAP1 may play an important role in the process of EBV release.

並列關鍵字

EBV ； IQGAP1 ； BGLF4 ； viral release

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