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National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 獸醫學系所 > 依資料類型分類 > 碩博士論文 >  麻疹病毒屬之病毒融合基因5′未轉譯區RNA之序列及功能分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/155299

標題: 麻疹病毒屬之病毒融合基因5′未轉譯區RNA之序列及功能分析
Sequence analysis and functional characterization of 5′ UTRs of morbillivirus fusion genes
作者: 宋光男
Chulakasian, Songkhla
Contributors: 張天傑
Tien-Jye Chang
獸醫學系暨研究所
關鍵字: 麻疹病毒;犬瘟熱病毒;小反芻獸病毒;融合基因;轉譯增進功能;5′未轉譯區
morbillivirus;canine distemper virus;peste des peptits ruminants virus;Fusion gene;translational enhancer
日期: 2013
Issue Date: 2013-11-21 11:50:11 (UTC+8)
Publisher: 獸醫學系暨研究所
摘要: 麻疹病毒屬中,各病毒的融合基因 (fusion gene, F) 及其五端未轉錄區 (5’ UTR) 的核酸長度及核酸序列具高度變異性。其中,犬瘟熱病毒 (CDV) 具有最長的訊息胜肽 (signal peptide, Fsp) 序列,且在不同犬瘟熱病毒株中具有高基因變異。 此外,小反芻獸病毒 (PPRV) 融合基因之 5’ UTR含有最長的高GC核酸序列。本論文即分別對此兩病毒之F基因五端未轉錄區之序列特性以及功能進行一系列之分析。

在本篇論文的第一部分(章節二):於少數的犬瘟熱病毒株中發現一高頻率變異區域,介於病毒基質基因 (matrix gene, M) 的三端未轉錄區 (3’UTR)與融合基因之間 (此區域命名為M-F UTR) ,針對此高變異區,我們建立了一個擴增阻礙性變異分析 (amplification refractory mutation analysis) 以應用於區別診斷不同的CDV分離株。頻繁的多型性序列現象散發存在於整個M-F UTR區域中:地方性病毒株與疫苗株的核酸序列只具有82.5% 到93.8% 相同性 (similarity) 。此現象可窺見於融合基因 (F gene) 起始密碼子下游第35個核酸的位置,三個連續的腺苷核醣核酸 (AAA) 被高度保留於三個疫苗株當中,然而於地方病毒株中則被取代為TGC序列;因此,可視為設計區別診斷引子的理想標的序列。基於此變異而建立的診斷方法可成功的區別台灣本土野外型犬瘟熱病毒株,以及不同的疫苗株型別。此方法提供了有力的臨床應用,例如確診犬瘟熱病毒感染,與評估疫苗免疫狀態和辨別目前野外循環病毒基因型別。

在第二部分(章節三)中,對小反芻獸病毒 (PPRV) F基因五端未轉錄區中具有高GC比例的區段進行序列分析,結果顯示未轉錄區的近末端 (proximal end, 85核苷酸) 存有一穩定的RNA二級結構,當中包含九個核糖核酸完全與18S rRNA互補的序列,推測可能藉由與mRNA和rRNA的交互作用來影響蛋白轉譯能力。基於這些特性,針對小反芻獸病毒五端未轉錄區在蛋白轉譯之調控,進行進實驗探討,同時亦與其他兩種麻疹病毒進行比較研析。報導基因分析結果顯示,小反芻獸病毒之融合基因的五端未轉錄區具強力的蛋白轉譯增進能力,且此功能不具細胞或基因專一性。以北方墨點分析轉染細胞中累積的報導基因RNA量及RNA穩定度,推論經由五端未轉錄區驅使造成的基因表現增高是由於mRNA量的增加和mRNA穩定度增進所形成。分析刪除未轉錄區的局部區段確認18S rRNA互補序列和遠端的核酸序列區域 (28-85核苷酸) 的存在是增進活性所必須,亦顯示出RNA的二級結構構型是一重要的因素。結論,於實驗中的結果,強烈顯示出小反芻獸病毒之融合基因的五端未轉譯區的蛋白轉譯增進功能,為藉由促進蛋白轉譯效率和mRNA的穩定度來達成。

總結,在此論文中描述了麻疹病毒屬中融合基因的基因多變性,演化上的流行病學和病毒的地理上的分布。另外,在麻疹病毒中存在的不同的轉譯調控現象,進一步顯示出病毒採用多變的策略來控制自身的基因表現。
The fusion (F) gene and 5′ untranslated region (UTR) of morbilliviruses have a high variation of nucleic acid sequence and length. Sequence analysis revealed that F gene of canine distemper virus (CDV) has a longest signal peptide (Fsp) sequence with the highest genetic variation among different CDV strains, while peste des peptits ruminants virus (PPRV) has a longest F 5′ UTR with the highest GC content amid morbilliviruses.

In the first part of this the thesis (Chapter 2) the high frequency variations have been found in the region spanning from the 3′ UTR of the matrix (M) gene to the F gene (designated M-F UTR) in different CDV strains. This highly variable region provides an advantage for establishing a differential diagnosis assay, an amplification refractory mutation analysis. Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the Taiwan isolates; that severed as target sequences for design of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.

In the second part of this thesis (Chapter 3) an unusual long 5′ UTR with a high GC content of PPRV-F gene was analyzed and results suggest that the proximal end of this UTR contains stable RNA secondary structure with a 9-nt sequence which could perfectly complement to 18S rRNA and might affect translation through mRNA-rRNA interaction. Based on these features, the functional role of the proximal PPRV-F 5′ UTR on translational efficiency compared with other two morbilliviruses was examined. By using reporter gene assays, PPRV-F 5′ UTR functioned as a strong enhancer of translational efficiency independent of cell- and gene-specificity. Northern blotting analysis of the accumulative RNA levels and mRNA stability suggested that elevated gene expression driven by PPRV-F 5′ UTR was accompanied by an increased mRNA level and enhanced mRNA stability. Deletion analysis identified the complementary sequence and distal nucleotides are necessary for the enhancing activity, and results suggest RNA structural conformation is important. Taken together, data obtained from this study strongly suggest that the proximal PPRV-F 5′ UTR functions as a translational enhancer by promoting translation efficiency and mRNA stability.

In summary, this thesis described the genetic diversity of morbillivirus-F gene, the evolutionary epidemiology and geographical distribution of virus. Moreover, the different translation regulatory mechanism among morbilliviruses further emphasizes their various strategies to control gene expression.
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