Mutational effects of the consensus aromatic residues in the mRNA capping domain of Bamboo mosaic virus on GTP methylation and virus accumulation and the establishment of BaMV replication system in Saccharomyces cerevisiae

竹嵌紋病毒為 alphavirus-like superfamily 中馬鈴薯 X 病毒屬之成員，為正意 RNA 病毒。其基因體含有五個主要的轉譯架構，轉譯架構 1 可轉譯出大小約 155 kDa 的複製酵素，由 N 端至 C 端可分為三個活性區域：戴帽酵素區、類解旋酵素區及 RNA 聚合酵素區。alphavirus-like superfamily 有著特異的戴帽流程，病毒的戴帽酵素功能會先產生 m7GTP (甲基轉移活性)，之後再將其中的 m7GMP 轉移至 5 端雙磷酸 RNA 上 (鳥苷轉移活性)。所形成的戴帽結構稱為 type 0 戴帽結構。竹嵌紋病毒戴帽酵素的 H68A 突變株，有著優於野生型酵素的甲基轉移活性，但卻失去鳥苷轉移活性；如此的特性，有助於研究瞭解甲基轉移活性。為了探討具有保留性的芳香族胺基酸對於受質 (AdoMet 和 GTP) 的結合及甲基轉移活性的重要性，本實驗以 H68A 為基礎，將芳香族胺基酸進行胺基酸定點突變。發現 Y126、F144、F161、Y192、Y203、Y213 這些芳香族胺基酸突變對 GTP 被甲基化的活性的影響和結合 AdoMet 的影響是彼此相關的。一般來說，這些突變株降低了 GTP 甲基化的活性，也損害竹嵌紋病毒在植物細胞中的累積。 有鑒於酵母菌運用在尋找病毒的宿主因子之方便性，本實驗企圖建立一個在酵母菌中可讓病毒複製的系統來研究竹嵌紋病毒的複製。pHGB 質體含有竹嵌紋病毒 cDNA，並利用 GAP (Glyceraldehyde-3-phosphate dehydrogenase) 啓動子在酵母菌中轉錄完整的病毒 RNA。將此質體送入不同的酵母菌株中，發現由病毒 RNA 中轉譯架構 5 的外鞘蛋白質可於這些酵母菌中累積。此外，將此質體中對病毒 RNA 聚合酵素活性重要的 GDD motif 剔除後，外鞘蛋白質的累積量急遽減少。但是，在病毒類解旋酵素中的重要胺基酸 GKS 突變卻不影響外鞘蛋白質的累積。因此，推測病毒在酵母菌中的外鞘蛋白質累積與 RNA 聚合酵素的功能性有關。此外，同為馬鈴薯病毒 X 屬的 PVX (Potato virus X) 及 FoMV (Foxtail mosaic virus) 等病毒則無法在此系統中累積其外鞘蛋白質。Bamboo mosaic virus (BaMV), a member of the Potexvirus of the alpaviurslike superfamily, is a positive-strand RNA virus. The genome of BaMV consists of five open reading frames (ORFs), and the ORF 1 encodes a 155-kDa replicase, which could be separated into a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain (RdRp) from N to C terminus. The alphavirus-like superfamily has a special pathway for cap formation, by which the capping enzyme will first methylate GTP to generate m7GTP (methyltransferase activity) and transfer the m7GMP moiety from m7GTP to the 5'-diphosphate end of RNA (guanylyltransferase activity). The H68A mutant of BaMV capping enzyme has an increased methyltransferase activity than wildtype, but lose the guanylyltransferase activity; therefore, it represents a better target for the study of methyltransferase. A number of aromatic residues are conserved among the capping enzyme of the alphavirus-like superfamily. In order to understand importance of the consensus residues in substrate affinity (AdoMet and GTP) and GTP methylation, each of the residues was mutated on the basis of H68A. The changes in GTP methylation are correlated with the changes in AdoMet affinity based on the mutation effects of Y126, F144, F161, Y192, Y203, and Y213. In general, most mutants decrease the activity of GTP methylation, and injure the viral accumulation in plant, too. Studying viral replication by using yeast as a host is a convenient way to find out the host factors; therefore, the system was attempted to be set up for studying the replication of BaMV. Plasmid, pHGB, containing the BaMV cDNA downstream the GAP (Glyceraldehyde-3-phosphate dehydrogenase) promoter, was constructed to drive the transcription of the complete genome RNA of BaMV in yeast. Several different yeast strains were transformed with the plasmid, and the coat protein (CP), which is encoded by the ORF 5 of the viral RNA, could be accumulated in these yeasts. Furthermore, deletion of the GDD motif, which is important for RdRp activity dramatically decreased the amount of CP. But the mutation of GKS, which is important residues for HLD, did not affect the accumulation of CP. The results implied that the accumulation of CP was related to the function of RdRp. However, PVX (Potato virus X) and FoMV (Foxtail mosaic virus) which both belong to the Potexvirus genus could not accumulate their CP in this system.第一章 序言 一、竹嵌紋病毒.............................................................................................1 二、病毒的戴帽途徑.....................................................................................1 三、關於竹嵌紋病毒戴帽酵素.....................................................................3 四、RNA 病毒在宿主細胞中的複製............................................................4 五、竹嵌紋病毒的宿主因子.........................................................................5 第二章 竹嵌紋病毒戴帽酵素中保留性芳香族胺基酸殘基突變對於鳥苷三 磷酸被甲基化的影響 第一節 前言..................................................................................................6 第二節 材料與方法 一、質體的構築...........................................................................................8 二、質體轉形...............................................................................................8 三、酵母菌表達戴帽酵素之培養及誘導..................................................9 四、戴帽酵素萃取及純化...........................................................................9 五、甲基轉移酶的活性分析.....................................................................10 六、鳥苷轉移酶活性分析.........................................................................11 七、偵測戴帽酵素對受質結合能力.........................................................11 八、菸草原生質體之轉染.........................................................................12 九、白蔾接種.............................................................................................14 十、蛋白質電泳分析.................................................................................15 十一、西方墨點法.....................................................................................15 十二、戴帽結構的確認.............................................................................15 第三節 實驗結果 一、甲基轉移酶活性對於甲基接受者的專一性.....................................17 二、對甲基轉移酶活性重要的芳香族胺基酸.........................................18 三、戴帽酵素突變株對受質的親和力.....................................................19 四、對於竹嵌紋病毒在植物細胞中累積的重要芳香族胺基酸.............20 五、確認 F384A 突變株酵素的鳥苷轉移酶活性....................................21 第四節 討論 一、GDP 甲基化的生理意義....................................................................22 二、芳香族胺基酸的重要性.....................................................................22 三、F384A 可能的角色.............................................................................23 第三章 在酵母菌中建立病毒複製系統 第一節 前言................................................................................................25 第二節 材料與方法 一、質體的構築.........................................................................................27 二、質體的轉形.........................................................................................27 三、酵母菌總蛋白質的萃取.....................................................................28 第三節 實驗結果 一、竹嵌紋病毒可在酵母菌中測得外鞘蛋白質.....................................29 二、pHGBG 及 pHGBGRz 在酵母菌中外鞘蛋白質累積量相同...........29 三、PVX 及 FoMV 無法在酵母菌中測得外鞘蛋白質............................29 四、竹嵌紋病毒重要胺基酸突變株.........................................................29 五、改造外鞘蛋白質的轉譯架構.............................................................30 第四節 討論 一、竹嵌紋病毒於酵母菌中的複製.........................................................31 二、外鞘蛋白質的改造 ............................................................................31 參考文獻...........................................................................................................5

Integrated OMICs Approach for the Group 1 Protease Mite-Allergen of House Dust Mite <i>Dermatophagoides microceras</i>

House dust mites (HDMs) are one of the most important allergy-causing agents of asthma. In central Taiwan, the prevalence of sensitization to Dermatophagoides microceras (Der m), a particular mite species of HDMs, is approximately 80% and is related to the IgE crossing reactivity of Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f). Integrated OMICs examination was used to identify and characterize the specific group 1 mite-allergic component (Der m 1). De novo draft genomic assembly and comparative genome analysis predicted that the full-length Der m 1 allergen gene is 321 amino acids in silico. Proteomics verified this result, and its recombinant protein production implicated the cysteine protease and α chain of fibrinogen proteolytic activity. In the sensitized mice, pathophysiological features and increased neutrophils accumulation were evident in the lung tissues and BALF with the combination of Der m 1 and 2 inhalation, respectively. Principal component analysis (PCA) of mice cytokines revealed that the cytokine profiles of the allergen-sensitized mice model with combined Der m 1 and 2 were similar to those with Der m 2 alone but differed from those with Der m 1 alone. Regarding the possible sensitizing roles of Der m 1 in the cells, the fibrinogen cleavage products (FCPs) derived from combined Der m 1 and Der m 2 induced the expression of pro-inflammatory cytokines IL-6 and IL-8 in human bronchial epithelium cells. Der m 1 biologically functions as a cysteine protease and contributes to the α chain of fibrinogen digestion in vitro. The combination of Der m 1 and 2 could induce similar cytokines expression patterns to Der m 2 in mice, and the FCPs derived from Der m 1 has a synergistic effect with Der m 2 to induce the expression of pro-inflammatory cytokines in human bronchial epithelium cells

Integrated OMICs Approach for the Group 1 Protease Mite-Allergen of House Dust Mite Dermatophagoides microceras

House dust mites (HDMs) are one of the most important allergy-causing agents of asthma. In central Taiwan, the prevalence of sensitization to Dermatophagoides microceras (Der m), a particular mite species of HDMs, is approximately 80% and is related to the IgE crossing reactivity of Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f). Integrated OMICs examination was used to identify and characterize the specific group 1 mite-allergic component (Der m 1). De novo draft genomic assembly and comparative genome analysis predicted that the full-length Der m 1 allergen gene is 321 amino acids in silico. Proteomics verified this result, and its recombinant protein production implicated the cysteine protease and &alpha; chain of fibrinogen proteolytic activity. In the sensitized mice, pathophysiological features and increased neutrophils accumulation were evident in the lung tissues and BALF with the combination of Der m 1 and 2 inhalation, respectively. Principal component analysis (PCA) of mice cytokines revealed that the cytokine profiles of the allergen-sensitized mice model with combined Der m 1 and 2 were similar to those with Der m 2 alone but differed from those with Der m 1 alone. Regarding the possible sensitizing roles of Der m 1 in the cells, the fibrinogen cleavage products (FCPs) derived from combined Der m 1 and Der m 2 induced the expression of pro-inflammatory cytokines IL-6 and IL-8 in human bronchial epithelium cells. Der m 1 biologically functions as a cysteine protease and contributes to the &alpha; chain of fibrinogen digestion in vitro. The combination of Der m 1 and 2 could induce similar cytokines expression patterns to Der m 2 in mice, and the FCPs derived from Der m 1 has a synergistic effect with Der m 2 to induce the expression of pro-inflammatory cytokines in human bronchial epithelium cells