EB 病毒蛋白質 Rta 對粒線體的影響

Role of Rta of Epstein-Barr virus in mitochondria

蔡曉涵(Hsiao-Han Tsai)

指導教授 ： 張麗冠

國立臺灣大學/生命科學院/生化科技學系/碩士(2016年)

https://doi.org/10.6342/NTU201600518

摘要

Epstein-Barr Virus (EB 病毒) 屬於人類疱疹病毒科，其生活史包括潛伏期 (latent life cycle) 與溶裂期 (lytic cycle)，而極早期蛋白質 Rta 是促使 EB 病毒進入溶裂期的關鍵轉錄因子之一。本實驗室在先前的研究中發現 Rta 蛋白質在 EB 病毒潛伏期時會分布於粒線體。粒線體參與了許多細胞生理途徑，如能量的產生、細胞死亡、宿主免疫反應的誘導和老化等；而粒線體自噬 (mitophagy) 為維持粒線體網絡健康的重要機制。本研究欲證實 Rta 蛋白質上是否具有粒線體導向序列 (mitochondria targeting sequence，MTS)，並初步探討 Rta 蛋白質位於粒線體上的意義及其扮演的角色。首先以具有病毒潛伏的 P3HR1 細胞、B95-8 細胞及 293-2089 細胞為材料，利用免疫螢光染色分析探討 Rta 於細胞中的分佈情形，結果證實 Rta 蛋白質位於粒線體；同時利用粒線體分離分析結果指出，細胞中 Rta 蛋白質在細胞質與粒線體中皆有表現。接著利用 293T 細胞為材料，轉染入不同 Rta 片段之建構質體，可藉由觀察其進粒線體與否推定出 MTS 為 Rta 蛋白質之 N 端第 68 至 90 胺基酸片段，並由生物資訊軟體預測出其具有 α 螺旋體結構。接著以共免疫沉澱分析證明了 Rta 蛋白質會與粒線體外膜蛋白導入受體 (import receptor) Tom20 結合。而後以 ATP 測定分析結果發現，在 293T 細胞表現 Rta 後會使細胞中的 ATP 產量提高。最後，以 carbonyl cyanide m-chlorophenyl hydrazine (CCCP) 處理 293T 細胞，誘導粒線體損害進而引發粒線體自噬，結果發現 Rta 蛋白質的表現可能會促進粒線體自噬。總和以上所述，本研究首度證實 Rta 含有一 α 螺旋體結構的 MTS，可位於粒線體並影響粒線體的功能。

關鍵字

EB 病毒 ； Rta ； 粒線體 ； 粒線體導向序列 ； 粒線體自噬

並列摘要

Epstein-Barr virus (EBV) is a human herpesvirus with two distinct life cycles, latent and lytic. Rta, an immediate-early protein, is an important transcription factor required for initiating the lytic cycle of EBV. Our preliminary study found that Rta is localized at mitochondria during EBV latency. Mitochondrium is a multifunctional organelle with diverse roles including energy production, apoptosis, elicitation of host immune response, and aging. Moreover, mitochondria autophagy (mitophagy) is a critical mechanism to maintain mitochondrial health and homeostasis. This study aims to investigate whether Rta contains mitochondria targeting sequence (MTS), and examine roles of Rta in mitochondria. First, immunofluorescence analysis indicated that Rta colocalizes with mitochondria in P3HR1 cells, B95-8 cells and 293-2089 cells during both EBV latency and lytic cycle. Mitochondria isolation assay was performed and indicated that Rta expresses both in cytosolic and mitochondrial fraction. Mutagenesis and bioinformatic analyses demonstrated that an MTS is localized between amino acid residues 68 to 90 in Rta, which forms an amphipathic α-helix structure. Co-immunoprecipitation assay further indicated that Rta binds to Tom20, an import receptor on mitochondrial outer membrane. ATP determination assay also revealed that overexpressing Rta increases ATP levels in 293T cells. Finally, treatment of carbonyl cyanide m-chlorophenyl hydrazine (CCCP) to 293T cells with expression of Rta affects mitophagy. Taken together, this study demonstrates that Rta is a mitochondrion-targeted protein that contains an amphipathic α-helical MTS, which may affect the functions of mitochondria.

並列關鍵字

Epstein-Barr virus ； Rta ； mitochondria ； mitochondria targeting sequence ； mitophagy

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